TW202536173A - Rna-guided nucleases and active fragments and variants thereof and methods of use - Google Patents

Abstract

Compositions and methods for binding to a target sequence of interest are provided. The compositions find use in cleaving or modifying a target sequence of interest, visualization of a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease （RGN） polypeptides, CRISPR RNA, guide RNA, and nucleic acid molecules encoding the same. Vectors and host cells comprising the nucleic acid molecules are also provided. Further provided are RGN systems for binding a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNA.

Description

RNA-guided nucleases, their active fragments and variants, and methods of use.

Cross-reference to related applications This application claims priority to U.S. Provisional Application No. 63/591,255, filed October 18, 2023, and U.S. Provisional Application No. 63/703,673, filed October 4, 2024, each of which is incorporated herein by reference in its entirety.

Reference to a Sequence List Submitted Electronically in XML File This application contains a sequence list submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on October 10, 2024, and is named L103438_1370WO_0313_1_SL, containing 3,630,310 bytes.

This invention relates to the fields of molecular biology and gene editing.

Targeted genome editing or modification is rapidly becoming an important tool in both basic and applied research. Early approaches involved engineered nucleases such as meganucleases, zinc finger fusion proteins, or TALENs, requiring the creation of chimeric nucleases with engineered, programmable, sequence-specific DNA-binding domains specific to each target sequence. RNA-guided nucleases (such as proteins in the CRISPR-Cas bacterial system, which uses clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) sequences) allow for specific sequence targeting by complexing the nuclease with guide RNA (which specifically hybridizes to a target sequence). Generating target-specific guide RNA is less costly and more efficient than creating chimeric nucleases for each target sequence. This RNA-guided nuclease can be used to selectively edit the genome via the introduction of a sequence-specific doublet break, which is repaired by error-prone non-homologous end-joining (NHEJ) to introduce a mutation at a specific genomic site. Alternatively, heterologous DNA can be introduced into the genomic site via homology-guided repair. RNA-guided nucleases (RGNs) can also be used for base editing when fused with deamineases.

This invention provides compositions and methods for binding to a target sequence of interest in a target nucleic acid molecule. These compositions can be used to cleave or modify a target nucleic acid molecule of interest, detect a target sequence of interest, and modify the expression of a gene of interest including the target sequence. The compositions include RNA-guided nuclease (RGN) peptides, CRISPR RNA (crRNA), guide RNA (gRNA), coding RNA-guided nuclease (RGN) peptides, CRISPR RNA (crRNA), guide RNA (gRNA), nucleic acid molecules including RNA-guided nuclease (RGN) peptides, CRISPR RNA (crRNA), guide RNA (gRNA), and vectors and host cells including the nucleic acid molecules. Also provided are an RGN system and ribonucleoprotein complex for binding to a target sequence of interest, wherein the RGN system and ribonucleoprotein complex include an RNA-guided nuclease polypeptide and one or more guide RNAs, wherein the RGN system and ribonucleoprotein complex are active without the need for tracrRNA. Thus, the method disclosed herein is selected for binding to a target sequence of interest in a target nucleic acid molecule, and in some embodiments, for cleaving or modifying the target nucleic acid molecule of interest, since it is active without the need for tracrRNA. The target nucleic acid molecule of interest can be modified, for example, by non-homologous terminal ligation, homology-guided repair of an introduced donor sequence, or base editing. The RGN polypeptide and RGN system described herein can bind to a site adjacent to and positioned at the 5' protospacer adjacent motif (PAM) site of the target nucleic acid molecule of interest.

Those skilled in the art to which this invention pertains, who will benefit from the teachings presented in the foregoing description and relational diagrams, will conceive of numerous modifications and other embodiments of the invention as explained herein. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended embodiments. Although specific terminology is used herein, such terminology is used only in a general and descriptive sense and not for limiting purposes. I. Summary

RNA-guided nucleases (RGNs) allow for targeted manipulation of one or more specific sites within the genome and are useful for therapeutic and research applications in the context of gene targeting. RGNs have been used in genome engineering in various organisms, including mammals, for example, by stimulating non-homologous terminal connections and homologous recombination.

In particular, in various embodiments, the RGNs and systems disclosed herein (e.g., RGNs and corresponding gRNAs) include V-H type clustered regularly spaced short palindromic repeat (CRISPR) enzymes and V-J type clustered regularly spaced short palindromic repeat (CRISPR) enzymes. The V-H and V-J types described herein are of particular interest, at least due to their smaller size (approximately 900 amino acids or less) and atypical prosepta adjacent motif (PAM) sequences compared to most characterized type II CRISPR enzymes. The V-H type system described herein uses an AT-rich PAM at the 5' end of the target sequence, compared to the type II system which uses a PAM at the 3' end of the target sequence. Similarly, the V-J type system described herein uses a T-rich PAM at the 5' end of the target sequence. The smaller size of type V nucleases allows for delivery via adeno-associated viral (AAV) vectors used in preclinical and clinical settings. Furthermore, because the type V system described herein uses only CRISPR RNA (crRNA) and does not require transactivating CRISPR RNA (tracrRNA), its associated guide RNA is relatively compact (approximately 40 nucleotides or less) compared to type II guide RNA. The smaller guide RNA of the type V editing system described herein allows for easier production of guide RNA with higher purity. In various embodiments, the type V enzymes described herein create overhangs (i.e., staggered) cuts in nucleic acid molecules, which are well-suited for the insertion of donor polynucleotides. Importantly, because the V-type guide RNAs described in this paper are self-processing and relatively short, these characteristics can be used to create constructs with multiple V-type guide RNAs in tandem arrangement for editing multiple targets. The V-type system described in this paper also functions in mammalian cells, which has not been demonstrated in the literature on the V-H type. In addition to their biochemical characteristics, the V-H subtype has attracted particular attention due to its relative rarity in sequenced genomes. Because annotating its CRISPR locus using the traditional CRISPR annotation pipeline is more challenging, this subtype is poorly described in the literature, with only one known case (Yan et al. (2019) Science 363(6422):88-91), which uses RTR PAM (especially TG-rich PAM) for dsDNA binding, and to the inventors' knowledge, its editing activity has not been reported. Therefore, the highly active V-H type enzyme and system described in this paper represent a significant contribution to this field.

The compositions and methods described herein are useful for creating breaks in polynucleotides, modifying polynucleotides, detecting specific sites within polynucleotides, or modifying the expression of specific genes. In certain embodiments, the compositions and methods described herein are useful for creating double-strand breaks in polynucleotides, modifying polynucleotides, detecting specific sites within polynucleotides, or modifying the expression of specific genes.

This paper discloses that RGNs can alter gene expression by modifying target nucleic acid molecules, including target sequences. In a specific embodiment, RGNs are guided to the target sequence (e.g., the target DNA sequence) via guide RNA (gRNA) as a part of the CRISPR RGN system. RGNs are considered "RNA-guided" because the guide RNA forms a complex with the RGN to guide the RGN to bind to the target sequence, and in some embodiments, a double-strand break is introduced at the target sequence (e.g., the target DNA sequence). After the target sequence is cleaved, the break can be repaired, so that the sequence of the target nucleic acid molecule is modified during the repair process. Thus, this paper provides a method for modifying target nucleic acid molecules in host cells using RGNs. For example, RGNs can be used to modify target sequences at genomic loci in eukaryotic or prokaryotic cells. II. RNA-guided nucleases

This article describes RNA-guided nucleases. The term "RNA-guided nuclease (RGN)" refers to a guide RNA molecule that binds to a specific target sequence (e.g., a target DNA sequence) in a sequence-specific manner and hybridizes with the target sequence by complexing with a polypeptide. While RGNs may be capable of cleaving the target sequence upon binding, the term RGN also includes nuclease-dead RGNs capable of binding to the target sequence but not cleaving it. Cleavage of the target sequence by an RGN can result in single-stranded or double-stranded breakage. RGNs capable only of cleaving the single strand in a double-stranded nucleic acid molecule are referred to herein as cleavage enzymes.

The RNA-guided nucleases disclosed in this article include LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, and LPG131. 00, LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, and LPG13116 RGN (the amino acid sequences of which are shown in SEQ ID NO: 1 to 4 and 1930 to 1956, respectively), and their active fragments or variants that retain the ability to bind to the target sequence in an RNA-guided sequence-specific manner. Among these implementation methods are LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, and LPG1310. 0. Active fragments or variants of LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, or LPG13116 RGN are capable of cleaving double-stranded target sequences. In some implementations, LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, and LPG13 100, LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, or The active variant of LPG13116 RGN includes an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in any of SEQ ID NO: 1 to 4 and 1930 to 1956.

In some embodiments, the active variant of LPG10238 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10238 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10238 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10238 RGN includes an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG10239 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10239 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10239 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10239 RGN includes an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG10240 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10240 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10240 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10240 RGN includes an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG10241 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10241 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10241 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10241 RGN includes an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13090 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1930, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13090 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1930, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13090 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1930, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13090 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1930 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13091 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1931, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13091 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1931, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13091 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1931, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13091 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1931 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13092 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1932, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13092 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1932, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13092 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1932, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13092 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1932 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13093 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1933, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13093 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1933, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13093 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1933, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13093 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1933 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13094 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1934, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13094 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1934, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13094 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1934, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13094 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1934 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13095 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1935, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13095 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1935, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13095 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1935, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13095 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1935 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13096 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1936, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13096 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1936, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13096 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1936, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13096 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1936 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13097 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1937, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13097 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1937, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13097 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1937, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13097 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1937 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13098 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1938, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13098 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1938, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13098 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1938, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13098 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1938 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13099 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1939, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13099 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1939, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13099 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1939, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13099 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1939 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13100 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1940, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13100 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1940, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13100 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1940, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13100 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1940 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13101 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1941, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13101 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1941, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13101 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1941, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13101 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1941 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13102 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1942, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13102 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1942, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13102 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1942, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13102 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1942 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13103 RGN comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence listed in SEQ ID NO: 1943, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13103 RGN comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence listed in SEQ ID NO: 1943, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13103 RGN comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence listed in SEQ ID NO: 1943, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13103 RGN comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence listed in SEQ ID NO: 1943, and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13104 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1944, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13104 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1944, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13104 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1944, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13104 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1944 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13105 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1945, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13105 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1945, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13105 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1945, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13105 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1945 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13106 RGN comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence listed in SEQ ID NO: 1946, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13106 RGN comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence listed in SEQ ID NO: 1946, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13106 RGN comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence listed in SEQ ID NO: 1946, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13106 RGN comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence listed in SEQ ID NO: 1946, and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13107 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1940, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13107 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1947, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13107 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1947, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13107 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1947 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13108 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1948, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13108 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1948, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13108 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1948, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13108 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1948 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13109 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1949, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13109 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1949, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13109 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1949, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13109 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1949 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13110 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1950, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13110 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1950, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13110 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1950, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13110 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1950 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13111 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1951, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13111 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1951, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13111 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1951, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13111 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1951 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13112 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1952, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13112 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1952, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13112 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1952, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13112 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1952 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13113 RGN includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1953, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13113 RGN includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1953, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13113 RGN includes an amino acid sequence that has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1953, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13113 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1953 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13114 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1954, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13114 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1954, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13114 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1954, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13114 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1954 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13115 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1955, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13115 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1955, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13115 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1955, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13115 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1955 and retains RNA-guided sequence-specific binding activity.

In some embodiments, the active variant of LPG13116 RGN includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1956, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13116 RGN includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1956, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13116 RGN includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1956, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG13116 RGN includes an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1956 and retains RNA-guided sequence-specific binding activity.

Retaining or possessing RNA-guided sequence-specific binding activity means that the RGN peptide is capable of binding to the target nucleic acid molecule in an RNA-guided sequence-specific manner, and can be measured by binding assays known in the art and described herein. In some embodiments, LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, and LPG131 are disclosed herein. LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, or LPG13116 The active variant or fragment of the RGN, or the RNA-guided sequence-specific binding activity of the active variant or fragment thereof, is approximately 80% to approximately 500%, approximately 80% to approximately 200%, or approximately 90% to approximately 150%, or approximately 95% to approximately 120%, or at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, or at least 102% of the RNA-guided sequence-specific binding activity of the reference RGN. At least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or higher.

The RGN peptides disclosed herein may retain or possess nuclease activity. Nuclease activity can be measured using methods known in the art and described herein. In some embodiments, LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, and LPG131 disclosed herein... 00, LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, or LPG13116 are active variants or fragments of RGN, or the nuclease activity of such active variants or fragments is the reference RGN. The nuclease activity of RGN is approximately 80% to approximately 500%, approximately 80% to approximately 200%, or approximately 90% to approximately 150%, or approximately 95% to approximately 120%, or at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or higher.

In some embodiments, the reference RGN has an amino acid sequence as shown in any of SEQ ID NO: 1 to 4 and 1930 to 1956. In some embodiments, the reference RGN is a variant of any of SEQ ID NO: 1 to 4 and 1930 to 1956 lacking a corresponding mutation of an active variant or fragment having, for example, RNA-guided sequence-specific binding activity or nuclease activity as described above. In some embodiments, the reference RGN is a distinct variant RGN. In some embodiments, the reference RGN is a distinct variant RGN sharing the same parental RGN. "Parental RGN" or "parental RGN polypeptide" refers to a parent polypeptide that can act as a template to introduce one or more mutations to produce the parent polypeptide. For example, LPG10238 is the parental RGN polypeptide of the mutant RGN polypeptides LPG10427 to LPG10821 and LPG12222, as discussed in Example 6. Other parental RGN polypeptides disclosed herein include LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, LPG13100, L... PG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, and LPG13116.

The active fragment of LPG10238 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1. The active fragment of LPG10239 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 2. The active fragment of LPG10240 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 3. The active fragment of LPG10241 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 4. The active fragment of LPG13090 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1930. The active fragment of LPG13091 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1931. The active fragment of LPG13092 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1932. The active fragment of LPG13093 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1933. The active fragment of LPG13094 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1934. The active fragment of LPG13095 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1935. The active fragment of LPG13096 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1936. The active fragment of LPG13097 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1937. The active fragment of LPG13098 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1938. The active fragment of LPG13099 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1939. The active fragment of LPG13100 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1940. The active fragment of LPG13101 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1941. The active fragment of LPG13102 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1942. The active fragment of LPG13103 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1943. The active fragment of LPG13104 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1944. The active fragment of LPG13105 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1945. The active fragment of LPG13106 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1946. The active fragment of LPG13107 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1947. The active fragment of LPG13108 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1948. The active fragment of LPG13109 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1949. The active fragment of LPG13110 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1950. The active fragment of LPG13111 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1951. The active fragment of LPG13112 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1952. The active fragment of LPG13113 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1953. The active fragment of LPG13114 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1954. The active fragment of LPG13115 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1955. The active fragment of LPG13116 RGN may include at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or more consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1956.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence (LPG10238) shown in SEQ ID NO: 1, wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 1. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are positively charged amino acid residues. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are arginine (R). In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of the RGN polypeptide are R. The RGN polypeptide of this disclosure may include an amino acid sequence shown in any of SEQ ID NO: 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 602, 707, 708, and 717.

When referring to a variant RGN that "the amino acid residues at one or more of the amino acid positions of the RGN polypeptide described therein differ from the corresponding amino acid residues of SEQ ID NO: ...", it is intended to indicate that, except that the amino acid residues at the listed positions differ from the corresponding amino acid residues of SEQ ID NO: ..., the variant RGN comprises an amino acid sequence identical to the amino acid sequence shown in SEQ ID NO: .... When referring to amino acid positions, the amino acid positions are numbered sequentially starting from the amino terminus of the given polypeptide. The first amino acid residue at the amino terminus of the polypeptide is designated as position 1. In some embodiments, the first amino acid residue is methionine. Then, the position number of each amino acid residue increases from the amino end of the polypeptide to the carboxyl end of the polypeptide, and the last amino acid position is the last amino acid residue at the carboxyl end of the polypeptide.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 552, 677, and 801 of the RGN polypeptide are different from the corresponding amino acid residues in SEQ ID NO: 1. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 552, 677, and 801 of the RGN polypeptide are positively charged amino acid residues. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 552, 677, and 801 of the RGN polypeptide are R. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 552, 677, and 801 of the RGN polypeptide are R. The RGN polypeptide disclosed herein may include amino acid sequences as shown in any of SEQ ID NO: 450, 463, 471, 485, 602 and 717.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R): (a) amino acid positions 517, 530, 552 and 677; (b) amino acid positions 530, 538, 552 and 801; (c) amino acid positions 530, 552, and 801. (d) Amino acid positions 530, 577, and 790; (e) Amino acid positions 517, 530, 538, 552, and 677; (f) Amino acid positions 530, 538, 677, and 801; (g) Amino acid positions 538, 552, 677, and 801; (h) Amino acid positions 517, 530, 538, and 552; (i) Amino acid position 517 and 530; (j) amino acid positions 530, 538, and 552; (k) amino acid positions 517, 530, 538, and 677; (l) amino acid positions 517, 552, 677, and 801; (m) amino acid positions 530, 677, and 801; (n) amino acid positions 517, 530, 677, and 801; (o) amino acid positions 517, 530, and 677; (p) amino acid positions 530, 538, 552, and 677; (q) amino acid positions 517, 552, and 677; and (r) amino acid positions 517, 530, 552, 677, and 801. In some embodiments, the RGN polypeptide includes, as shown in SEQ ID NO: The amino acid sequence shown in 1 has an amino acid sequence with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R): (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; (c) amine (d) Amino acid positions 530, 552, 677 and 801; (e) Amino acid positions 517, 530, 538, 552 and 677; (f) Amino acid positions 530, 538, 677 and 801; (g) Amino acid positions 538, 552, 677 and 801; (h) Amino acid positions 517, 530, 538 and 552; (i) Amino acid position 517 and 530; (j) amino acid positions 530, 538 and 552; (k) amino acid positions 517, 530, 538 and 677; (l) amino acid positions 517, 552, 677 and 801; (m) amino acid positions 530, 677 and 801; (n) amino acid positions 517, 530, 677 and 801; (o) amino acid positions 517, 530 and 677; (p) amino acid positions 530, 538, 552 and 677; (q) amino acid positions 517, 552 and 677; and (r) amino acid positions 517, 530, 552, 677 and 801. The RGN polypeptide disclosed herein may include amino acid sequences as shown in any of SEQ ID NO: 1072, 1080, 1082, 1105, 1084, 1081, 1083, 1069, 1034, 1059, 1070, 1078, 1064, 1074, 1051, 1079, 1056 and 1087.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R): (a) amino acid positions 517, 530, 552 and 677; (b) amino acid positions 530, 538, 552 and 801; and (c) amino acid positions 530, 552, 677 and 801. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R): (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; and (c) amino acid positions 530, 552, 677, and 801. The RGN polypeptide of this disclosure may include the amino acid sequence shown in any one of SEQ ID NO: 1072, 1080, and 1082.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence (LPG10241) shown in SEQ ID NO: 4, wherein one or more amino acid residues at one or more of the amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of the amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are positively charged amino acid residues. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of the amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are R. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of the amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of the RGN polypeptide are R. The RGN polypeptide of this disclosure may include an amino acid sequence shown in any of SEQ ID NO: 809, 810, 866, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 1012, and 1019.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein one or more amino acid residues at one or more of amino acid positions 377, 625, 638, 691, 698, and 747 of the RGN polypeptide are different from the corresponding amino acid residues in SEQ ID NO: 4. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein one or more amino acid residues at one or more of amino acid positions 377, 625, 638, 691, 698, and 747 of the RGN polypeptide are positively charged amino acid residues. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 625, 638, 691, 698, and 747 of the RGN polypeptide are R. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 625, 638, 691, 698, and 747 of the RGN polypeptide are R. The RGN polypeptide disclosed herein may include amino acid sequences as shown in any of SEQ ID NO: 809, 898, 909, 960, 966 and 1012.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R): (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid positions 377, 625, and 691; (g) amino acid positions 377, 625, and 691; Amino acid positions 377, 625, 698 and 747; (h) amino acid positions 377, 638, 691 and 698; (i) amino acid positions 377, 625 and 638; (j) amino acid positions 377, 638, 698 and 747; (k) amino acid positions 377, 625, 638, 691 and 698; (l) amino acid positions 625, 638 and 698; (m) amino acid positions 377, 625, 691 and 698; (n) amino acid positions 377, 638 and 698; (o) amino acid positions 625, 691, 698 and 747; (p) amino acid positions 638 and 698; and (q) amino acid positions 377 and 691. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R): (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid positions 377, 638, 691, 698, and 747; Amino acid positions 377, 625, and 691; (g) Amino acid positions 377, 625, 698, and 747; (h) Amino acid positions 377, 638, 691, and 698; (i) Amino acid positions 377, 625, and 638; (j) Amino acid positions 377, 638, 698, and 747; (k) Amino acid positions 377, 625, 638, 691, and 698; (l) Amino acid positions 625, 638, and 698; (m) Amino acid positions 377, 625, 691, and 698; (n) Amino acid positions 377, 638, and 698; (o) Amino acid positions 625, 691, 698, and 747; (p) Amino acid positions 638 and 698; and (q) Amino acid positions 377 and 691. The RGN polypeptide disclosed herein may include amino acid sequences as shown in any of SEQ ID NO: 1123, 1158, 1159, 1148, 1160, 1122, 1146, 1147, 1121, 1149, 1156, 1132, 1144, 1126, 1154, 1116 and 1108.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R): (a) amino acid positions 377, 625 and 698; (b) amino acid positions 377, 625, 638, 698 and 747; and (c) amino acid positions 377, 625, 691, 698 and 747. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R): (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; and (c) amino acid positions 377, 625, 691, 698, and 747. The RGN polypeptide of this disclosure may include the amino acid sequence shown in any one of SEQ ID NO: 1123, 1158, and 1159.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the amino acid residues at one or more of the amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residues at one or more of the amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are positively charged amino acid residues. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residues at one or more of the amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are R or lysine (K). In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at one or more of the amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide is R or K.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at the amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide is arginine (R) and/or lysine (K): (a) amino acid positions 736, 743, 752, 753, 755, 757, 758 and 760; (b) amino acid positions 740, 747, 751, 753, 755, 760, 764 and 766; (c) amino acid positions 685, 691 and 698; and (d) amino acid positions 685, 692, 695, 702 and 707. The RGN polypeptide disclosed herein may include amino acid sequences as shown in any of SEQ ID NO: 1168, 1171, 1169 and 1170.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the RGN polypeptide includes the deletion of amino acid residues corresponding to amino acid residues 751 to 769 of SEQ ID NO: 4. In some embodiments, the RGN polypeptide disclosed herein may include an amino acid sequence shown in SEQ ID NO: 1173.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more of the amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596 and 862 of the RGN polypeptide include substitution or insertion of one or more glycine (G) in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more of the amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596, and 862 of the RGN polypeptide include one or more substitutions or insertions of G at the N-terminal position, at the amino acid position, or at the C-terminal position adjacent to the amino acid position. In some embodiments, the substitution or insertion of one or more Gs includes the substitution or insertion of one, two, or three Gs. In some embodiments, one or more G substitutions or insertions are selected from: (a) a three-G insertion between amino acid positions 293 and 294; (b) a two-G insertion between amino acid positions 359 and 360; (c) a G substitution at amino acid position 361; (d) a one-G insertion between amino acid positions 402 and 403; (e) a G substitution at amino acid position 401; (f) a G substitution at amino acid position 596; and (g) a G substitution at amino acid position 862. The RGN polypeptide disclosed herein may include the amino acid sequence represented by any one of SEQ ID NO: 1304, 1322, 1324, 1328, 1330, 1361, and 1400.

In the context of substitution or insertion of one or more Gs in an RGN polypeptide, the terms "immediately N-terminal to" and "immediately C-terminal to" refer to substitution or insertion at the first amino acid position relative to the listed amino acid position in the N-terminal direction or at the first amino acid position relative to the listed amino acid position in the C-terminal direction, respectively.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more of the amino acid positions 566, 567, and 584 of the RGN polypeptide include substitution or insertion of one or more glycine (G) at a position immediately adjacent to the amino acid position in the N-terminal direction, at the amino acid position, or at a position immediately adjacent to the amino acid position in the C-terminal direction. In some embodiments, the RGN polypeptide includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more of the amino acid positions 566, 567, and 584 of the RGN polypeptide include substitution or insertion of one or more G at a position immediately adjacent to the amino acid position in the N-terminal direction, at the amino acid position, or at a position immediately adjacent to the amino acid position in the C-terminal direction. In some embodiments, the substitution or insertion of one or more Gs includes the substitution or insertion of one, two, or three Gs. In some embodiments, the substitution or insertion of one or more Gs is selected from: (a) the substitution of G at amino acid position 584 and the substitution of G at amino acid position 567; and (b) the substitution of G at amino acid position 584. The RGN polypeptide of this disclosure may include an amino acid sequence selected from: (a) the amino acid sequence shown in SEQ ID NO: 1223; and (b) the amino acid sequence shown in SEQ ID NO: 1228.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the variant LPG10238 RGN polypeptide. The variant LPG10238 RGN polypeptide includes SEQ ID NO: The amino acid sequence of any one of the following: 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1304, 1322, 1324, 1328, 1330, 1361, 1400, and 2686, wherein the RGN polypeptide has RNA-guided sequence-specific binding activity. In some embodiments, the RGN polypeptide disclosed herein includes the amino acid sequence represented by any one of SEQ ID NO: 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1304, 1322, 1324, 1328, 1330, 1361, 1400, and 2686.

The RGN polypeptide disclosed herein may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the variant LPG10241 RGN polypeptide. The variant LPG10241 RGN polypeptide includes SEQ ID NO: The amino acid sequence of any one of 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228 and 2687, wherein the RGN polypeptide has RNA-guided sequence-specific binding activity. In some embodiments, the RGN polypeptide disclosed herein includes an amino acid sequence as shown in any of SEQ ID NO: 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, and 2687.

The RGN of this disclosure is bound to a target sequence (e.g., such as the target sequence disclosed herein). In some embodiments, the RGN of this disclosure, or an active variant or fragment thereof, identifies a target sequence at its 5' end on a non-targeted strand with a protoseptal adjacent motif (PAM) containing a common nucleotide sequence of AYG (where Y is C or T/U). In some embodiments, an RGN (LPG10238) having an amino acid sequence as shown in SEQ ID NO: 1, or an active variant or fragment thereof, identifies a target sequence at its 5' end on a non-targeted strand with a PAM containing a common nucleotide sequence as illustrated in Figure 3A.

In some embodiments, the RGN of this disclosure or its active variant or fragment identifies a PAM (where N is A, C, T/U, or G) containing a common nucleotide sequence at the 5' of the target sequence on its non-target strand. In some embodiments, the RGN (LPG10239) having the amino acid sequence shown in SEQ ID NO: 2 or its active variant or fragment identifies a PAM containing a common nucleotide sequence as illustrated in Figure 3B at the 5' of the target sequence on its non-target strand.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G; and V is A, G, or C). In some embodiments, the RGN (LPG10240) having the amino acid sequence shown in SEQ ID NO: 3 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3C.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG10241) having the amino acid sequence shown in SEQ ID NO: 4 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3D.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13090) having the amino acid sequence shown in SEQ ID NO: 1930 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3E.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of STTN at the 5' of the target sequence on its non-target strand (where S is C or G; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13091) having the amino acid sequence shown in SEQ ID NO: 1931 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of the target sequence on its non-target strand at the 5' of the target sequence as illustrated in Figure 3F.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13092) having the amino acid sequence shown in SEQ ID NO: 1932 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand, as illustrated in Figure 3G.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of ATTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13093) having the amino acid sequence shown in SEQ ID NO: 1933 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of ATTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3H.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13094) having the amino acid sequence shown in SEQ ID NO: 1934 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3I.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTH at the 5' of the target sequence on its non-target strand (where H is A, C, or T/U). In some embodiments, the RGN (LPG13095) having the amino acid sequence shown in SEQ ID NO: 1935 or its active variant or fragment thereof identified a PAM containing a common nucleotide sequence of the target sequence on its non-target strand at the 5' of the target sequence as illustrated in Figure 3J.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13096) having the amino acid sequence shown in SEQ ID NO: 1936 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3K.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of RTTN at the 5' of the target sequence on its non-target strand (where R is A or G; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13097) having the amino acid sequence shown in SEQ ID NO: 1937 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of RTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3L.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand (where V is A, G, or C; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13098) having the amino acid sequence shown in SEQ ID NO: 1938 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3M.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13099) having the amino acid sequence shown in SEQ ID NO: 1939 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand, as illustrated in Figure 3N.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13100) having the amino acid sequence shown in SEQ ID NO: 1940 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3O.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13101) having the amino acid sequence shown in SEQ ID NO: 1941 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3P.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13102) having the amino acid sequence shown in SEQ ID NO: 1942 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3Q.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand (where V is A, G, or C; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13103) having the amino acid sequence shown in SEQ ID NO: 1943 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3R.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of ATTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13104) having the amino acid sequence shown in SEQ ID NO: 1944 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of ATTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3S.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand (where V is A, G, or C; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13105) having the amino acid sequence shown in SEQ ID NO: 1945 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3T.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13106) having the amino acid sequence shown in SEQ ID NO: 1946 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand, as illustrated in Figure 3U.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand (where V is A, G, or C; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13107) having the amino acid sequence shown in SEQ ID NO: 1947 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3V.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13108) having the amino acid sequence shown in SEQ ID NO: 1948 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3W.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand (where V is A, G, or C; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13109) having the amino acid sequence shown in SEQ ID NO: 1949 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of VTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3X.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of RTTN at the 5' of the target sequence on its non-target strand (where R is A or G; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13110) having the amino acid sequence shown in SEQ ID NO: 1950 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of RTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3Y.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of RTTN at the 5' of the target sequence on its non-target strand (where R is A or G; and N is A, C, T/U, or G). In some embodiments, the RGN (LPG13111) having the amino acid sequence shown in SEQ ID NO: 1951 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of RTTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3Z.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13112) having the amino acid sequence shown in SEQ ID NO: 1952 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand, as explained in Figure 3AA.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of ATG at the 5' of the target sequence on its non-target strand. In some embodiments, the RGN (LPG13113) having the amino acid sequence shown in SEQ ID NO: 1953 or its active variant or fragment identified has a PAM containing a common nucleotide sequence of ATG at the 5' of the target sequence on its non-target strand.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13114) having the amino acid sequence shown in SEQ ID NO: 1954 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3CC.

In some embodiments, the RGN or its active variant or fragment thereof identified in this disclosure has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand (where N is A, C, T/U, or G). In some embodiments, the RGN (LPG13115) having the amino acid sequence shown in SEQ ID NO: 1955 or its active variant or fragment thereof has a PAM containing a common nucleotide sequence of TTN at the 5' of the target sequence on its non-target strand as illustrated in Figure 3DD.

In some embodiments, the RGN of this disclosure or its active variant or fragment identifies a PAM (where N is A, C, T/U, or G) containing a common nucleotide sequence at the 5' of the target sequence on its non-target strand. In some embodiments, the RGN (LPG13116) having the amino acid sequence shown in SEQ ID NO: 1956 or its active variant or fragment identifies a PAM containing a common nucleotide sequence as illustrated in Figure 3EE at the 5' of the target sequence on its non-target strand.

As used in this paper with respect to the PAM recognition nucleotide motif, Y refers to cytosine (C) or thymine/uracil (T/U); N refers to adenine (A) or cytosine (C) or thymine/uracil (T/U) or guanine (G); V refers to adenine (A) or cytosine (C) or guanine (G); S refers to cytosine (C) or guanine (G); H refers to adenine (A) or cytosine (C) or thymine/uracil (T/U); and R refers to adenine (A) or guanine (G). In some embodiments, the active fragment or variant of the RGN that recognizes these PAM sequences is capable of binding to the target sequence, while in other embodiments, it is capable of cleaving or nicking the target sequence.

The RGNs provided herein may include at least one nuclease domain (e.g., DNase, RNase domain) and at least one RNA recognition domain and/or RNA binding domain to interact with guide RNA. In some embodiments, the type V RGNs disclosed herein include only one nuclease domain. In some embodiments, the nuclease domain of the type V RGN is a RuvC domain. In some embodiments, the type V RGNs disclosed herein do not include an HNH nuclease domain. Other domains that may be found in the RGNs provided herein include, but are not limited to: a DNA binding domain, a helicase domain, a protein-protein interaction domain, and a dimerization domain. In some embodiments, the RGN provided herein may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with one or more of the DNA binding domain, helicase domain, protein-protein interaction domain and dimerization domain of type V RGN.

The RGN peptide disclosed herein may include one or more of the following domains: a PAM interaction (PI) domain, a Rec domain (RecI, RecII), an oligo-binding domain (OBD), a zinc-binding domain (e.g., including a zinc finger), or a RuvC nuclease domain. The RuvC domain may include RuvCI, RuvCII, RuvCIII domains, or combinations thereof. The Rec or recognition domain mediates nucleic acid binding through multiple Rec domains (e.g., RecI to III). The general domain of the RGN peptide can be determined by structural comparison with an RGN peptide having a defined domain.

LPG10241 RGN (SEQ ID NO: 4) or its variants LPG10241 RGN (SEQ ID NO: 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228 and... Non-limiting examples of the domains within (2687) include: PI with amino acid residues 1 to 59; RecI with amino acid residues 74 to 216; OBD with amino acid residues 216 to 370; RuvC-1 with amino acid residues 370 to 445; RecII with amino acid residues 445 to 594; RuvC-II with amino acid residues 594 to 681; zinc-bonded domains (ZF) with amino acid residues 681 to 710; and RuvC-III with amino acid residues 710 to 770. The domain organization of the labeled LPG10241 is shown in Figure 4A.

LPG10238 RGN (SEQ ID NO: 1) or its variant LPG10238 RGN (SEQ ID NO: 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 108 Non-limiting examples of the domains within (7, 1105, 1304, 1322, 1324, 1328, 1330, 1361, 1400, and 2686) include: RecI with amino acid residues 66 to 160; RuvC-1 with amino acid residues 473 to 527; RecII with amino acid residues 527 to 667; RuvC-II with amino acid residues 667 to 725; and RuvC-III with amino acid residues 732 to 889. The domain configuration of the labeled LPG10238 is shown in Figure 4B.

The RGN or its active variants or fragments disclosed herein may have a PAM interaction (PI) domain, which contributes to the recognition of PAM sites in target nucleic acid molecules. The PI domain may include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acid residues. In some embodiments, the PI domain of the RGN or its active variants or fragments disclosed herein is located within the N-terminal region of the RGN. The N-terminal region of the PI domain of the RGN or its active variants or fragments, including the contents of this disclosure, may include: 100 amino acid residues at the N-terminus (i.e., amino acid residues 1 to 100 starting from the N-terminus of the RGN), 90 amino acid residues at the N-terminus, 80 amino acid residues at the N-terminus, 70 amino acid residues at the N-terminus, 60 amino acid residues at the N-terminus, 50 amino acid residues at the N-terminus, 40 amino acid residues at the N-terminus, 30 amino acid residues at the N-terminus, 20 amino acid residues at the N-terminus, or 10 amino acid residues at the N-terminus. In some embodiments, the PI domain of the RGN or its active variant or fragment thereof disclosed herein is within or contains amino acid residues 1 to 59 of the RGN (i.e., amino acid residues 1 to 59 starting from the N-terminus). In some embodiments, the PI domain of the RGN or its active variant or fragment thereof having the amino acid sequence shown in SEQ ID NO: 2 is within or contains amino acid residues 1 to 59 of the RGN. In some embodiments, the PI domain of the RGN or its active variant or fragment thereof having the amino acid sequence shown in SEQ ID NO: 3 is within or contains amino acid residues 1 to 59 of the RGN. In some embodiments, the PI domain of an RGN having an amino acid sequence as shown in SEQ ID NO: 4 or an active variant or fragment thereof is within amino acid residues 1 to 59 of the RGN or comprises amino acid residues 1 to 59 of the RGN.

The active variants or fragments of the LPG10238 RGN disclosed herein may include those with SEQ ID NO: 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1 The amino acid residues 1 to 472 of any one of 105, 1304, 1322, 1324, 1328, 1330, 1361, 1400 and 2686 have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identical, and may include amino acid residues that are 100% sequence identical to amino acid residues 473 to 889 of the responder LPG10238 RGN or have 1, 2, 3 or 4 amino acid residues different from amino acid residues 473 to 889.

The active variants or fragments of the LPG10241 RGN disclosed herein may include those with SEQ ID NO: 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160. The amino acid residues 1 to 369 of any one of 1168, 1169, 1170, 1171, 1173, 1223, 1228 and 2687 have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identical, and may include amino acid residues that are 100% sequence identical to amino acid residues 370 to 770 of the corresponding LPG10241 RGN or have 1, 2, 3 or 4 amino acid residues different from amino acid residues 370 to 770.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in AYG. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 5 or 1631, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in AYG.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 6, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 3, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 7, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 3, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 4, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 8 and 1617 to 1620, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 4, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1930, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1957, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1930, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1931, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in STTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1958, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1931, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in STTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1932, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1959, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1932, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1933, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1960, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1933, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1934, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1961, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1934, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1935, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in TTH. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1962, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1935, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in TTH.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1936, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1963, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1936, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1937, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1964, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1937, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1938, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1965, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1938, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1939, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1966, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1939, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1940, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1967, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1940, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1941, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1968, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1941, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1942, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1969, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1942, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1943, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1970, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1943, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1944, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1971, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1944, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1945, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1972, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1945, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1946, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1973, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1946, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1947, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1974, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1947, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1948, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1975, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1948, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1949, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1976, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1949, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1950, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1977, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1950, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1951, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTYN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1978, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1951, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTYN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1952, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1979, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1952, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1953, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in ATG. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1980, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1953, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in ATG.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1954, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1981, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1954, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1955, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1982, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1955, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1956, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1983, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1956, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In various embodiments, the target sequence is bound to the RGN provided herein. In those cases where the target sequence is double-stranded (e.g., double-stranded DNA), the target strand of the target sequence hybridizes with the guide RNA associated with the RGN. If the polypeptide has nuclease activity, the target strand and/or non-target strand of the target sequence (e.g., the target DNA sequence) may subsequently be cleaved by the RGN. The term "cleave" or "cleavage" refers to the hydrolysis of at least one phosphodiester bond within one or both strands of the backbone of the double-stranded target sequence (e.g., the target DNA sequence), which may result in the breakage of a single or double strand within the target sequence. The RGN disclosed in this invention can cleave nucleotides within a polynucleotide, acting as an endonuclease. Alternatively, the RGN disclosed in this invention can be an exonuclease, removing unbroken nucleotides from the ends (5' and/or 3' ends) of the polynucleotide. In some embodiments, the disclosed RGN can cleave nucleotides of the target polynucleotide at any position within the polynucleotide, thereby acting as both an endonuclease and an exonuclease. The cleavage of the target polynucleotide by the RGN disclosed in this invention can result in staggered breaks or blunt ends. A blunt cut produces two blunt ends, such that each blunt end of the cleaved nucleic acid molecule has two strands of equal length, that is, neither strand of the blunt end contains an unpaired base.

In some embodiments, the V-type RGN disclosed herein produces staggered cuts in nucleic acid molecules. Staggered cuts in nucleic acid molecules result in two sticky ends or overhanging ends, and are formed when the nuclease cleaves each strand of the nucleic acid molecule such that the cuts are not directly opposite each other. For each sticky end of the cleaved nucleic acid molecule, one strand (i.e., the overhanging strand) is longer than the other (usually by at least several nucleotides), such that the longer strand has unpaired bases. The longer strand of the protruding end of the cleaved nucleic acid molecule may have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more unpaired nucleotides. In some embodiments, the longer strand of the protruding end of the cleaved nucleic acid molecule may have one unpaired nucleotide. The protruding end of the cleaved nucleic acid molecule may be a 3' protrusion or a 5' protrusion. In some embodiments, the protruding end of the cleaved nucleic acid molecule is a 3' protrusion. In some embodiments, the protruding end of the cleaved nucleic acid molecule is a 5' protrusion. In some embodiments, the RGN of this disclosure or its active variant or fragment cleaves the target nucleic acid molecule to form an interleaved cut, wherein the interleaved cut creates a 3' protrusion with an unpaired nucleotide.

An RGN polypeptide (LPG10238) having the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, can cleave a double-stranded nucleic acid molecule to produce a 3' protrusion with one unpaired nucleotide. An RGN polypeptide (LPG10239) having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, can cleave a double-stranded nucleic acid molecule to produce a 5' protrusion with 10 unpaired nucleotides. An RGN polypeptide (LPG10240) having the amino acid sequence shown in SEQ ID NO: 3, or an active variant or fragment thereof, can cleave a double-stranded nucleic acid molecule to produce a 5' protrusion with 5 unpaired nucleotides. An RGN polypeptide (LPG10241) having the amino acid sequence shown in SEQ ID NO: 4, or an active variant or fragment thereof, can cleave a double-stranded nucleic acid molecule to produce a 5' protrusion with 12 unpaired nucleotides.

The RGN disclosed in this invention may be a wild-type sequence obtained from bacteria, viruses, or archaea. Alternatively, the RGN may be a variant or fragment of a wild-type polypeptide. For example, a wild-type RGN may be modified to alter nuclease activity or PAM specificity. In some embodiments, the RGN is not naturally occurring.

In some embodiments, RGNs can function as nickases, cleaving only one strand of a double-stranded target sequence (e.g., a target DNA sequence). These RGNs have a single-function nuclease domain. In certain embodiments, the nickase is capable of cleaving either the target strand or the untarget strand of a double-stranded target sequence (e.g., a target DNA sequence). In embodiments using nickases, to achieve efficient double-strand cleavage of a double-stranded target sequence (e.g., a target DNA sequence), two nickases are required, each nicking a single strand within the double-stranded target sequence.

In other embodiments, the RGN completely lacks nuclease activity and is referred to herein as catalytically dead, nuclease-dead, or nuclease-inactive. In some embodiments, for the type V RGN disclosed herein, which comprises only a RuvC nuclease domain, the enzyme in a catalytically dead, nuclease-dead, or nuclease-inactive form is used to modify nucleic acids, such as in base editing as described herein. Any known method in the art for introducing mutations into amino acid sequences (e.g., PCR-mediated mutagenesis and site-directed mutagenesis) can be used to produce nickase-free or nuclease-deactive RGNs. See, for example, U.S. Publication No. 2014/0068797 and U.S. Patent No. 9,790,490; each of these U.S. Publication and U.S. Patent is incorporated herein by reference in its entirety. For example, mutation of any of the three RuvC catalytic residues in the V-type RGN disclosed herein could produce an enzyme with no catalytic activity.

A V-type RGN comprising the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, may further comprise at least one of the following amino acid mutations to produce a non-catalytically active enzyme: D474A, E672A, D759A, wherein the indicated mutant amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 1. A V-type RGN comprising the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, may further comprise two or three of the following amino acid mutations to produce a non-catalytically active enzyme: D474A, E672A, D759A, wherein the indicated mutant amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 1. In some embodiments, the non-catalytically active RGN polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 2686.

A type V RGN, including the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, may further include at least one of the following amino acid mutations to produce a non-catalytically active enzyme: D474R, E672R, D759R, wherein the indicated mutated amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 1. In some embodiments, the non-catalytically active RGN polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in any of SEQ ID NO: 408, 597, and 678. V-type RGNs, including the amino acid sequence shown in SEQ ID NO: 1, or their active variants or fragments, may further include two or three of the following amino acid mutations to produce an enzyme without catalytic activity: D474R, E672R, D759R, wherein the indicated mutant amino acid residue corresponds to the amino acid residue of the sequence shown in SEQ ID NO: 1.

A V-type RGN comprising the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, may further comprise at least one of the following amino acid mutations to produce a non-catalytically active enzyme: D389A, E600A, D690A, wherein the indicated mutant amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 2. A V-type RGN comprising the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, may further comprise two or three of the following amino acid mutations to produce a non-catalytically active enzyme: D389A, E600A, D690A, wherein the indicated mutant amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 2.

A V-type RGN comprising the amino acid sequence shown in SEQ ID NO: 3, or an active variant or fragment thereof, may further comprise at least one of the following amino acid mutations to produce a non-catalytically active enzyme: D413A, E636A, D725A, wherein the indicated mutant amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 3. A V-type RGN comprising the amino acid sequence shown in SEQ ID NO: 3, or an active variant or fragment thereof, may further comprise two or three of the following amino acid mutations to produce a non-catalytically active enzyme: D413A, E636A, D725A, wherein the indicated mutant amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 3.

A V-type RGN comprising the amino acid sequence shown in SEQ ID NO: 4, or an active variant or fragment thereof, may further comprise at least one of the following amino acid mutations to produce a non-catalytically active enzyme: D396A, E622A, D711A, wherein the indicated mutant amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 4. A V-type RGN comprising the amino acid sequence shown in SEQ ID NO: 4, or an active variant or fragment thereof, may further comprise two or three of the following amino acid mutations to produce a non-catalytically active enzyme: D396A, E622A, D711A, wherein the indicated mutant amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 4. In some embodiments, the non-catalytically active RGN polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 2687.

A type V RGN, including the amino acid sequence shown in SEQ ID NO: 4, or an active variant or fragment thereof, may further include at least one of the following amino acid mutations to produce a non-catalytically inactive enzyme: D396R, E622R, D711R, wherein the indicated mutated amino acid residue corresponds to an amino acid residue of the sequence shown in SEQ ID NO: 4. In some embodiments, the non-catalytically inactive RGN polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in any of SEQ ID NO: 827, 896, and 979. V-type RGNs, including the amino acid sequence shown in SEQ ID NO: 4, or their active variants or fragments, may further include two or three of the following amino acid mutations to produce an enzyme without catalytic activity: D396R, E622R, D711R, wherein the indicated mutant amino acid residue corresponds to the amino acid residue of the sequence shown in SEQ ID NO: 4. III. Fusion proteins including RNA-guided nucleases.

This disclosure provides: fusion proteins, including the RGN polypeptide disclosed herein that is operatively fused to at least one heteropeptide; and polynucleotides that encode the fusion protein. When used to refer to the linking (by fusion or insertion) of two protein coding regions, even if one is inserted into the other, the terms "operatively linked" or "operatively fused" are still intended to indicate that the coding regions are within the same reading frame. In some embodiments, a polypeptide that is "operatively fused" or "operatively linked" means that the structure and/or biological activity of each individual peptide is also present in the fusion body.

As used herein with respect to a polypeptide heterologous to another polypeptide (e.g., an RGN polypeptide), "heterologous" refers to a polypeptide that does not operatively fuse with the RGN polypeptide described herein in nature. Heterologous polypeptides may originate from foreign species or from the same species. Heterologous polypeptides may be in their native form or may be substantially modified from their native form in constituents and/or genomic loci through deliberate human intervention. Heterologous polypeptides may be any polypeptide, including but not limited to localization signals, cell-penetrating domains, detectable labels (e.g., fluorescent proteins), purification tags, base-editing polypeptides (e.g., deaminers), or polymerase-editing polypeptides (e.g., DNA polymerases, reverse transcriptases). Heterogeneous peptides may contain effector domains, which may include, for example, cleavage domains, deaminase domains, or expression modulator domains, as further described herein.

The heterologous peptide or effector domain may be directly or indirectly located at the N-terminus, C-terminus, or internal address of the RGN peptide, either directly or indirectly via peptide-linkers. Alternatively, the heterologous peptide or effector domain may be directly or indirectly located at the internal address of the RGN peptide (e.g., a base-editing peptide such as a deaminase or a polymerase-editing peptide such as a DNA polymerase or reverse transcriptase).

RGNs (including RGNs with or without nuclease activity) can be used to deliver fused peptides, polynucleotides, or small molecule payloads to specific genomic addresses. In some of these embodiments, the RGN peptide or guide RNA may be fused with a detectable tracer or purified tracer to allow the detection of a specific sequence. As a non-limiting example, an RGN without nuclease activity may be fused with a detectable tracer (e.g., fluorescent protein) and targeted to a specific sequence associated with a disease to allow the detection of disease-related sequences. As another non-limiting example, an RGN with nuclease activity may be fused with a detectable tracer (e.g., fluorescent protein) and targeted to a specific sequence associated with a disease to allow the detection of disease-related sequences. Detectable traces or purified traces can be directly or indirectly located at the N-terminus, C-terminus, or internal site of the RGN via a linker peptide. In some embodiments, the RGN of the fusion protein is configured as a nuclease-free RGN. In some embodiments, the RGN of the fusion protein is configured as a nickase-active RGN.

The detectable tracer is a molecule that is observable or otherwise observable. The detectable tracer can be fused with RGN to form a fusion protein (e.g., a fluorescent protein), or it can be a small molecule coupled to an RGN peptide that is observable or detectable by other means. Detectable tracers that can be fused with the RGN disclosed in this invention to form a fusion protein include any detectable protein domain, including but not limited to fluorescent proteins or protein domains detectable by specific antibodies. Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreen1) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellow1). Non-limiting examples of small molecule detectable tracers include radioactive tracers such as ^3H and ^35S .

The RGN peptides disclosed herein may also include purified tracers, which are any molecules that can be used to isolate proteins or fusion proteins from mixtures (e.g., biological samples, culture media). Non-limiting examples of purified tracers include biotin, myc, maltose-binding protein (MBP), glutathione-S-transferase (GST), and 3X FLAG tracers.

Alternatively, nuclease-free RGNs can target specific genomic sites to alter the expression of a desired gene (i.e., the target gene). In some embodiments, the binding of a nuclease-free RGN to the target sequence leads to a reduction in the expression of the target gene by interfering with the binding of RNA polymerases or transcription factors within the targeted genomic region. In some embodiments, the RGN (e.g., a nuclease-free RGN) or its complex guide RNA further includes an expression regulator that, upon binding to the target sequence within the target gene, inhibits or activates the expression of the target gene.

The effector domain may include expression regulators. In some embodiments, the expression regulator of the fusion protein includes a transcriptional repressor domain that interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to reduce or terminate the transcription of at least one gene. Transcriptional repressor domains are known in the art and include, but are not limited to, Sp1-like repressors, IκB, and Krüppel cassette (KRAB) domains.

In some embodiments, the expression regulator of the fusion protein includes a transcriptional activation domain that interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerase and transcription factors, to increase or activate the transcription of at least one gene. Transcriptional activation domains are known in the art and include, but are not limited to, the herpes simplex virus VP16 activation domain and the NFAT activation domain.

In some embodiments, the expression regulator of the fusion protein includes an epigenetic modification domain that regulates the expression of a target sequence or a regulated gene through an epigenetic mechanism. In some embodiments, the expression regulator covalently modifies DNA or histones to alter histone structure and/or chromosomal structure without altering the DNA sequence, resulting in changes in gene expression (e.g., upregulation or downregulation). Non-limiting examples of epigenetic modification include acetylation or methylation of lysine residues, arginine methylation, phosphorylation of serine and threonine, ubiquitination and sumoylation of histones, and methylation and hydroxymethylation of cytosine residues in DNA. Non-restrictive examples of expression regulators include histone acetyltransferases, histone deacetyltransferases, histone methyltransferases, histone demethylases, DNA methyltransferases, and DNA demethylases.

The effector domain may include a deaminerase domain. In some embodiments, a nuclease-free RGN or a nickase-active RGN may target a specific genomic site to modify the sequence of the target polynucleotide by fusing with a base-editing polypeptide (e.g., a deaminerase polypeptide) or its active variant or fragment (which directly chemically modifies the nucleotide base (e.g., directly deamines the nucleotide)), resulting in the conversion from one nucleotide base to another. A deaminerase domain refers to a polypeptide or part of a polypeptide that can chemically modify a nucleotide base (e.g., deamines the nucleotide), resulting in the conversion from one nucleotide base to another. Deaminers or base-editing polypeptides may include a deaminerase domain. In some embodiments, the deaminerase and the deaminerase domain may be the same polypeptide. Base-editing peptides can be fused to RGN on either the amino-terminal (N-terminal) or carboxyl-terminal (C-terminal) side. Alternatively, base-editing peptides can be fused to RGN via peptide-linking molecules. A "base editor" is a fusion protein comprising a DNA-targeting peptide (such as RGN) and a base-editing peptide (such as a deaminase). Non-limiting examples of deaminases or base editors useful for these compositions and methods include cytosine deaminases, adenine deaminases, or base editors (e.g., the adenine deaminases or base editors described in Gaudelli et al. (2017) Nature 551:464-471, U.S. Patents No. 2017/0121693 and 2018/0073012, and International Patent No. WO/2018/027078, or WO 2020/139783, WO 2022/056254, WO 2022/204093, and WO 2020/139783). The deaminase or base editor disclosed in International Patent No. 2024/095245 (each of which is incorporated herein by reference in its entirety). In some embodiments, the deaminase useful to these compositions and methods disclosed herein is the deaminase disclosed in Table 17 of International Patent No. WO 2020/139783, which is incorporated herein by reference in its entirety. Furthermore, certain fusion proteins known in the art between RGN and base-editing enzymes (e.g., cytosine deaminers) may also include at least one uracil-stabilizing polypeptide that, through deaminerase, increases the mutation rate of cytidine, deoxycytidine, or cytosine into thymidine, deoxythymidine, or thymine in nucleic acid molecules. Non-limiting examples of uracil-stabilizing polypeptides include those disclosed in PCT patents WO 2021/217002 and WO 2022/015969, each of which is incorporated herein by reference in its entirety. The disclosed uracil-stabilizing peptide comprises a USP2 domain and a uracil glycosylase inhibitor (UGI) domain, which can improve base editing efficiency. Therefore, a base editor may include the RGN or a variant thereof described herein, a deaminase, and optionally at least one uracil-stabilizing peptide, such as UGI or USP2.

In some embodiments, RGN may be fused with a polymerase or a prime editing polypeptide (e.g., DNA polymerase or reverse transcriptase (RT)). Polymerase or prime editing is a precise, versatile genome editing method that uses a nucleic acid programmable DNA-binding protein that works in conjunction with a polymerase to directly write new gene information into a specified DNA site (described, for example, in US 11,447,770B1, WO2021072328, WO2021226558, WO2020156575, WO2021042047, and US11193123, each of which is incorporated herein by reference in its entirety). A polymerase or lead editing system uses an RGN that is a nicking enzyme and a DNA polymerase (e.g., RT), and the system is programmed with polymerase or lead editing (PE) guide RNA (“PEgRNA”). PEgRNA is a guide RNA that includes a primer binding site (PBS) and a DNA synthesis template, which acts as a template for polymerization, including editing of alternative strands. PEgRNA specifies a target sequence, and the DNA synthesis template for PEgRNA is provided by means of an extension engineered to the guide RNA backbone (e.g., at the 5' or 3' end, or at an internal portion of the guide RNA backbone).

As used herein, "polymerase editor" or "PE" refers to a protein or multiple proteins comprising a DNA polymerase (e.g., reverse transcriptase) and an RNA-binding polypeptide (e.g., RGN polypeptide) that replaces a target sequence by using a DNA synthesis template as a template for the DNA polymerase (e.g., reverse transcriptase). The PE, along with PEgRNA, is capable of editing double-stranded polynucleotides. The PEgRNA includes an extension containing a primer-binding site (PBS) and a DNA synthesis template containing the desired editing. A PE comprising an RGN nickase/DNA polymerase (i.e., reverse transcriptase) fusion is guided to the target sequence by PEgRNA and creates nicks in the non-target strand upstream of the sequence to be edited and downstream of the PAM, thereby creating a 3' lobe on the non-target strand. The PBS is complementary to the 3' lobe of the non-standard strand, and the hybridization of the PBS with the 3' lobe of the non-standard strand allows for the polymerization of the edited alternative strand using a DNA synthesis template. In some embodiments, the length of the PBS is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the PEgRNA comprises PBS with a length of at least 5 nucleotides (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, or 20 nucleotides). In some embodiments, the PEgRNA may comprise PBS with a length of at least 8 nucleotides. In some embodiments, the PEgRNA may comprise PBS with a length of 9, 11, 12, 13, or 15 nucleotides. Hybridization of PBS with the 3′ lobe of the non-standard strand allows for the polymerization of edited alternative strands using a DNA synthesis template in the extension of the PEgRNA. The length of the DNA synthesis template can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. In some embodiments, the PEgRNA comprises a DNA synthesis template having a length of at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the PEgRNA comprises a DNA synthesis template having a length of 19, 22, 23, 24, 25, 26, 29, 30, 32, 34, 37, 38, 39, 40, 42, or 46 nucleotides. DNA synthesis templates include desired edits, which can be the substitution, deletion, or addition of one or more nucleotides.

PEgRNA elongation can be formed from either RNA or DNA. In the case of RNA elongation, the polymerase or leader editor polymerase can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of DNA elongation, the polymerase or leader editor polymerase can be a DNA-dependent DNA polymerase.

The substitution strand containing the desired edit (e.g., substitution, deletion, or addition) shares the same sequence (excluding the desired edit) with the non-target strand of the target sequence to be edited. Through DNA repair and/or replication mechanisms, the non-target strand of the target sequence is replaced by a newly synthesized substitution strand containing the desired edit. In some cases, because the polymerase or leader editor not only searches for and locates the desired target sequence to be edited, but also simultaneously writes a substitution strand containing the desired edit and installed as a replacement for the corresponding non-target strand of the target sequence, polymerase or leader editing can be considered a "search-and-replace" genome editing technique. Therefore, in some embodiments, the guide RNA of this disclosure includes an extension comprising an editing template for polymerase or leader editing. In some embodiments, the polymerase or lead editing polypeptide that can be fused with the RGN comprises a DNA polymerase. In some embodiments, the DNA polymerase is a reverse transcriptase. In some embodiments, the RGN is a nicking enzyme.

In some embodiments, the RGN peptide and polymerase-edited peptide (e.g., RT) of the polymerase editor of this disclosure may be provided in trans form rather than as a fusion protein, wherein the RGN peptide and polymerase-edited peptide (e.g., RT) are separate peptides. In some embodiments, the RGN peptide and polymerase-edited peptide (e.g., RT) are transcribed together and contain a sequence of a self-cleaving peptide (e.g., the 2A peptide of P2A), such that transcription results in two separate peptides. Any self-cleaving peptide known in the art may be used in these embodiments, including but not limited to 2A peptides, which are a class of peptides 18 to 22 amino acids long that can function via ribosomal skipping during transcription. Non-limiting examples of 2A peptides are T2A, P2A, E2A, and F2A.

To reduce the likelihood of edits introduced by the polymerase editor being removed due to mismatch repair of the edited strand, nicking guide RNA (nick guide RNA) can be used. Nick guide RNA is a type of guide RNA that targets sequences within the unedited strand near and opposite the original nick site, guiding the RGN nicking enzyme of the PE system to this unedited strand to introduce a single-strand nick. Nick guide RNA can be programmed to match the edited sequence introduced by PEgRNA, but not the original unedited sequence, to ensure that the nick occurs after an edit event on the non-targeted strand.

The effector domain may include a splicing domain. In some embodiments, the RGN fusion protein includes a splicing domain, which is any domain capable of cleaving polynucleotides (i.e., RNA, DNA, or RNA/DNA hybrids) and includes, but is not limited to, restriction endonucleases and homing endonucleases (e.g., IIS-type endonucleases) (e.g., FokI ) (see, for example, Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).

RGNs fused to heterologous peptides or domains can be separated or linked by conjugates. As used herein, a conjugate refers to a chemical group or molecule that links two molecules or parts (e.g., the binding and cleavage domains of a nuclease). In some embodiments, the conjugate links the gRNA binding domain of an RGN to a base-editing peptide, such as a deaminase. In some embodiments, the conjugate links a nuclease-free RGN or an RGN nickase to a deaminase. Typically, the conjugate is located between or on either side of two groups, molecules, or other parts and is covalently linked to each group, molecule, or other part, thereby linking them. In some embodiments, the conjugate is an organic molecule, group, polymer, or chemical part. In some embodiments, the conjugate is an amino acid or multiple amino acids (e.g., a peptide or protein conjugate). In some embodiments, the linker has a length of 5 to 100 amino acids, for example, lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 150, or 150 to 200 amino acids. Longer or shorter linkers are also conceived. In some embodiments, the peptide linker includes at least one NLS. In some embodiments, the peptide linker includes two NLSs. The peptide conjugate can be operatively fused to the N-terminus, C-terminus, or both of the N-terminus and C-terminus of an RGN peptide or a heterologous peptide (e.g., a base-editing peptide or a polymerase-editing peptide). In some embodiments, the peptide conjugate comprises one or more copies of the amino acid sequence GSSG (SEQ ID NO: 1928). In some embodiments, the peptide conjugate comprises an amino acid sequence as shown in GSSGGSSG (SEQ ID NO: 34). In some embodiments, the peptide conjugate has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher of the sequence in SEQ ID NO: 1676 or 1677. In some embodiments, the peptide linker has the amino acid sequence of SEQ ID NO: 1676 or 1677.

The RGN disclosed herein may include at least one nuclear localization signal (NLS) to enhance the transport of the RGN to the cell nucleus. Nuclear localization signals are known in the art and typically include a basic amino acid (see, for example, Lange et al., J. Biol. Chem. (2007) 282:5101-5105). In some embodiments, the RGN includes 2, 3, 4, 5, 6, or more nuclear localization signals. The nuclear localization signals (one or more) may be heterologous NLS. Non-limiting examples of nuclear localization signals useful to the RGN disclosed herein are nuclear localization signals of SV40 large T-antigen, nucleoplasmin, and c-Myc (see, for example, Ray et al. (2015) Bioconjug Chem26 (6):1004-7). In some embodiments, the NLS has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 33, 35, 407, or 1927. In some embodiments, the RGN includes the NLS sequence as shown in SEQ ID NO: 33, 35, 407, or 1927. The RGN may include one or more NLS sequences at its N-terminus, C-terminus, or both. For example, the RGN may include two NLS sequences in the N-terminal region and four NLS sequences in the C-terminal region.

Other known localization signal sequences in this field that localize peptides to (one or more) specific subcellular addresses can also be used to target RGNs, including but not limited to plasmid localization sequences, mitochondrial localization sequences, and dual-targeting signal sequences targeting both plasmids and mitochondria (see, for example, Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19; Kunze and Berger (2015) Front Physiol dx.doi.org/10.3389/fphys.2015.00259; Herrmann and Neupert (2003) IUBMB Life 55:219-225; Soll (2002) Curr Opin Plant Biol 5:529-535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al. (2009) FEBS J 276:1187-1195; Silva-Filho (2003) Curr Opin Plant Biol 6:589-595; Peeters and Small (2001) Biochim Biophys Acta 1541:54-63; Murcha et al. (2014) J Exp Bot 65:6301-6335; Mackenzie (2005) Trends Cell Biol 15:548-554; Glaser et al. (1998) Plant Mol Biol 38:311-338.

The RGN disclosed in this invention may include at least one cell-penetrating domain that promotes cellular uptake of the RGN. Cell-penetrating domains are known in the art and generally include multiple segments of positively charged amino acid residues (i.e., polycationic cell-penetrating domains), alternating polar amino acid residues, and nonpolar amino acid residues (i.e., amphiphilic cell-penetrating domains), or hydrophobic amino acid residues (i.e., hydrophobic cell-penetrating domains) (see, for example, Milletti F. (2012) Drug Discov Today 17:850-860). A non-limiting example of a cell-penetrating domain is the trans-activating transcription activator (TAT) from human immunodeficiency virus 1.

Nuclear localization signals, plasmid localization signals, mitochondrial localization signals, dual-target localization signals, and/or cell-penetrating domains can be located at the amino terminus (N-terminus), carboxyl terminus (C-terminus), or internal address of the RGN. IV. Guide RNA

This disclosure provides guide RNA and coding guide RNA to target associated RNA-guided nucleases (RGNs) to polynucleotides of a target sequence. The term "guide RNA" refers to a nucleotide sequence that is sufficiently complementary to the target nucleotide sequence to hybridize with the target sequence and to guide the associated RGN to bind specifically to the target nucleotide sequence. More specifically, when the target nucleotide sequence is double-stranded, as is the case with DNA, the target nucleotide sequence consists of a target strand and a non-target strand (including PAM sequences). In these embodiments, the guide RNA is sufficiently complementary to the target strand of the double-stranded target sequence (e.g., the target DNA sequence) such that the guide RNA hybridizes with the target strand and guides the associated RGN to bind specifically to the target sequence (e.g., the target DNA sequence). Therefore, in some embodiments, the guide RNA contains a spacer with the same sequence as the non-target strand, except that uracil (U) replaces thymidine (T) in the guide RNA. In embodiments using multiplex gene editing and with multiple guide RNAs, each of one or more guide RNAs is sufficiently complementary to the target strand of a specific target sequence and is capable of hybridizing with the target strand of that target sequence. Thus, the "corresponding target sequence" of the guide RNA refers to the target sequence to which the guide RNA is sufficiently complementary and capable of hybridizing.

Each RGN's guide RNA is one or more RNA molecules (usually one or two) that bind to the RGN and guide the RGN to a specific target nucleotide sequence. In embodiments where the RGN has nicking or nuclease activity, it also cleaves the target strand and/or the non-target strand. Generally, guide RNAs include CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA), but some RGNs do not require tracrRNA. Specifically, the disclosed RGNs and RGN systems contain guide RNAs that do not require tracrRNA. Therefore, in some embodiments, the guide RNA binding to the RGN or included in the RGN system disclosed herein is crRNA.

The guiding RNA (e.g., crRNA) disclosed herein may include at least one chemical modification. The at least one chemically modified locking nucleic acid includes: bridged nucleic acid (BNA) modification; 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluorine (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-phosphorothioate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and phosphorothioate (PS) modification; or combinations thereof. In some embodiments, BNA includes 2',4' BNA modification. In some embodiments, the 2',4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA ^NC [N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, 2',4' BNA is LNA modification. In some embodiments, 2',4' BNA is cET modification. In some embodiments, at least one chemical modification includes BNA modification, 2'-O-Me modification, or PS modification. Chemical modifications of the spacer, crRNA repeat, crRNA, tracrRNA and guide RNA are described in WO 2024/042489, which is incorporated herein by reference in its entirety.

This disclosure provides CRISPR RNA (crRNA) or coding CRISPR RNA, a polynucleotide that targets an associated RGN to a target sequence. The crRNA comprises a spacer and a CRISPR repeater. The "spacer" has a nucleotide sequence that is directly hybridized to a target strand of the target sequence of interest (e.g., a target DNA sequence). The spacer is engineered to be fully or partially complementary to the target strand of the target sequence of interest. In some embodiments, the spacer may comprise from about 8 nucleotides to about 30 nucleotides or more. For example, the length of the spacer may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. In some embodiments, the length of the spacer is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. In some embodiments, the length of the spacer is about 10 to about 26 nucleotides, or about 12 to about 30 nucleotides. In some embodiments, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the spacer and the target strand of the target sequence (e.g., the target DNA sequence) is between 50% and 99% or higher, including but not limited to about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher. In certain embodiments, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the spacer and the target strand of the target sequence (e.g., the target DNA sequence) is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. In some embodiments, the spacer may be identical in sequence to a non-target strand of the target sequence. In some embodiments where the target sequence is the target DNA sequence, the spacer may be identical in sequence to a non-target strand of the target DNA sequence, except that thymidine (T) in the non-target strand is replaced by uracil (U) in the spacer. In a particular embodiment, the septum does not contain secondary structures that can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, for example, Zuker and Stiegler (1981) Nucleic Acids Res. 9:133-148) and RNAfold (see, for example, Gruber et al. (2008) Cell 106(1):23-24).

The crRNA disclosed in this invention includes a spacer capable of targeting a bound RGN polypeptide to a target DNA sequence. In some embodiments, the spacer has a nucleotide sequence as shown in any one of SEQ ID NO: 294 to 396, 1789 to 1796, 1798 to 1880, 1890 to 1893, and 2082 to 2125. In some embodiments, the target sequence is in the genome of a eukaryotic cell. The eukaryotic cell may be present in vitro, in vivo, or in vivo. In some embodiments, the target DNA sequence has a nucleotide sequence as shown in any one of SEQ ID NO: 183 to 285, 1684 to 1691, 1693 to 1775, 1785 to 1788, and 2038 to 2081. In some embodiments, the target sequence is in the genome of a prokaryotic cell.

In addition to the spacer, crRNA further includes a CRISPR RNA (crRNA) repetition. Generally, a crRNA repetition comprises a nucleotide sequence that, alone or in combination with a hybrid tracrRNA, forms a structure recognized by the RGN polypeptide. In this disclosure, the crRNA repetition can be recognized and bound by the RGN of this disclosure, and the crRNA repetition does not necessarily need to be hybridized with tracrRNA. For type V guiding crRNA, the spacer is located at the 3' end of the crRNA repetition. Therefore, when considering type V crRNA, the crRNA repetition begins at the 5' end of the crRNA, followed by the spacer at the 3' end of the crRNA repetition. The backbone is the portion of the guiding RNA excluding the spacer. As used herein, the terms "crRNA repetition" and "crRNA backbone" are interchangeable. In various implementations, the crRNA repeat (i.e., the crRNA backbone) may contain from about 8 nucleotides to about 40 nucleotides or more. For example, the length of the crRNA repeat (i.e., the crRNA backbone) may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40 or more nucleotides. In certain embodiments, the crRNA repeat (i.e., the crRNA backbone) has a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides. In some embodiments, the crRNA repeat (i.e., the crRNA backbone) of this disclosure has a total length of 25 to 40 nucleotides (nt). In some embodiments, the crRNA repeats (i.e., the crRNA backbone) disclosed herein have a total length of up to 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt. The crRNA repeats (i.e., the crRNA backbone) disclosed herein may be included in the common sequence of the CRISPR array found in the genome of the organism that obtained the crRNA repeats.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) comprises a nucleotide sequence or an active variant or fragment thereof as shown in any of SEQ ID NOs: 5 to 8, 1617 to 1620, 1631 and 1957 to 1983, capable of guiding the sequence-specific binding of the associated RGN provided herein to the target sequence of interest. In some embodiments, the active crRNA replicon (i.e., the crRNA backbone) variant of the wild-type sequence comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of the nucleotide sequences shown in SEQ ID NOs: 5 to 8, 1617 to 1620, 1631 and 1957 to 1983. In some embodiments, the active crRNA repeat of the wild-type sequence (i.e., the crRNA backbone) fragment contains at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 consecutive nucleotides of any of the nucleotide sequences shown in SEQ ID NO: 5 to 8, 1617 to 1620, 1631 and 1957 to 1983.

In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 5 or differs from SEQ ID NO: 5 by 1 to 5 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 5 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 4 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 3 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 5.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 6 or differs from SEQ ID NO: 6 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 6.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 7 or differs from SEQ ID NO: 7 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 7.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 8 or differs from SEQ ID NO: 8 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 8.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1617 or differs from SEQ ID NO: 1617 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1617.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1618 or differs from SEQ ID NO: 1618 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1618.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1619 or differs from SEQ ID NO: 1619 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1619.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1620 or differs from SEQ ID NO: 1620 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1620.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1631 or differs from SEQ ID NO: 1631 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1631.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1957 or differs from SEQ ID NO: 1957 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1957.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1958 or differs from SEQ ID NO: 1958 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1958.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1959 or differs from SEQ ID NO: 1959 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1959.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1960 or differs from SEQ ID NO: 1960 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1960.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1961 or differs from SEQ ID NO: 1961 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1961.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1962 or differs from SEQ ID NO: 1962 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1962.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1963 or differs from SEQ ID NO: 1963 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1963.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1964 or differs from SEQ ID NO: 1964 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1964.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1965 or differs from SEQ ID NO: 1965 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1965.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1966 or differs from SEQ ID NO: 1966 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1966.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1967 or differs from SEQ ID NO: 1967 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1967.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1968 or differs from SEQ ID NO: 1968 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1968.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1969 or differs from SEQ ID NO: 1969 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1969.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1970 or differs from SEQ ID NO: 1970 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1970.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1971 or differs from SEQ ID NO: 1971 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1971.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1972 or differs from SEQ ID NO: 1972 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1972.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1973 or differs from SEQ ID NO: 1973 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1973.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1974 or differs from SEQ ID NO: 1974 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1974.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1975 or differs from SEQ ID NO: 1975 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 2 nucleotides. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by one nucleotide. In some embodiments, the crRNA repeater (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1975.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1976 or differs from SEQ ID NO: 1976 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1976.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1977 or differs from SEQ ID NO: 1977 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1977.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1978 or differs from SEQ ID NO: 1978 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1978.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1979 or differs from SEQ ID NO: 1979 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1979.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1980 or differs from SEQ ID NO: 1980 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1980.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1981 or differs from SEQ ID NO: 1981 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1981.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1982 or differs from SEQ ID NO: 1982 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1982.

In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence as shown in SEQ ID NO: 1983 or differs from SEQ ID NO: 1983 by 1 to 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 5 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 4 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 3 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 2 nucleotides. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by one nucleotide. In some embodiments, the crRNA replicon (i.e., the crRNA backbone) has the nucleotide sequence shown in SEQ ID NO: 1983.

In some implementations, the crRNA is not naturally occurring. In some of these implementations, the specific crRNA repeat (i.e., the crRNA backbone) is not linked to the engineered spacer in nature, and the crRNA repeat (i.e., the crRNA backbone) is considered heterologous to the spacer. In some implementations, the spacer is an engineered sequence that is not naturally occurring.

In some embodiments, since the guide RNA of the V-H, V-I, or V-J type RGN or RGN system does not require tracrRNA to function, crRNA is the guide RNA. The guide RNA (i.e., crRNA) disclosed herein may have the nucleotide sequence or its active variant or fragment shown in any of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658. In some embodiments, the guide RNA (i.e., crRNA) disclosed herein may have a nucleotide sequence or an active variant or fragment thereof as shown in any of SEQ ID NO: 39 to 49, 51, 53, 54, 58, 61, 62, 64, 66, 68, 69, 78, 80, 81, 83, 84, 86, 88, 91, 96, 98, 99, 101, 109 to 128, 130, 132, 133, 135, 137 to 139, 147 to 153, 159, 163, 165, 168, 170, 173, 175, 176, 178, 179 and 182. The guide RNA (i.e., crRNA) disclosed herein may have a total length of 35 to 250 nucleotides (nt), or 35 to 220 nt, or 35 to 170 nt, or 35 to 125 nt, or 40 to 100 nt, or 50 to 70 nt, or 40 to 70 nt, or 40 to 60 nt. In some embodiments, the guide RNA (i.e., crRNA) disclosed herein may have a maximum length of 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt, 83 nt. nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 160 nt, 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt, 180 nt, 190 nt, 200 nt, 205 nt, 210 nt, 215 nt, 220 nt, 225 nt, 230 nt, 235 nt, 240 nt, 245 nt or 250 nt total length.

The guide RNA (i.e., crRNA) disclosed herein may include at least one crRNA backbone (bb) and at least one spacer. The at least one crRNA bb may include two or more crRNA bbs, and the two or more crRNA bbs may be different. In some embodiments, the differences include different lengths of the crRNA bbs. In some embodiments, the differences include different sequences of the crRNA bbs. In some embodiments, the differences include different lengths and sequences of the crRNA bbs. The at least one crRNA bb may include two or more crRNA bbs, and the two or more crRNA bbs may be identical. In some embodiments, the at least one crRNA bb may include two or more crRNA bbs, and the two or more crRNA bbs may be of the same length. In some embodiments, the at least one crRNA bb may include two or more crRNA bbs, and the two or more crRNA bbs may have the same sequence. In some embodiments, at least one crRNA bb may comprise two or more crRNA bbs, and the two or more crRNA bbs may have the same length and sequence. At least one crRNA bb may comprise two or more crRNA bbs, and the two or more crRNA bbs may comprise a mixture of some identical crRNA bbs and some different crRNA bbs. In some embodiments, at least one crRNA bb may comprise two or more crRNA bbs, and the two or more crRNA bbs may comprise a mixture of some crRNA bbs having the same length and/or sequence and some crRNA bbs having different lengths and/or sequences.

In some embodiments, the guide RNA (i.e., crRNA) is in the form of crRNA bb-septum from 5' to 3'. In some embodiments, the guide RNA (i.e., crRNA) may include two crRNA bbs and one septum, in the form of crRNA bb-septum-crRNA bb from 5' to 3'. In some embodiments, the guide RNA (i.e., crRNA) may include three crRNA bbs, a first septum, and a second septum, in the form of crRNA bb-first septum-crRNA bb-second septum-crRNA bb from 5' to 3'. Those skilled in the art may consider forms with additional crRNA bbs and septums, for example, various forms of crRNA bb-septum arranged end-to-end with crRNA bb as the terminal. Some guide RNA forms are shown in Figure 13A.

The V-type guide RNAs described herein are self-processing and relatively short in length. These advantages allow for the creation of constructs with multiple guide RNAs arranged in tandem for editing multiple targets. Tandem arrays can be created as described in Example 11, wherein two or more nucleotide sequences, each encoding a crRNA, are arranged end-to-end and operatively linked to a promoter. Tandem arrays of single polynucleotide-coding crRNAs are transcribed into single transcripts and then processed by the V-H, V-I, or V-J type RGN system disclosed herein into multiple single guide RNAs (i.e., crRNAs), which can then target multiple dissimilar sequences.

Two polynucleotide sequences are considered substantially complementary when they hybridize with each other under strict conditions. Similarly, if the guide RNA bound to an RGN binds to a target sequence under strict conditions, the RGN is considered to bind to the specific target sequence in a sequence-specific manner. The terms "strict conditions" or "strict hybridization conditions" are intended to mean that the detectability of two polynucleotide sequences hybridizing with each other is higher than the detectability of hybridization with other sequences (e.g., at least 2 times higher than background). Strict conditions are sequence-dependent and will vary under different environments. Typically, stringent conditions would be those involving a pH of 7.0 to 8.3, a salt concentration of less than about 1.5 M Na ions (typically about 0.01 to 1.0 M Na ions) (or other salts), and a temperature of at least about 30°C for short sequences (e.g., 10 to 50 nucleotides) and at least about 60°C for long sequences (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding a destabilizing agent such as methylphenidate. Exemplary low-strict conditions include hybridization at 37°C in a buffered solution of 30 to 35% methylamine, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing at 50 to 55°C in 1X to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate). Exemplary medium-strict conditions include hybridization at 37°C in 40 to 45% methylamine, 1.0 M NaCl, and 1% SDS, followed by washing at 55 to 60°C in 0.5X to 1X SSC. Exemplary high-strict conditions include hybridization at 37°C in 50% methylamine, 1 M NaCl, and 1% SDS, followed by washing at 60 to 65°C in 0.1X SSC. Optionally, the wash buffer may include about 0.1% to about 1% SDS. Hybridization duration is generally less than about 24 hours, typically about 4 to about 12 hours. The washing duration will be at least sufficient to reach equilibrium.

Tm is the temperature at which 50% of the complementary target sequence hybridizes with the perfectly matched sequence (under defined ionic strength and pH). For DNA-DNA hybrids, Tm can be roughly estimated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in DNA, % form is the percentage of methylamine in the hybrid solution, and L is the length of the hybrid in the base pair. Generally, stringent conditions are chosen to be approximately 5°C lower than the thermal melting point (Tm) of the specific sequence and its complementary sequences at defined ionic strength and pH. However, extremely stringent conditions may be employed by hybridization and/or washing at 1, 2, 3, or 4°C lower than the specific melting point (Tm); moderately stringent conditions may be employed by hybridization and/or washing at 6, 7, 8, 9, or 10°C lower than the specific melting point (Tm); and low-stringent conditions may be employed by hybridization and/or washing at 11, 12, 13, 14, 15, or 20°C lower than the specific melting point (Tm). Those skilled in the art will understand that variations in the strictness of hybridization and/or washing solutions are described using the equation, the hybrid and washing compositions, and the desired Tm based on intrinsic properties. Broad standards for nucleic acid hybridization can be found in Tijssen (1993), Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual (Second Edition, Cold Spring Harbor Laboratory Press, Plainview, New York).

The term "sequence specificity" can also refer to the fact that RGN peptides bind to the target sequence with higher affinity than to the randomized background sequence.

Guide RNA can be chemically synthesized or synthesized via in vitro transcription. Assays for determining the sequence-specific binding between RGN and guide RNA are known in the art and include, but are not limited to, in vitro binding assays for expressed RGN and guide RNA, which can be labeled with a detectable tracer (e.g., biotin) and used in pull-down assays, wherein the guide RNA:RGN complex is captured by a detectable tracer (e.g., using streptavidin beads). Guide RNA with a sequence or structure unrelated to the guide RNA can be used as a negative control for the non-specific binding of RGN to RNA.

Guide RNA can be introduced into target cells, organelles, or embryos as an RNA molecule. In some embodiments, the nucleotide sequence encoding the guide RNA is introduced into the target cells, organelles, or embryos. In some embodiments, the nucleotide sequence encoding the guide RNA is operatively linked to a promoter (e.g., the RNA polymerase III promoter). The promoter can be a native promoter or heterologous to the nucleotide sequence encoding the guide RNA. In some embodiments, the guide RNA can be introduced into target cells, organelles, or embryos as a ribonucleoprotein complex, as described herein, wherein the guide RNA is bound to an RGN polypeptide.

By hybridizing the guide RNA with the target sequence, the guide RNA directs the associated RGN to the specific target nucleotide sequence. The target sequence can be bound to the RGN in vitro or in cells (and in some embodiments, cleaved). The target sequence is within a target polynucleotide and can include DNA, RNA, or a combination of both, and can be single-stranded or double-stranded. The target sequence can be genomic DNA (i.e., chromosomal DNA), plasmid DNA, or RNA molecules (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). In embodiments where the target sequence is a chromosomal sequence, the chromosomal sequence can be a nuclear, plasmid, or mitochondrial chromosome sequence. In the compositions and methods disclosed in this invention, the target sequence is within a double-stranded target nucleic acid molecule (e.g., a target DNA sequence). In some embodiments, the target sequence is unique within the target genome. In some embodiments, the target sequence includes a target share and a non-target share, and the target sequence (i.e., the sequence on the non-target share) has a nucleotide sequence as shown in any of SEQ ID NO: 183 to 285, 1684 to 1691, 1693 to 1775, 1785 to 1788, and 2038 to 2081.

The target sequence is adjacent to the original spacer adjacent motif (PAM), and the non-target strand of the target sequence is a strand that includes the PAM. The PAM is adjacent to the target sequence and often includes N, where N represents any nucleotide. In some embodiments, the PAM includes about 1 to about 10 N, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 N. In a particular embodiment, the PAM includes 1 to 10 N, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 N. Generally, the PAM may be at the 5' or 3' of the target sequence on its non-target strand. The PAM of the RGN disclosed in this invention is adjacent to the 5' of the target sequence on its non-target strand. Generally, PAM is a common sequence of about 3 to 4 nucleotides, but in certain embodiments, its length can be 2, 3, 4, 5, 6, 7, 8, 9 or more nucleotides.

In some embodiments, the RGN binds to a guide RNA comprising a crRNA repeat (i.e., the crRNA backbone) having a nucleotide sequence as shown in any of SEQ ID NOs: 5 to 8, 1617 to 1620, 1631, and 1957 to 1983, or an active variant or fragment thereof. The RGN system will be further described in Examples 1 to 14 and Tables 1 to 32 of this specification.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in AYG. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 5 or 1631, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in AYG.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 6, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 3, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 7, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 3, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 4, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 8 and 1617 to 1620, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 4, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1930, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1957, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1930, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1931, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in STTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1958, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1931, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in STTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1932, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1959, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1932, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1933, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1960, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1933, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1934, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1961, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1934, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1935, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in TTH. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1962, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1935, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in TTH.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1936, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1963, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1936, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1937, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1964, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1937, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1938, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1965, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1938, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1939, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1966, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1939, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1940, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1967, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1940, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1941, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1968, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1941, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1942, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1969, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1942, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1943, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1970, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1943, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1944, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1971, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1944, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in ATTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1945, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1972, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1945, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1946, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1973, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1946, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1947, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1974, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1947, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1948, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1975, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1948, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1949, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1976, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1949, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in VTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1950, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1977, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1950, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1951, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTYN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1978, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1951, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of the PAM sequence shown in RTYN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1952, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1979, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1952, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1953, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in ATG. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1980, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1953, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in ATG.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1954, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1981, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1954, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1955, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1982, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1955, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1956, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN. In some embodiments, when binding to a guide RNA comprising a crRNA repeat (i.e., crRNA backbone) sequence shown in SEQ ID NO: 1983, or an active variant or fragment thereof, an RGN having the amino acid sequence shown in SEQ ID NO: 1956, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 3' end of a PAM sequence shown in TTN.

As is well known in the art, the specificity of PAM sequences for a given nuclease enzyme is affected by enzyme concentration (see, for example, Karvelis et al. (2015) Genome Biol 16:253), which can be modified by altering the amount of ribonucleoprotein complexes used to express RGN or delivered to the target cell, organelle, or embryo.

When recognizing a corresponding PAM sequence, an active RGN can cleave one or both strands of the target sequence at a specific cleavage site. As used herein, a cleavage site includes a specific nucleotide within the target sequence that is cleaved by the RGN in the target strand, non-target strand, or both strands. A cleavage site may include the 1st and 2nd, 2nd and 3rd, 3rd and 4th, 4th and 5th, 5th and 6th, 7th and 8th, or 8th and 9th nucleotides from the PAM in the 5' or 3' direction. The V-H, V-I, and V-J type RGNs of this disclosure recognize 5' PAM cleavage sites, and therefore the cleavage site may include the 1st and 2nd, 2nd and 3rd, 3rd and 4th, 4th and 5th, 5th and 6th, 7th and 8th, or 8th and 9th nucleotides from the PAM in the 3' direction. In some embodiments, the cleavage site may be more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM towards the 5' or 3' direction. For the V-H, V-I, and V-J type RGNs and RGN systems disclosed herein, the cleavage site may be more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides from the PAM towards the 3' direction. Because RGNs can cleave target sequences that result in staggered ends, in some embodiments, the cleavage site on the non-target strand of the target sequence is defined based on a distance of two nucleotides from the PAM, while for the target strand, it is defined based on a distance of two nucleotides from the complement of the PAM. In some embodiments, the V-type RGN of this disclosure cleaves approximately 18 nucleotides from PAM on the target strand. In some embodiments, the V-type RGN of this disclosure cleaves approximately 23 nucleotides from the complement of PAM on the target strand.

Unbound by any single theory, the distance between the cleavage site and the PAM site in type V RGN could potentially allow for several re-cuttings after non-homologous end-joint (NHEJ) but before complete disruption of the PAM/septum binding. This could allow for more opportunities for homology-dependent repair when donor polynucleotides are introduced as part of gene editing. V. Coding RNA-guided nucleases and/or polynucleotides guiding RNA.

This disclosure provides polynucleotides including the guide RNA (i.e., CRISPR RNA (crRNA) disclosed herein) and polynucleotides including nucleotide sequences encoding the RNA-guided nuclease (RGN) and/or gRNA (i.e., crRNA) disclosed herein. In various embodiments, the guide RNA (gRNA) bound to the RGN or included in the RGN system of this disclosure is crRNA, because tracrRNA molecules are not required. The polynucleotides disclosed in this invention comprise or encode a CRISPR RNA (crRNA) repeater (i.e., the crRNA backbone), the CRISPR RNA (crRNA) repeater (i.e., the crRNA backbone) having a nucleotide sequence as shown in any of SEQ ID NO: 5 to 8, 1617 to 1620, 1631 and 1957 to 1983, or an active variant or fragment thereof, which, when included within a guide RNA, is capable of guiding the sequence-specific binding of an associated RGN to a target sequence of interest. Polynucleotides encoding an RGN are also provided, the RGN having a sequence as shown in SEQ ID: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 10 The amino acid sequences and their active fragments or variants shown in 84, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 retain the ability to bind to the target sequence in an RNA-guided sequence-specific manner.

The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit the content of this disclosure to polynucleotides including DNA. Those skilled in the art will recognize that polynucleotides can include ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. These include peptide nucleic acids (PNAs), PNA-DNA chimers, locked nucleic acids (LNAs), and phosphate-thioester linked sequences. The polynucleotides disclosed herein also encompass all forms of sequences, including but not limited to: single-stranded forms, double-stranded forms, DNA-RNA hybrids, triplex structures, stem-and-loop structures, circular RNA, etc.

In some embodiments of the compositions and methods disclosed herein, including the writing of a polynucleotide encoding an RGN, the polynucleotide is an mRNA (messenger RNA) molecule. mRNA refers to any polynucleotide that encodes the polypeptide of interest and is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or in vitro. In some embodiments, the basic components of the mRNA molecule include at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly-A tail. The 5' UTR, located at the 5' end of the coding sequence and transcribed into a portion of the mRNA, can include various regulatory elements, including, for example, those controlling the initiation of mRNA translation: a 5' cap structure, a G-quadruplex structure (G4), a stem-loop structure, and an internal ribosome entry site (IRES). The 3' UTR, located at the 3' end of the coding sequence and transcribed into a portion of the mRNA, can participate in numerous regulatory processes, including transcript splicing, stability and polyadenylation, translation, and mRNA localization. The 3' UTR can serve as a binding site for a wide range of proteins and small non-coding RNAs (e.g., microRNAs). The 5' UTR and/or 3' UTR that are heterologous to the mRNA originate from an organism or species different from the mRNA, or if they originate from the same organism or species as the mRNA, then the 5' UTR and/or 3' UTR have been substantially modified from their natural form in the constituents and/or genome loci by intentional human intervention.

In some embodiments, incorporating heterologous 5' UTRs and/or 3' UTRs of the mRNA of the RGN peptide disclosed in the codebook modifies RGN peptide synthesis from mRNA in tissues (e.g., liver, or in vitro cells such as stem cells, hepatocytes, or lymphocytes). The heterologous 5' UTRs and/or 3' UTRs can increase protein synthesis, for example, by prolonging the time mRNA is retained in the translational polyribosome (information stability) and/or increasing the rate at which ribosomes initiate translation on mRNA (information translation efficiency). Therefore, incorporating heterologous 5' UTRs and/or 3' UTRs of the mRNA of the RGN peptide disclosed in the codebook can induce prolonged and/or increased RGN peptide synthesis, thereby enabling modification of the cleavage or modification of the nucleic acid molecule targeted by the RGN peptide. In some implementations, the enhancement of RGN peptide synthesis from mRNA occurs in a tissue-specific manner. Heterologous UTR sequences are described, for example, in US 2023/0050143 and US 2017/0252461.

In some embodiments, the mRNA encoding the RGN useful in the methods and compositions disclosed in this invention may contain one or more structural and/or chemical modifications or alterations that endow the polynucleotide with useful properties. For example, useful properties of the mRNA may include inducing an innate immune response in the cell to which the mRNA is introduced. A "structural" feature or modification is the insertion, deletion, duplication, inversion, or randomization of two or more linked nucleotides in the mRNA without significant chemical modification of the nucleotides themselves. Because the chemical bonds will necessarily be broken and reformed to produce the structural modification, the structural modification is chemical in nature and therefore a chemical modification. However, the structural modification will result in a different nucleotide sequence. Chemical modification of mRNA may involve incorporating 5-methylcytosine, N1-methyl-pseuuridine, pseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxyuridine, 2'-fluoroguanidine, 2'-fluorouridine, 5-bromouridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3(1-E-propenylamino)]uridine, α-thiocytidine, N6-methyladenosine, 5-methylcytidine, N4-acetylcytidine, 5-methacin, or combinations thereof into the mRNA.

Nucleic acid molecules that write RGNs can be codon-optimized for performance in the organism of interest. A "codon-optimized" coding sequence is a polynucleotide coding sequence whose codon usage frequency is designed to mimic a better codon usage frequency or the transcriptional conditions of a specific host cell. Performance in a specific host cell or organism is enhanced by changes in one or more codons at the nucleic acid level, without altering the translated amino acid sequence. Nucleic acid molecules can be codon-optimized either wholly or partially. Codon tables and other references providing information on preferences across a wide range of organisms are available in this field (see, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11, a discussion of codon usage preferences in plants). Methods for optimizing coding sequences of RGN codons for synthetic plant-preferred genes or mammalian (e.g., human) codons are available in this art. See, for example, U.S. Patents 5,380,831 and 5,436,391 and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference. Non-limiting examples of codon-optimized coding sequences of RGNs disclosed in this invention are shown in SEQ ID NO: 29 to 32 and 2659 to 2685.

The polynucleotides of the RGN and/or gRNA (i.e., crRNA) provided herein may be provided in an expression cassette for expression in vitro or in cells, organelles, embryos, or organisms of interest. The cassette contains 5' and 3' regulatory sequences operatively linked to the polynucleotides of the RGN and/or gRNA (i.e., crRNA) provided herein that allow for polynucleotide expression. The cassette may additionally contain at least one additional gene or gene element for co-transformation into an organism. If additional genes or elements are included, these components are operatively linked. The term "operatively linked" is intended to express a functional link between two or more elements. For example, an operatively linked promoter to a region of interest (e.g., a region encoding RGN and/or gRNA (i.e., crRNA)) is a functional link that allows the region of interest to be expressed. The operatively linked elements can be continuous or discontinuous. When used to refer to the link between two protein coding regions, "operatively linked" or "operatively fused" means that the coding regions are within the same reading frame. In some embodiments, an "operatively fused" peptide means that the structure and/or biological activity of each individual peptide is also present in the fusion. Alternatively, additional genes or elements may be provided on multiple expression casks. For example, the nucleotide sequence of the RGN disclosed in the codebook invention can be located on an expression cassette, while the nucleotide sequence of the codebook guiding RNA (i.e., crRNA) can be located on a separate expression cassette. This expression cassette is provided with multiple restriction sites and/or recombination sites to allow transcriptional regulation of the polynucleotide insertion regulated region. The expression cassette may additionally contain a selectable marker gene.

The expression cassette will contain, in the 5' to 3' direction of transcription, a transcription (or, in some embodiments, translation) initiation region (i.e., promoter), a polynucleotide encoded by the RGN- and/or gRNA (i.e., crRNA) of the present invention, and a transcription (or, in some embodiments, translation) termination region (i.e., termination region) that functions in the cell or organism of interest. The promoter of the present disclosure is capable of guiding or driving the expression of the encoded sequence in the host cell. Regulatory regions (e.g., promoter, transcription regulatory region, and translation termination region) may be endogenous or heterologous to the host cell, or endogenous or heterologous to each other. As used herein, “heterologous” means a sequence derived from a foreign species, or, if from the same species, a sequence substantially modified from its natural form in the constituents and/or genomic loci through deliberate human intervention. Chimeric genes, as used herein, include coding sequences operatively linked to transcription start regions heterologous to those coding sequences.

Suitable termination regions may include termination regions derived from simian virus (SV40), human growth hormone (hGH), bovine growth hormone (BGH), and rabbit beta-globin (RbGlob). See also Proudfoot (1991) Cell 64:671-674; Munroe et al. (1990) Gene 91:151-158; Schek et al. (1992) Molecular and Cellular Biology 12(12):5386-5393; Gil and Proudfoot (1987) Cell 49(3):399-406; Goodwin and Rottman (1992) The Journal of Biological Chemistry 267(23):16330-16334; and Lanoix and Acheson (1988) EMBO J. 7(8):2515-2522. Additional termination regions can be obtained from Ti plasmids of Agrobacterium tumefaciens , such as octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Additional regulatory signals include, but are not limited to: transcriptional initiation start site, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See, for example, U.S. Patents 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992), Molecular Cloning: A Laboratory Manual, edited by Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), hereinafter referred to as "Sambrook 11"; Davis et al. (1980), Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) and the references cited therein.

During the preparation of the presentation cassette, various DNA fragments can be manipulated to provide the DNA sequence in the appropriate orientation and at the appropriate time within the appropriate reading frame. For this purpose, transposons or connectors can be used to link the DNA fragments, or other manipulations may be involved to provide the sequence for appropriate restriction sites, removal of excess DNA, removal of restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, and resubstitution (e.g., transition and transversion) may be employed.

Many promoters can be used in the implementation of this disclosure. Promoters can be selected based on the desired outcome. Nucleic acids can be combined with persistent, inducible, growth-phase specific, cell-type specific, tissue-biased, tissue-specific promoters, or other promoters to express themselves in the organism of interest. See, for example, WO Initiators shown in Chinese Patent No. 99/43838 and U.S. Patent Nos. 8,575,425, 7,790,846, 8,147,856, 8,586,832, 7,772,369, 7,534,939, 6,072,050, 5,659,026, 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, and 6,177,611, which are incorporated herein by reference.

Exemplary persistent promoters used for expression in the cells of this disclosure include: the SV40 early promoter; the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter; the cytomegalovirus (CMV) promoter (e.g., the CMV immediate early promoter region (CMVIE)); the rous sarcoma virus (RSV) promoter; the human ubiquitin C promoter (UBC); and the human U6 small nuclear promoter. promoters (U6); enhanced U6 promoter; human H1 promoter from RNA polymerase III (H1); human elongation factor 1α promoter (EF1A); human beta-actin promoter (ACTB); human or mouse phosphoglycerate kinase 1 promoter (PGK); chicken β-actin promoter coupled with CMV early enhancer (CAGG); yeast transcription elongation factor promoter (TEF1); and so on. See, for example, Miyagishi et al. (2002) Nature Biotechnology 20:497-500; Xia et al. (2003) Nucleic Acids Res. 31(17):e100-e100; Pasleau et al. (1985) Gene 38:227–232; Martin-Gallardo et al. (1988) Gene 70: 51–56; Oellig and Seliger (1990) J Neurosci Res 26: 390–396; Manthorpe et al. (1993) Hum Gene Ther 4: 419–431; Yew et al. (1997) Hum Gene Ther 8: 575–584; Xu et al. (2001) Gene 272: 149–156; Nguyen et al. (2008) J Surg Res 148: 60–66; Costa et al. (2005) Nat Meth. 2:259–260; Lam and Truong (2020) ACS Synth. Biol. 9(10):2625–2631.

For expression in plants, persistent promoters also include the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); and MAS (Velten et al. (1984) EMBO J. 3:2723-2730).

Examples of induced promoters include: stress-regulated promoters, such as the Adh1 promoter, Hsp70 promoter, and Hsp90 promoter (Wurm et al. (1986) Proc. Natl. Acad. Sci. USA. 83:5414–5418; Nover L. Heat Shock Response. CRC Press; Boca Raton, FL, USA: 1991); light-inducible promoters, such as the PPDK promoter and the pepcarboxylase promoter; and metal-regulated promoters (Mayo et al. (1982) Cell. 29:99–108; Searle et al. (1985) Mol. Cell. Biol.). 5:1480–1489); hormone-responsive promoters, including glucocorticoid-responsive promoters (Hynes et al. (1981) Proc. Natl. Acad. Sci. USA. 78:2038–2042; Klock et al. (1987) Nature. 329:734–736). Chemoregulatory promoters include: the In2-2 promoter, which is safener-induced (US Patent No. 5,364,780); the Axig1 promoter, which is auxin-induced and tapetum-specific but also active in callus (PCT US 01/22169); and steroid-responsive promoters (see, for example, Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J). Estrogen-induced ERE promoters and glucocorticoid-induced promoters in 14(2):247-257; tetracycline-induced and tetracycline-inhibiting promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237; Gossen et al. (1993) Trends Biochem Sci. 18:471–475; Gossen and Bujard (1992) Proc. Natl Acad. Sci. USA 89:5547–5551; Zhou et al. (2006) Gene Ther. 13:1382–1390; and U.S. Patents No. 5,814,618 and No. 5,789,156; isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter; and lactose-regulated promoter. Induced behavior can be obtained using manipulator systems comprising: AlcR/acetaldehyde, ArgR/L-arginine, BirA/biotinyl-AMP, CymR/cumate, EthR/2-phenylethylbutyrate, HdnoR/6-hydroxynicotine oxidase, HucR/uric acid, MphR(A)/macrolide, PIP/streptogramin, Rex/NADH, RheA/heat, ScbR/SCB1, TraR/3-sideoxy-C8-HSL, and TtgR/phloretin; see, for example, U.S. Patent No. 8,728,759B2; U.S. Patent No. 7,745,592B2; Weber and Fussenegger. (2004) Methods Mol. Biol. 267:451–466; Hartenbach et al. (2007) Nucleic Acids Res. 35:e136; Weber et al. (2009) Metab. Eng. 11:117–124; Weber et al. (2008) Proc. Natl. Acad. Sci. USA. 105:9994–9998; Malphettes et al. (2005) Nucleic Acids Res. 33:e107; Kemmer et al. (2010) Nat. Biotechnol. 28:355–360; Weber et al. (2002) Nat. Biotechnol. 20:901–907; Fussenegger et al. (2000) Nat. Biotechnol. 18:1203–1208; Weber et al. (2006) Metab. Eng. 8:273–280; Weber et al. (2003) Nucleic Acids Res. 31:e69; Weber et al. (2003) Nucleic Acids Res. 31:e71; Neddermann et al. (2003) EMBO Rep. 4:159–165; and Gitzinger et al. (2009) Proc. Natl. Acad. Sci. USA. 106:10638–10643. Induced phenotypes can be obtained using protein-protein interaction systems, including: rapamycin-induced interaction between FKBP12 (FK506-binding protein 12) and mTOR (Rivera et al. (1996) Nat. Med. 2:1028-1032; Belshaw et al. (1996) Proc. Natl. Acad. Sci. USA. 93:4604-46077); the interaction between abscisic acid (ABA)-regulated PYL1 (abscisic acid receptor) and ABI1 (protein phosphatase 2C56) (Liang et al. (2011) Sci. Signal. 4(164):rs2-rs2); and light-induced protein-protein interaction systems (Wang et al. (2012) Nat. Methods). 9:266–269; Yamada et al. (2018) Cell. Rep. 25:487–500.

Tissue-specific or tissue-preferred promoters can be used to target the expression of performance architectures within a specific tissue. In some embodiments, tissue-specific or tissue-preferred promoters are active in mammalian tissues. Tissue-specific or tissue-preferred promoters include promoters that preferentially initiate transcription in certain tissues such as white blood cells (e.g., CD4 T cells), the heart, kidneys, liver, CNS, eyes, pancreas, skeletal muscle, and testes. In some embodiments, tissue-specific or tissue-preferred promoters are active in plant tissues. Examples of developmentally controlled promoters in plants include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, fruits, seeds, or flowers. "Tissue-specific" promoters are promoters that initiate transcription only in certain tissues. Unlike persistent gene expression, tissue-specific expression is the result of interactions between several levels of gene regulation. Thus, promoters from homologous or closely related plant species can preferentially initiate transcription in specific tissues for efficient and reliable expression of transgenic genes. In some implementations, expression includes tissue-preferred promoters. "Tissue-preferred" promoters are promoters that preferentially initiate transcription in, but not necessarily entirely in, or only in, certain tissues.

In some embodiments, the nucleic acid molecules encoding RGN and/or gRNA (i.e., crRNA) include cell type-specific promoters. A "cell type-specific" promoter is a promoter that primarily drives expression in certain cell types within one or more organs. Examples of cell type-specific promoters that primarily possess certain active cell types include, for example, primary cells, neurons, glial cells, adipocytes, cardiomyocytes, smooth muscle cells, photoreceptor cells, and retinal ganglia cells. Cell type-specific promoters that function primarily in plants can be examples of active plant cells, including, for example, BETL cells, vascular cells, stalk cells, and stem cells in roots and leaves. Nucleic acid molecules can also contain cell type-biased promoters. "Cell type-biased" promoters are those that primarily drive the expression of certain cell types, mostly but not necessarily entirely, or only in one or more organs. Examples of cell type-biased promoters that favor active cells include, for example, primary cells, neurons, adipocytes, cardiomyocytes, smooth muscle cells, and photoreceptor cells. The promoters that favor cell type preferences that function in plants may favor some examples of active plant cells, including, for example, BETL cells, vascular bundle cells, stalk cells, and stem cells in roots and leaves.

The nucleic acid sequence encoding RGN and/or gRNA (i.e., crRNA) can be operatively linked to a promoter sequence recognized by bacteriophage RNA polymerase, for example, for in vitro mRNA synthesis. In this embodiment, the in vitro transcribed RNA can be purified for use in the methods described herein. For example, the promoter sequence can be a T7, T3, or SP6 promoter sequence, or a variation of the T7, T3, or SP6 promoter sequence. In this embodiment, the expressed protein and/or RNA can be purified for use in the genome modification methods described herein.

RNA polymerase II promoters can be operatively linked to polynucleotides of the RGN polypeptide disclosed in the codebook for expression of the RGN polypeptide. For example, RNA pol II promoters useful for expressing the RGN polypeptide disclosed herein include CMV pol II promoters, CMV pol II promoters + CMV upstream enhancers, and CBh pol II promoters. In some embodiments, pol II promoters useful for expressing the RGN polypeptide include amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the amino acid sequences shown in SEQ ID NO: 1674 or 1675. In some embodiments, pol III promoters useful for expressing one or more gRNAs (i.e., crRNAs) include amino acid sequences shown in SEQ ID NO: 1674 or 1675.

In some embodiments, the polynucleotide encoding RGN and/or gRNA (i.e., crRNA) may be linked to a polyadenylation signal (e.g., the SV40 polyA signal and other signals that function in plants) and/or at least one transcription termination sequence. In some embodiments, the polyadenylation signal useful to this disclosure includes an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 1678. In some embodiments, the polyadenylation signal useful to this disclosure includes the amino acid sequence shown in SEQ ID NO: 1678. In addition, the sequence that writes the RGN may also be linked to one or more sequences that write at least one nuclear localization signal, at least one cell penetration domain and/or at least one signal peptide capable of transporting the protein to a specific subcellular address, as described elsewhere herein.

Polynucleotides encoding RGN and/or gRNA (i.e., crRNA) can be present in one or more vectors. A "vector" refers to a polynucleotide composition used to transfer, deliver, or introduce nucleic acids into a host cell. Suitable vectors include plasmid vectors, phage particles, myxosomes, artificial/microchromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated virus vectors, baculovirus vectors). Vectors may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylated sequences, transcription termination sequences), selective marker sequences (e.g., antibiotic resistance genes), replication origins, etc. Further information can be found in "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 3rd edition, 2001.

The vector may also include selective marker genes for selecting transformed cells. Selective marker genes are used for the selection of transformed cells or tissues. Marker genes include: genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT); and genes accreting resistance to herbicides such as glyphosate, bromobenzonitrile, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). Marker genes may include genes that allow selection for growth on specific nutrients or substances, such as dihydrofolate reductase (DHFR; Simonsen and Levinson (1983) Proc. Natl. Acad. Sci. USA 80:2495-2499), histidinol dehydrogenase (hisD; Hartman and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051), puromycin-N-acetyl transferase (pac or puro; de la Luna et al. (1988) Gene 62:121-126), and thymidine kinase (TK; Littlefield (1964) Science 145:709-710) and xanthine-guanine phosphoribosyltransferase (XGPRT or gpt; Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA 78:2072-2076).

The expression cartridge or vector containing the polynucleotide encoding the RGN polypeptide may further include a polynucleotide encoding gRNA (i.e., crRNA). The polynucleotide sequence encoding the gRNA (i.e., crRNA) may be operatively linked to at least one transcriptional control sequence for expression of the gRNA (i.e., crRNA) in the organism of interest or host cell. For example, the polynucleotide encoding the gRNA (i.e., crRNA) may be operatively linked to a promoter sequence recognized by RNA polymerase III (Pol III). Examples of suitable pol III promoters include, but are not limited to, mammalian U6, U3, H1, and 7SK RNA promoters and rice U6 and U3 promoters, such as the RNA pol III U6 promoter shown in SEQ ID NO: 38, and the promoter disclosed in WO 2022/261394, which is incorporated herein by reference in its entirety. In some embodiments, pol III promoters useful for expressing one or more gRNAs (i.e., crRNAs) include amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identical to the amino acid sequences shown in SEQ ID NO: 1672 or 1673. In some embodiments, the pol III promoter, which is useful for expressing one or more gRNAs (i.e., crRNAs), includes an amino acid sequence as shown in SEQ ID NO: 1672 or 1673.

As noted, expression constructs, including nucleotide sequences encoding RGN and/or gRNA (i.e., crRNA), can be used to transform the organism of interest. Methods for transformation involve introducing the nucleotide construct into the organism of interest. "Introduction" is defined as introducing the nucleotide construct into the host cell in such a way that the construct can enter the host cell. The method of the present invention does not require a specific method for introducing the nucleotide construct into the host organism, but only for the nucleotide construct to enter at least one cell of the host organism. The host cell can be a eukaryotic or prokaryotic cell. In some embodiments, the eukaryotic host cell is a plant cell, mammalian cell, avian cell, or insect cell. In some embodiments, eukaryotic cells including or representing the RGN disclosed in this invention, or modified with the RGN disclosed in this invention, are human cells. In some embodiments, eukaryotic cells including or representing the RGN disclosed in this invention, or modified with the RGN disclosed in this invention, are primary cells. The term "primary cells" refers to cells directly isolated from a multicellular organism. Primary cells typically undergo very little population doubling and are therefore more representative of the major functional components of the tissue from which they originate compared to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and therefore cannot be cultured in vitro for extended periods. In some embodiments, the primary cells are primary T cells. In some embodiments, eukaryotic cells including or representing the RGN disclosed in this invention, or modified with the RGN disclosed in this invention, are hematopoietic cells, such as immune cells (i.e., cells of the innate or adaptive immune system), including but not limited to: B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells. In some embodiments, the eukaryotic cells that include or represent the RGN disclosed in this invention or the RGN modified by this invention are eye cells, muscle cells (e.g., skeletal muscle cells), epithelial cells (e.g., lung epithelial cells), or pathological cells (e.g., tumor cells).

Methods for introducing nucleotide constructs into plant and other host cells are known in the art, including but not limited to: stable transformation methods, transient transformation methods and virus-mediated methods.

This method yields transformed organisms, such as plants, including whole plants and plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, and their offspring. Plant cells can be differentiated or undifferentiated (e.g., wound tissue, suspension cultured cells, protoplasts, leaf cells, root cells, phloem cells, pollen). In some embodiments, the method disclosed in this invention can result in transformed organisms or cell lines derived from these transformed cells.

"Transgenic organism,""transformedorganism," or "stable transformed" organism, cell, or tissue refers to an organism that has incorporated or integrated polynucleotides of RGN and/or gRNA (i.e., crRNA) containing the contents disclosed in the codebook. It should be recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments can also be incorporated into the host cell. Agrobacterium and gene gun-mediated transformation remain the two main methods used for transforming plant cells. However, the transformation of host cells can be achieved through infection, transfection, microinjection, electroporation, microprojection, gene gun or particle bombardment, electroporation, silicon dioxide/carbon fiber, ultrasound-mediated, PEG-mediated, calcium phosphate co-precipitation, polycationic DMSO technology, DEAE dextran program, and virus-mediated, liposome-mediated, etc. Virus-mediated introduction of polynucleotides encoding RGN and/or gRNA (i.e., crRNA) includes introduction and expression mediated by retrotranscribers, lentiviruses, adenoviruses, and adeno-associated viruses, as well as the use of cauliflower mosaic viruses (e.g., cauliflower mosaic virus), geminiviruses (e.g., bean golden yellow mosaic virus or maize streak virus), and RNA plant viruses (e.g., tobacco mosaic virus).

Transformation procedures and procedures for introducing polypeptide or polynucleotide sequences into plants can vary depending on the type of host cell targeted for transformation (e.g., monocot or dicotyledonous plant cells). Methods for transformation are known in the art and include those described in U.S. Patents 8,575,425, 7,692,068, 8,802,934, and 7,541,517, each of which is incorporated herein by reference. See also Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4:1-12; Bates, GW (1999) Methods in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107:1041-1047; Jones et al. (2005) Plant Methods 1:5.

Transformation can result in the stable or transient integration of nucleic acids into the cell. "Stable transformation" refers to the integration of introduced nucleotide constructs into the host cell's genome, which can be inherited by offspring. "Temporary transformation" refers to the introduction of polynucleotides into the host cell without integration into the host cell's genome.

Methods for chloroplast transformation are known in this field. See, for example, Svab et al. (1990) Proc. Nail. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. These methods rely on particle gun delivery of DNA containing selective markers and targeting the DNA to the plastid genome via homologous recombination. Alternatively, plastid transformation can be accomplished by transactivating silencing transgenes carried by plastids through tissue-biased expression of nucleocoding and plastid-guided RNA polymerases. This system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

Transformed cells can be grown into transgenic organisms, such as plants, in a conventional manner. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same or different transgenic strains to identify hybrids exhibiting a persistent phenotypic phenotype. Two or more generations can be grown to ensure that the polynucleotides encoding RGN and/or gRNA (i.e., crRNA) are stably maintained and inherited. Seeds are then harvested to ensure that the polynucleotides encoding RGN and/or gRNA (i.e., crRNA) are preserved. In this way, this disclosure provides a transformed plant or plant part having a nucleotide structure (e.g., an expression box of this disclosure) stably incorporated into its genome. Seeds having an expression box of this disclosure stably incorporated into their genome may also be referred to as "transgenic seeds".

Alternatively, transformed cells can be introduced into the organism. These cells can be derived from the organism, wherein the cells are transformed in vitro. These cells can be autologous (derived from and returned to the same individual) or allogeneic (the donor and recipient individuals belong to the same species).

The sequences provided in this article can be used to transform any plant species, including but not limited to monocots and dicots. Examples of plants of interest include, but are not limited to: corn, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and rapeseed, Brassica sp., alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, alligator, fig, guava, mango, olive, papaya, cashew, macadamia nut, almond, oats, vegetables, ornamental plants, and conifers.

Vegetables include, but are not limited to: tomatoes, lettuce, mung beans, lima beans, peas, and members of the genus *Curcumis* such as cucumbers, cantaloupes, and honeydew melons. Ornamental plants include, but are not limited to: azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, Christmas flowers, and chrysanthemums. In certain embodiments, the plants of this invention are crops (e.g., corn, sorghum, wheat, sunflowers, tomatoes, cruciferous plants, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

As used herein, the term "plant" includes: plant cells, plant protoplasts, plant cell tissue cultures from which regenerated plants can be derived, plant callus, plant clumps, and complete plant cells in a plant or its parts (e.g., embryo, pollen, ovule, seed, leaf, flower, branch, fruit, kernel, spike, rachis, husk, stem, root, root tip, anther, etc.). Grains are intended to represent mature seeds produced by commercial growers for purposes other than the growth or propagation of a species. Progeny, variants, and mutants of regenerated plants are also included within the scope of this invention, provided that these parts include the introduced polynucleotides. Further, processed plant products or byproducts retaining the sequences disclosed herein are provided, including, for example, soybean meal.

Writing RGN and/or gRNA (i.e., crRNA) or polynucleotides including gRNA (i.e., crRNA) can also be used to transform any eukaryotic species, including but not limited to: animals (e.g., mammals, humans, insects, fish, birds and reptiles), plants, fungi, amoebae, algae and yeast. In some embodiments, writing RGN and/or gRNA (i.e., crRNA) or polynucleotides including gRNA (i.e., crRNA) can also be used to transform any prokaryotic species, including but not limited to: archaea and bacteria (e.g., Bacillus, Klebsiella sp., Streptococcus, Rhizobium, Escherichia sp., Pseudomonas, Salmonella, Shigella, Vibrio, Yersinia, Mycobacterium, Agrobacterium, Lactobacillus).

Traditional viral and nonviral gene transfer methods can be used to introduce nucleic acids into mammalian, insect, or avian cells or target tissues. This method can be used to deliver nucleic acids encoded by RGN systems into cultured cells or cells in a host organism. Nonviral vector delivery systems include: DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery agents such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which, upon delivery to cells, possess an appendage-type or integrated genome. Non-limiting examples include vectors of cauliflower mosaic virus (e.g., cauliflower mosaic virus), geminivirus (e.g., legume golden yellow mosaic virus or corn stripe virus), and RNA plant virus (e.g., tobacco mosaic virus). For a review of gene therapy procedures, see Anderson, Science 256: 808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddadada et al., in Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

Non-viral delivery methods for nucleic acids include liposome transfection, nuclear transfection, microinjection, gene gun, virions, liposomes, immunoliposomes, polycations, or lipid-mediated nucleic acid conjugates, naked DNA, artificial viral particles, and drug-enhanced uptake of DNA. Liposome transfection is described in, for example, U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and the liposome transfection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Suitable for use in receptor-recognition lipofection of polynucleotides, including those cations and neutral lipids in Feigner's WO 91/17424 and WO 91/16024. Delivery can be to cells (e.g., in vitro or ex vivo) or to the target tissue (e.g., in vivo). The preparation of lipid-nucleic acid complexes (including targeted liposomes, such as immunoliposome complexes) is well known to those of ordinary skill in the art (see, for example, Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820). (1992); U.S. Patents No. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028 and 4,946,787.

Systems based on RNA or DNA viruses are used to deliver nucleic acids through highly evolved processes that target viruses to specific cells within the body and deliver the viral payload to the cell nucleus. Viral vectors can be administered directly to patients (in vivo) or used for in vitro cell treatment, and modified cells can be optionally administered to patients (ex vivo). Traditional virus-based systems may include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible using retroviruses, lentiviruses, and adeno-associated viruses for gene transfer, often resulting in long-term expression of the inserted transgenic gene. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues.

The tropism of retrotransmitters can be altered by incorporating foreign mantle proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retrotransmitter vectors capable of transducing or infecting non-dividing cells and typically producing high viral potency. Therefore, the choice of retrotransmitter gene transfer system depends on the target tissue. Retrotransmitter vectors consist of cis-acting long telomeres, which have the capacity to package 6-10 kb of foreign sequences. Minimal cis-acting LTRs are sufficient to replicate and package the vector, which is then used to integrate the therapeutic gene into the target cells to provide permanent transgenic expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibberish leukemia virus (GaLV), simultaneous immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66:1635-1640 (1992); Sommnerfelt et al., J. Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., J. Viral. 65:2220-2224 (1991); PCT/US94/05700).

In applications where short-term performance is preferred, adenovirus-based systems can be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types without requiring cell division. High potency and high performance levels have been achieved with such vectors. These vectors can be mass-produced in relatively simple systems. Adeno-associated virus (“AAV”) vectors can also be used, for example, in the in vitro generation of nucleic acids and peptides to transduce cells with targeted nucleic acids, and in in vivo and in vitro gene therapy programs (see, for example, West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93/24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)).

As used herein, the terms "adeno-associated virus" or "AAV" refer to a member of the genus dependoparvovirus, which belongs to the family Parvoviridae and is associated with this name. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11, numbered sequentially, have been described. Non-limiting exemplary serotypes useful in the compositions and methods disclosed herein include any of the 11 serotypes (e.g., AAV2, AAV5, AAV6, AAV8) or variant serotypes, such as AAV-DJ. AAVs are superior to other viral vectors in in vivo gene delivery (e.g., coding gene editing) because they typically do not integrate into the host genome, due to their low virulence and low probability of inducing insertional mutagenesis. AAV has a packaging limit of approximately 4.5 to 4.75 kb.

The construction of reconstructed AAV vectors has been described in numerous publications, including U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., I. Viral. 63:03822-3828 (1989). Packaging cells are commonly used to form viral particles capable of infecting host cells. Such cells include 293 cells packaging adenoviruses and ψJ2 cells or PA317 cells packaging retroviruses.

The carrier of this disclosure may include a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with any of the nucleotide sequences in SEQ ID NO: 1679 to 1683. In some embodiments, the carrier of this disclosure includes a nucleotide sequence as shown in any of SEQ ID NO: 1679 to 1683.

Viral vectors used in gene therapy are typically generated by creating cell lines that encapsulate nucleic acid vectors within viral particles. These vectors usually contain the minimum viral sequences required for encapsulation and subsequent integration into the host; other viral sequences are replaced by expression capsules containing one or more polynucleotides to be expressed. Missing viral functions are typically provided trans-present by the encapsulating cell line. For example, AAV vectors used in gene therapy typically only possess the ITR sequence derived from the AAV genome required for encapsulation and integration into the host genome. Viral DNA is encapsulated in cell lines containing helper plasmids (rep and cap) that encode other AAV genes, but lack the ITR sequence. Adenoviruses can also be used as helpers to infect the cell lines. Adjuvant viruses promote the replication of AAV vectors and the expression of AAV genes derived from adjuvant plasmids. Due to the lack of an ITR sequence, adjuvant plasmids were not packaged in significant quantities. Adenovirus contamination can be reduced by, for example, heat treatment; adenovirus is more sensitive to heat treatment than AAV. Additional methods for delivering nucleic acids into cells are known to those skilled in the art. See, for example, US20030087817, which is incorporated herein by reference.

In some embodiments, host cells are transiently or non-transiently transfected using one or more vectors described herein. In some embodiments, cells are transfected while they are naturally present in an individual. In some embodiments, the transfected cells are taken from an individual.

In some implementations, the transfected cells are eukaryotic cells. In some implementations, the eukaryotic cells are animal cells (e.g., mammals, humans, insects, fish, birds, and reptiles). In some implementations, the transfected cells are human cells. In some implementations, the transfected cells are hematopoietic cells, such as immune cells (i.e., cells of the innate or adaptive immune system), including but not limited to: B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells.

In some embodiments, the cells are derived from cells taken from an individual, such as a cell line. In some embodiments, the cells or cell lines are prokaryotic. In some embodiments, the cells or cell lines are eukaryotic. In further embodiments, the cells or cell lines are derived from insects, birds, plants, or fungi. In some embodiments, the cells or cell lines may be mammals, such as humans, monkeys, mice, cows, pigs, goats, hamsters, rats, cats, or dogs. Many cell lines used for tissue culture are known in the art. Examples of cell lines include, but are not limited to: C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TFl, CTLL-2, CIR, Rat6, CVI, RPTE, AlO, T24, 182, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388Dl, SEM-K2, WEHI-231, HB56, TIB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4. COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial cells, BALB/3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-Ll, 132-d5 human fetal fibroblasts, 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3, C3H-10Tl/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-Kl, CHO-K2, CHO-T, CHO Dhfr-/-, COR-L23, COR-L23/CPR, COR-L235010, CORL23/ R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA 2. HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, LY cells, K562 cells, Ku812, KCL22, KGl, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR/ 0.2R, MONO-MAC 6. MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell line, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR and their transgenic variants. Cell lines can be obtained from a variety of sources known to the general knowledge of the art (see, for example, the American Type Culture Collection (ATCC) (Manassas, Virginia)).

In some embodiments, new cell lines comprising one or more vector-derived sequences are established by transfecting cells with one or more nucleic acid molecules or vectors described herein. In some embodiments, new cell lines comprising cells containing modified sequences but lacking any other foreign sequences are established by briefly transfecting cells with components of an RGN system as described herein (e.g., by brief transfection with one or more vectors, or by RNA transfection) and then modifying the activity of the RGN system. In some embodiments, one or more test compounds are evaluated using cells briefly or non-briefly transfected with one or more vectors described herein, or cell lines derived from such cells.

In some embodiments, one or more vectors described herein are used to generate non-human transgenic animals or plants. In some embodiments, the transgenic animal is an insect. In further embodiments, the insect is a pest, such as a mosquito or tick. In some examples, the insect is a plant pest, such as a corn root borer or fall armyworm. In some embodiments, the transgenic animal is a bird, such as a chicken, turkey, goose, or duck. In some embodiments, the transgenic animal is a mammal, such as a human, mouse, rat, hamster, monkey, ape, rabbit, pig, cow, horse, goat, sheep, cat, or dog. VI. Variations and fragments of polypeptides and polynucleotides

This disclosure provides information with SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, etc. The amino acid sequences shown in 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 represent the active variants and fragments of RNA-guided nucleases (RGN), as well as the corresponding CRISPR repeaters and gRNAs. In some embodiments, the active variants or fragments of the RGN disclosed herein include V-H, V-I, or V-J type RGNs. In some embodiments, this disclosure provides: having the following characteristics as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, Reactive variants and fragments of RGN of any of the amino acid sequences shown in 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687; CRISPR RNA repeaters (i.e., crRNA backbones) comprising active variants and fragments of nucleotide sequences as shown in any of SEQ ID NO: 5 to 8, 1617 to 1620, 1631 and 1957 to 1983; gRNAs (i.e., crRNAs) comprising active variants and fragments of sequences as shown in any of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658; and polynucleotides with the same coding scheme.

Although the activity of a variant or fragment may change compared to the polynucleotide or peptide of interest, the variant or fragment should retain the functionality of the polynucleotide or peptide of interest. For example, when compared to the polynucleotide or peptide of interest, the variant or fragment may have increased activity, decreased activity, a different activity profile, or any other change in activity.

Having, for example, SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, Fragments and variants of RGN polypeptides of any of the amino acid sequences shown in 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 (such as those disclosed herein) will retain sequence-specific, RNA-guided DNA-binding activity. In some embodiments, fragments and variants of the RGN peptide disclosed herein retain nuclease activity. In some embodiments, fragments and variants of the RGN peptide disclosed herein bind to guide RNA that does not contain tracrRNA.

Some implementations include, for example, SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 11 The active variant or fragment of an RGN polypeptide with an amino acid sequence shown in any of the following sequences is a reference RGN (reference RGN). The sequence-specific, RNA-guided DNA-binding activity of the RNA (RGN) is approximately 80% to approximately 500%, approximately 80% to approximately 200%, or approximately 90% to approximately 150%, or approximately 95% to approximately 120%, or at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, or at least... 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or higher. Sequence-specific, RNA-guided DNA-binding activity assays for indirectly or directly measuring RGN peptides are known to those of ordinary skill in the art and include: T7 endonuclease assays, chromatin immunoprecipitation assays, gel migration translocation assays, DNA pull-down assays, reporter assays, microdisk capture, detection assays, competitive binding assays, single-molecule fluorescence microscopy, and next-generation sequencing (NGS) with targeted analysis.

In some embodiments, including SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1 The active variants or fragments of RGN polypeptides with amino acid sequences shown in any of the following sequences are characterized by nuclease activity: 083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687. The activity is approximately 80% to approximately 500%, approximately 80% to approximately 200%, or approximately 90% to approximately 150%, or approximately 95% to approximately 120% of the nuclease activity of the reference RGN, or at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, up to At least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or higher. Nuclease activity can be measured by assays known to those of ordinary skill in the art, including but not limited to: tracking of indels by decomposition (TIDE) analysis; flow cytometry; and in vitro or in vivo shear assays, wherein the shearing is confirmed by PCR, sequencing, or gel electrophoresis with or without the attachment of appropriate tracers (e.g., radioisotopes, fluorescent substances) to the target sequence to facilitate the detection of degradation products. In some embodiments, nicking-triggered exponential amplification reaction (NTEXPAR) assays can be used (see, for example, Zhang et al. (2016) Chem. Sci. 7:4951-4957). In vivo cleavage can be assessed using the Surveyor assay (Guschin et al. (2010) Methods Mol Biol 649:247-256). In some embodiments, the efficiency of cleavage of the target sequence is assessed by measuring the percentage change in the expression of the target sequence or the polypeptide encoded by the target sequence in cells containing the target sequence or in cells containing the target sequence. In some embodiments, the expression is measured using quantitative PCR, microarrays, RNA-seq, flow cytometry, immunooblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or combinations thereof. In some embodiments, the target sequence encodes proteins expressed on the cell surface, and when measured by flow cytometry, the efficiency of target sequence cleavage is assessed by measuring the percentage of cells with reduced protein expression on the cell surface.

In some embodiments, the reference RGN has the following SEQ ID NOs: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, The amino acid sequence of any one of 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687. In some embodiments, reference RGN is SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 10 A variant of any of the following: 87, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687, lacking a corresponding mutation in its active variant or fragment, and possessing, for example, RNA-guided sequence-specific binding activity or nuclease activity as described above. In some embodiments, the reference RGN is a different variant of the RGN. In some embodiments, a reference RGN is a different variant of the same parent RGN. "Parent RGN" or "parent RGN polypeptide" refers to a parent polypeptide that can serve as a template to introduce one or more mutations to produce the parent polypeptide. For example, LPG10238 is the parent RGN polypeptide of the mutant RGN polypeptides LPG10427 to LPG10821 and LPG12222, as discussed in Example 6. Other parental RGN peptides disclosed herein include LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, and LPG13100. LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115 and LPG13116.

This article discloses that fragments and variants of CRISPR RNA (crRNA) repeaters (i.e., the crRNA backbone), when used as part of guide RNA, retain the ability to bind to the RGN (complexed with the guide RNA) in a sequence-specific manner and guide the RGN to the target sequence (e.g., the target DNA sequence).

The term "fragment" refers to a portion of the polynucleotide or polypeptide sequence disclosed herein. A "fragment" or "bioactive portion" comprises a polynucleotide comprising a sufficient number of consecutive nucleotides to retain biological activity (i.e., when included within guide RNA, it binds to the RGN in a sequence-specific manner and guides the RGN to the target nucleotide sequence). A "fragment" or "bioactive portion" comprises a polypeptide comprising a sufficient number of consecutive amino acid residues to retain biological activity (i.e., when complexed with guide RNA, it binds to the target sequence in a sequence-specific manner). Fragments of the RGN protein include those shorter than the full-length sequence due to the use of alternative downstream initiation sites. The bioactive portion of the RGN protein may include, for example, SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 11 A polypeptide of 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 or more consecutive amino acid residues from any of the following: 05, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687. Such bioactive portions can be prepared using recombination techniques and their sequence specificity and RNA-guided DNA binding activity can be evaluated. The bioactive fragment of the crRNA repeat sequence (i.e., the crRNA backbone) may include at least eight consecutive amino acids from any of SEQ ID NOs: 5 to 8, 1617 to 1620, 1631, and 1957 to 1983. The bioactive portion of the CRISPR repeat sequence (i.e., the crRNA backbone) may be a polynucleotide comprising, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive nucleotides from any of SEQ ID NOs: 5 to 8, 1617 to 1620, 1631, and 1957 to 1983. The biologically active fragment of gRNA (i.e., crRNA) may include at least 30 consecutive nucleotides of any one of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658. The biologically active fragment of gRNA (i.e., crRNA) may include at least 30 consecutive nucleotides of any one of SEQ ID NO: 39 to 49, 51, 53, 54, 58, 61, 62, 64, 66, 68, 69, 78, 80, 81, 83, 84, 86, 88, 91, 96, 98, 99, 101, 109 to 128, 130, 132, 133, 135, 137-139, 147 to 153, 159, 163, 165, 168, 170, 173, 175, 176, 178, 179 and 182. The biologically active portion of gRNA (i.e., crRNA) can be a polynucleotide comprising 30, 35, 40, 45, 50, 55 or more consecutive nucleotides, such as any of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658. The biologically active portion of gRNA (i.e., crRNA) can be a polynucleotide comprising, for example, any of the following: SEQ ID NO: 39 to 49, 51, 53, 54, 58, 61, 62, 64, 66, 68, 69, 78, 80, 81, 83, 84, 86, 88, 91, 96, 98, 99, 101, 109 to 128, 130, 132, 133, 135, 137 to 139, 147 to 153, 159, 163, 165, 168, 170, 173, 175, 176, 178, 179, and 182, 30, 35, 40, 45, 50, 55, or more consecutive nucleotides.

Generally, a "variant" is intended to express substantially similar sequences. For polynucleotides, variants include the deletion and/or addition of one or more nucleotides at one or more sites within a natural polynucleotide and/or the substitution of one or more nucleotides at one or more sites within a natural polynucleotide. As used herein, "natural" or "wild-type" polynucleotides or polypeptides respectively comprise naturally occurring nucleotide or amino acid sequences. For polynucleotides, conservative variants include those sequences that, due to degeneracy of the genetic code, encode the natural amino acid sequence of the gene of concern. Such naturally occurring variants can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as described below. Variant polynucleotides also include synthetically obtained polynucleotides, such as those produced by site-directed mutagenesis that still encode the polypeptide or polynucleotide of concern. Generally, variants of a particular polynucleotide disclosed herein have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the particular polynucleotide as determined by the sequence alignment procedures and parameters described elsewhere herein. The specific polypeptide variants disclosed herein have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the specific polynucleotide as determined by the sequence alignment procedures and parameters described elsewhere herein.

The variants of the specific polynucleotides (i.e., the reference polynucleotides) disclosed herein can also be assessed by comparing the percentage of sequence similarity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The sequence alignment procedures and parameters described elsewhere herein can be used to calculate the percentage of sequence similarity between any two polypeptides. When assessing any given polynucleotide pair disclosed herein by comparing the percentage of sequence similarity shared by two polypeptides encoded by any given polynucleotide pair disclosed herein, the percentage of sequence similarity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

The present invention discloses a polynucleotide encoding an RGN polypeptide that is active without the need for tracrRNA. In some embodiments, the present invention discloses an RGN polypeptide that encodes a V-H, V-I, or V-J type RGN. In some embodiments, the present invention discloses a polynucleotide encoding an RGN polypeptide, wherein the RGN polypeptide includes those such as SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975. 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132 The amino acid sequences shown in 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 have an amino acid sequence with at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology.

Bioactive variants of the RGN peptide disclosed herein may differ by as few as about 1-15 amino acid residues, as few as about 1-10 (e.g., about 6-10), as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. In some embodiments, the peptide may include an N-terminal or C-terminal truncation, which may include the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300 or more amino acids from the N-terminus or C-terminus of the peptide.

In some embodiments, the polynucleotides disclosed in this invention include or encode crRNA repeaters (i.e., crRNA backbones), which include nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleotide sequences shown in any of SEQ ID NO: 5 to 8, 1617 to 1620, 1631 and 1957 to 1983.

The variants of guide RNA (i.e., crRNA) disclosed in this article include guide RNA (i.e., crRNA) with modified nucleotides, sugars, phosphate backbones and/or nucleobases. Variant guiding RNA (i.e., crRNA) may contain modifications, including: 2'-O-methyl (2'-O-Me) modification; 2'-fluoro (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphosilicate (MSP; 2'-O-methyl 3'-thioPACE) modification; 2'-O-methyl 3'-phosphosilicate (MP) modification; thiophosphate (PS) modification; and bridged nucleic acid (BNA) modifications (e.g., 2',4'BNA, close-labeled nucleic acid (LNA), N-methyl-substituted bridged nucleic acid BNA ^NC). [N-Me], 2'-O,4'-C-ethylene-bridging nucleic acids (2',4'-ENA) and S-restricted ethyl (cEt); or combinations thereof. Chemical modifications of spacers, crRNA repeats, crRNA, tracrRNA and guide RNA are described in WO 2024/042489, which is incorporated herein by reference in its entirety.

The bioactive variants of the guide RNA (i.e., crRNA) disclosed herein may differ by as few as about 1 to 15 nucleotides, or as few as about 1 to 10, such as about 6 to 10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 nucleotide. In some embodiments, the polynucleotide may include a 5' or 3' truncation, which may include the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides counting from the 5' or 3' end of the polynucleotide.

It is understood that the RGN polypeptides, crRNA repeaters (i.e., the crRNA backbone), and gRNAs (i.e., crRNAs) provided herein can be modified to create variant proteins and polynucleotides. Artificially designed modifications can be introduced through the application of site-directed mutagenesis techniques. Alternatively, naturally occurring, unknown, or unidentified polynucleotides and/or polypeptides that are structurally and/or functionally related to the sequences disclosed herein are also considered to fall within the scope of this invention. Conserved amino acid substitutions can be performed in non-conserved regions that do not alter the function of the RGN protein. Alternatively, modifications can be made to improve RGN activity.

Variant polynucleotides and proteins also encompass sequences and proteins obtained from mutagenesis and recombination procedures such as DNA shuffling. Using such procedures, one or more different RGN proteins disclosed herein (e.g., SEQ ID NO: 1 to 4) are manipulated to create new RGN proteins possessing desired properties. In this manner, the recombinant polynucleotide library is generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and are capable of homologous recombination in vitro or in vivo. For example, using this method, a sequence motif encoding the domain of interest can be shuffled between the RGN sequence provided herein and other known RGN genes to obtain a new gene encoding a protein with improved properties of interest (e.g., increased Km in the case of enzymes). Such DNA shuffling strategies are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Patents 5,605,793 and 5,837,458. "Mixed" nucleic acids are nucleic acids generated by a mixing program (such as any mixing program described herein). Nucleic acids in a hybrid arrangement are generated by recombining two or more nucleic acids (or strings) in a manner that is, for example, artificial and optionally recursive (physical or virtual). Generally, one or more screening steps are used during the hybrid arrangement process to identify the nucleic acids of interest; this screening step can be performed before or after any recombination step. In some (but not all) hybrid arrangement implementations, it is desirable to perform multiple rounds of recombination before selection to increase the diversity of the pool to be screened. The entire recombination and selection process can optionally be repeated recursively. Depending on the context, hybrid arrangement may refer to the entire recombination and selection process, or alternatively, only the recombination portion of the entire process.

As used herein, “sequence similarity” or “identity” in the context of two polynucleotide or polypeptide sequences refers to the presence of identical residues on two sequences within a specified comparison window when aligned to obtain maximum correspondence. It is recognized that the differences in the positions of dissimilar residues often arise from the substitution of conserved amino acids, where an amino acid residue is substituted by another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. Protein sequences that differ due to such conserved substitutions are said to have “sequence similarity” or “identity.” The means used to measure sequence similarity are well known to those skilled in the art. Typically, this involves assessing conserved substitutions as partial mismatches rather than complete mismatches. Therefore, for example, if a score of 1 is given for the same amino acid and a score of zero is given for non-conservative substitutions, then a score between 0 and 1 is given for conservative substitutions. For example, the score for conservative substitutions is calculated as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

As used herein, "sequence identity percentage" refers to a value determined by comparing two best-aligned sequences in a comparison window, where the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (excluding additions or deletions) used for the best-alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleotide or amino acid residues appear in the two sequences; dividing the number of matching positions by the total number of positions in the comparison window; and multiplying the result by 100 to obtain the sequence identity percentage.

Unless otherwise stated, the sequence identity/similarity values provided herein refer to values obtained using GAP version 10 with the following parameters: % identity and similarity of nucleotide sequences using a GAP weight of 50 and a length weight of 3 and an nwsgapdna.cmp rating matrix; % identity and similarity of amino acid sequences using a GAP weight of 8 and a length weight of 2 and a BLOSUM62 rating matrix; or any equivalent program. "Equivalent program" means any sequence comparison program that, when compared to the corresponding alignment generated by GAP version 10 for any two sequences involved, produces alignments with the same nucleotide or amino acid residue matches and the same percentage of sequence identity.

When similarity is assessed by comparing two sequences using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty, and gap extension penalty, the two sequences are considered "best aligned" to achieve the highest possible score for that sequence pair. The use of amino acid substitution matrices and their application in quantifying the similarity between two sequences is well-known in the field and has been described, for example, in Dayhoff et al. (1978), "Atlas of Protein Sequence and Structure," Vol. 5, Supplement 3 (MO Dayhoff, ed.), pp. 345-352, "A model of evolutionary change in proteins," Natl. Biomed. Res. Found., Washington, DC; and Henikoff et al. (1992), Proc. Natl. Acad. Sci. USA 89:10915-10919. The BLOSUM62 matrix is often used as the default scoring substitution matrix in sequence alignment procedures. A gap presence penalty is applied for introducing a single amino acid gap in one of the aligned sequences, while a gap extension penalty is applied for each additional empty amino acid position inserted into an opened gap. Alignments are defined by the amino acid positions of each sequence at the start and end of the alignment, and optionally by inserting one or more gaps in one or two sequences, to achieve the highest possible score. While best alignment and scoring can be performed manually, this process can be facilitated by computer-implemented alignment algorithms, such as the gapped BLAST 2.0 described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402 and publicly available on the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov on the Global Information Network). Best alignments involving multiple alignments can be prepared using, for example, PSI-BLAST, which is available via www.ncbi.nlm.nih.gov on the Global Information Network and described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

Regarding the amino acid sequence that best aligns with the reference sequence, the amino acid residue "corresponds" to the position in the reference sequence that it pairs with during alignment. The "position" is represented by a number, which sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Because deletions, insertions, truncations, fusions, etc., must be considered when determining the best alignment, the number of amino acid residues in the test sequence, usually determined by simply counting from the N-terminus, does not necessarily have to be the same as the number of their corresponding positions in the reference sequence. For example, in the case of a deletion in the aligned test sequence, there will be no amino acid corresponding to the deletion site in the reference sequence. If there is an insertion in the aligned reference sequence, the insertion will not correspond to any amino acid position in the reference sequence. In the case of truncation or fusion, the reference sequence or the sequence being compared may contain amino acid stretches that do not correspond to any amino acid in the corresponding sequence. VII. Antibodies

This also includes antibodies against the RGN peptide or ribonucleoproteins comprising the RGN peptide as disclosed herein, wherein the RGN peptide comprises having the following characteristics: SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, RGN polypeptides containing the amino acid sequences or their active variants or fragments shown in any of the following: 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687. Methods for generating antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; and U.S. Patent No. 4,196,265). These antibodies can be used in kits for the detection and separation of RGN peptides or ribonucleoprotein complexes. Therefore, this disclosure provides kits comprising antibodies that specifically bind to the peptides or ribonucleoprotein complexes described herein, the peptides or ribonucleoprotein complexes comprising, for example, having a SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081 RGN polypeptides with amino acid sequences shown in any of the following sequences: 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687. VIII. RGN systems for binding target sequences and ribonucleoprotein complexes and methods for their preparation.

This disclosure provides an RNA-guided nuclease (RGN) system for binding to a target sequence of interest (e.g., a target DNA sequence), wherein the RGN system comprises at least one RGN polypeptide or a polynucleotide sequence encoding at least one RGN polypeptide and one or more guide RNAs (i.e., one or more crRNAs) capable of forming a complex (ribonucleoprotein complex) with the RGN polypeptide. The guide RNA (i.e., crRNA) hybridizes with the target strand of the target sequence of interest and also forms a complex with the RGN polypeptide, thereby guiding the RGN polypeptide to bind to the target sequence. In some embodiments, the target sequence comprises a nucleotide sequence as shown in any of SEQ ID NO: 183 to 285, 1684 to 1691, 1693 to 1775, 1785 to 1788, and 2038 to 2081. In some embodiments, the RGN is capable of recognizing and binding to the target sequence via a PAM adjacent to its 5' end. In some embodiments, the RGN disclosed herein is active without the need for tracrRNA. In some embodiments, the RGN disclosed herein comprises a V-H type RGN. In some embodiments, the RGN disclosed herein comprises a V-I type RGN. In some embodiments, the RGN disclosed herein comprises a V-J type RGN. In some of these embodiments, the RGN includes, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1 The amino acid sequence or its active variant or fragment represented by any one of 082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687. In some embodiments, the guide RNA includes a crRNA repeater (i.e., a crRNA backbone) having a nucleotide sequence or an active variant or fragment thereof as shown in any of SEQ ID NOs: 5 to 8, 1617 to 1620, 1631, and 1957 to 1983. The guide RNA (i.e., crRNA) of the RGN system disclosed herein may include spacers hybridized with eukaryotic target sequences. In some embodiments, the eukaryotic target sequence includes a mammalian target sequence. In some embodiments, the guide RNA (i.e., crRNA) includes a nucleotide sequence or an active variant or fragment thereof as shown in any of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658. In some embodiments, the guide RNA (i.e., crRNA) comprises a nucleotide sequence or an active variant or fragment thereof as shown in any of SEQ ID NO: 39 to 49, 51, 53, 54, 58, 61, 62, 64, 66, 68, 69, 78, 80, 81, 83, 84, 86, 88, 91, 96, 98, 99, 101, 109 to 128, 130, 132, 133, 135, 137 to 139, 147 to 153, 159, 163, 165, 168, 170, 173, 175, 176, 178, 179, and 182. In some embodiments, the RGN system comprises an RGN polypeptide heterologous to the guide RNA, wherein the RGN polypeptide is not found to be complexed with (i.e., bound to) the guide RNA in nature.

The RGN system provided herein for binding to a target sequence of interest may comprise a ribonucleoprotein (RNP) complex, which is at least one molecule of RNA that binds to at least one protein. The RNP complex provided herein comprises at least one guide RNA (i.e., crRNA) as RNA and an RGN polypeptide as a protein. The guide RNA (i.e., crRNA) of the RNP complex disclosed herein may include a spacer hybridized with a eukaryotic target sequence. In some embodiments, the eukaryotic target sequence includes a mammalian target sequence. Such RNP complexes may be purified from cells or organisms naturally expressing RGN polypeptides and have been engineered to express a specific guide RNA (i.e., crRNA) specific to the target sequence of interest. In some embodiments, the RNP complex can be purified from one or more polynucleotides encoding an RGN polypeptide (e.g., mRNA encoding the RGN polypeptide) and guide RNA (or a polynucleotide including the guide RNA) and cultured under conditions that allow expression of the RGN polypeptide and guide RNA (i.e., crRNA). Therefore, methods for producing RGN polypeptides or RNP complexes are provided. Such methods include culturing cells comprising a polynucleotide sequence encoding an RGN polypeptide (and, in some embodiments, a polynucleotide sequence encoding or including the guide RNA) under conditions that express an RGN polypeptide (and, in some embodiments, guide RNA). The RGN polypeptide or RNP complex can then be purified from lysates of the cultured cells. In some embodiments, the polynucleotide sequence encoding the RGN polypeptide comprises mRNA (messenger RNA). In some embodiments, the method for assembling an RNP complex includes, under conditions suitable for forming an RNP complex, combining one or more of the guide RNA (i.e., crRNA) disclosed herein with one or more of the RGN polypeptide disclosed herein.

Methods for purifying RGN peptides or RNP complexes from lysates of biological samples are known in the art (e.g., size exclusion and/or affinity chromatography, 2D-PAGE, HPLC, reverse phase chromatography, immunoprecipitation). In a particular method, the RGN peptide is generated by recombination and includes purification traces that facilitate its purification. These purification traces include, but are not limited to: glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) traces, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG (e.g., 3x FLAG traces), HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 10xHis, biotinylate carboxyl group carrier protein (BCCP), and calcium-modulating protein. Generally, labeled RGN peptides or RNP complexes are purified using immobilized metal affinity chromatography. It should be understood that other similar methods known in the art, including other forms of chromatography or, for example, immunoprecipitation, may be used alone or in combination.

"Isolated" or "purified" polypeptides or their bioactive portions substantially or essentially do not contain the components typically associated with or interacting with the polypeptide in its natural environment. Therefore, isolated or purified polypeptides, when generated by recombination, substantially do not contain other cellular material or culture medium, or, when chemically synthesized, substantially do not contain chemical precursors or other chemicals. Proteins substantially free of cellular material comprise protein preparations having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminated proteins. When the proteins of the present invention or their bioactive portions are generated by recombination, the optimal culture medium presents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein chemicals of concern. Similarly, "isolated" polynucleotides or nucleic acid molecules are removed from their native environment. When chemically synthesized, isolated polynucleotides do not actually contain chemical precursors or other chemicals, or have been removed from the locus of the genome by the breaking of phosphodiester bonds. Isolated polynucleotides can be part of a carrier, a component of a substance, or can be contained within a cell (provided the cell is not the original environment of the polynucleotide).

The specific methods provided herein for binding and/or cleaving target nucleic acid molecules, including the target sequence of interest, involve the use of in vitro assembled RNP complexes. In vitro assembly of RNP complexes can be performed using any method known in the art, wherein the RGN peptide is brought into contact with the guide RNA (i.e., crRNA) under conditions that allow the RGN peptide to bind to the guide RNA. As used herein, “contact,” “contacting,” or “contacted” means bringing together the components to be reacted under conditions suitable for the desired reaction; or bringing one entity into contact with one or more other entities, with or without any intermediate means. In some embodiments, "contact," "contacting," or "contacted" includes, but is not limited to, placing entities in the same container or solution, or transfection, transformation, viral delivery, or LNP delivery of one entity and one or more other entities. RGN peptides can be purified from biological samples, cell lysates, or culture media, generated by in vitro transcription, or chemically synthesized. Guide RNA (i.e., crRNA) can be purified from biological samples, cell lysates, or culture media, transcribed in vitro, or chemically synthesized. Contact between RGN peptides and guide RNA in solution (e.g., a buffered saline solution) can be made to allow for in vitro assembly of RNP complexes.

Some embodiments of this disclosure provide kits that include one or more elements of the RGN system described herein, comprising: guide RNA (i.e., crRNA); an RGN polypeptide or a polynucleotide encoding it; a cell; and the complete RGN system. In some embodiments, the kit includes suitable reagents, buffers, and/or instructions for use of one or more elements of the RGN system, for example, for in vitro or in vivo nucleic acid editing. Reagents may be provided in any suitable container (e.g., vials, bottles, or tubes). Reagents may be used in processes employing one or more elements of the RGN system. For example, restriction enzymes may be included for cloning polynucleotides encoding RGN into a vector. In some embodiments, the kit includes instructions for designing and using suitable guide RNA (i.e., crRNA) to target the editing sequence. The reagent may be available in a form suitable for a specific assay, or in a form requiring the addition of one or more other components prior to use (e.g., as a concentrate or freeze-dried form). The buffer may be any buffer, including but not limited to: sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10.

A kit containing one or more elements of the RGN system disclosed herein is useful in a wide variety of applications, including the modification (e.g., excision of a portion of a target polynucleotide, deletion, insertion of a donor polynucleotide, translocating, inactivation, activation, base editing, polymerase editing) of target sequences in various cell types. Thus, a kit containing one or more elements of the RGN system disclosed herein can be useful in, for example, gene therapy, drug screening, disease diagnosis, and prognosis.

In some embodiments, the kit of this disclosure includes a pharmaceutical kit containing the pharmaceutical composition described herein. In some embodiments, the pharmaceutical kit may include: (a) a container containing the composition of this disclosure in a freeze-dried form; and (b) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent may be used to reconstitute or dilute the freeze-dried compound of this disclosure. Alternatively, associated with (one or more) such containers may be a notification in the form prescribed by a governmental agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, reflecting approval by the manufacturer, user, or seller for human administration. IX. Methods for binding, cleaving, or modifying target nucleic acid molecules.

This disclosure provides methods for binding to, cleaving, and/or modifying a target nucleic acid molecule of interest (e.g., target DNA) that includes a target sequence. The methods include delivering an RGN system to a target sequence or a cell, organelle, or embryo that includes the target sequence. The RGN system includes at least one guide RNA (i.e., crRNA) or a polynucleotide encoding it; and at least one RGN polypeptide or a polynucleotide encoding it. In some embodiments, the target sequence includes a nucleotide sequence as shown in any of SEQ ID NOs: 183 to 285, 1684 to 1691, 1693 to 1775, 1785 to 1788, and 2038 to 2081. In some embodiments, the RGN is capable of recognizing and binding to the target sequence at its 5' PAM. In some embodiments, the RGN disclosed herein is active without tracrRNA. In some embodiments, the RGN disclosed herein comprises V-H, V-I, or V-J type RGNs. In some embodiments of these embodiments, the RGN polypeptide includes, for example, SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1 The amino acid sequence or its active variant or fragment represented by any one of 082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687. In some embodiments, the guide RNA comprises a crRNA repeat (i.e., the crRNA backbone) having a nucleotide sequence as shown in any of SEQ ID NOs: 5 to 8, 1617 to 1620, 1631 and 1957 to 1983, or an active variant or fragment thereof. In some embodiments, the guide RNA (i.e., crRNA) comprises a nucleotide sequence as shown in any of SEQ ID NOs: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658, or an active variant or fragment thereof. In some embodiments, the guide RNA (i.e., crRNA) comprises a nucleotide sequence or an active variant or fragment thereof as shown in any of SEQ ID NO: 39 to 49, 51, 53, 54, 58, 61, 62, 64, 66, 68, 69, 78, 80, 81, 83, 84, 86, 88, 91, 96, 98, 99, 101, 109 to 128, 130, 132, 133, 135, 137 to 139, 147 to 153, 159, 163, 165, 168, 170, 173, 175, 176, 178, 179, and 182. Methods for binding, cleaving, and/or modifying a target nucleic acid molecule of interest (e.g., target DNA) including the target sequence can be performed in vitro or ex vivo. In some implementations, the methods used to bind, cut, and/or modify the target nucleic acid molecule (e.g., target DNA) are not methods for treating human or animal bodies, or processes for modifying human germline genetic identity.

The RGN of the system can be a nuclease-free RGN, a nicking enzyme-like RGN, or a fusion polypeptide. In some embodiments, the fusion polypeptide includes a base-editing polypeptide, such as a cytosine deaminase or an adenine deaminase. In some embodiments, the fusion peptide includes peptides that recruit members of functional nucleic acid repair complexes, such as members of nucleotide excision repair (NER) or transcription-coupled nucleotide excision repair (TC-NER) pathways (Wei et al., 2015, PNAS USA 112(27):E3495-504; Troelstra et al., 1992, Cell 71:939-953; Marnef et al., 2017, J Mol Biol 429(9):1277-1288), as described in U.S. Provisional Patent Application No. 63/332,486 filed April 19, 2022, which is incorporated herein by reference in its entirety. In some embodiments, the RGN fusion protein includes CSB (van den Boom et al., 2004, J Cell Biol 166(1):27-36; van Gool et al., 1997, EMBO J 16(19):5955-65; examples of which are shown in SEQ ID NO: 397), which is a member of the TC-NER (nucleotide excision repair) pathway and functions in the recruitment of other members. In a further embodiment, the RGN fusion protein includes the active domain of CSB, such as the acidic domain of CSB including amino acid residues 356 to 394 of SEQ ID NO: 397 (Teng et al., 2018, Nat Commun 9(1):4115).

In a particular implementation, the RGN and/or guide RNA (i.e., crRNA) is heterologous to the cell, organelle, or embryo in which the RGN and/or guide RNA (i.e., crRNA) (or a polynucleotide encoding at least one of the RGN and guide RNA) is introduced.

The delivery of polynucleotides, including coding guide RNA (i.e., crRNA) and/or RGN polypeptides, to cells or embryos can be in vitro, in vivo, or in vivo. Cells or embryos can be cultured under conditions that express guide RNA (i.e., crRNA) and/or RGN polypeptides. In some embodiments, the method includes contacting the target nucleic acid molecule with the RNP complex. Contact can be in vitro, in vivo, or in vivo. The RNP complex may include RGNs with no nuclease activity or with nickase activity. In some embodiments, the RGN of the RNP complex is a fusion polypeptide comprising a base-editing polypeptide. In some embodiments, the method includes introducing the RNP complex into cells, organelles, or embryos containing the target nucleic acid molecule. RNP complexes can be RNP complexes that have been purified from biological samples, generated by recombination, and subsequently purified or assembled in vitro, as described herein. In those embodiments in which the RGN ribonucleoprotein complex has been assembled in vitro before contact with the target nucleic acid molecule, cell, organelle, or embryo, the method may further include assembling the complex in vitro prior to contact with the target nucleic acid molecule, cell, organelle, or embryo. The purified or in vitro assembled RGN ribonucleoprotein complex can be introduced into cells, organelles, or embryos using any method known in the art (e.g., electroporation). In some embodiments, the delivery of polynucleotides encoding guide RNA (i.e., crRNA) and/or RGN polypeptides to cells or embryos is not a method of treating a human or animal body by means of therapy, or a process that does not modify the phylogenetic genetic identity of a human. In some implementations, the method of bringing the target nucleic acid molecule into contact with the RNP complex is not a method of treating a human or animal, or a process that modifies a human's phylogenetic genetic identity. In some implementations, the embryo is a non-human embryo.

Upon delivery to or contact with a target nucleic acid molecule or a cell, organelle, or embryo containing the target nucleic acid molecule, a guiding RNA (i.e., crRNA) guides an RGN polypeptide, base editor, or PE to bind to the target sequence within the target nucleic acid molecule in a sequence-specific manner. In embodiments where the RGN possesses nuclease activity, the RGN polypeptide cleaves the target sequence of interest upon binding. Subsequently, the target sequence (e.g., the target DNA sequence) can be modified via endogenous repair mechanisms such as non-homologous end ligation or homology-directed repair using provided donor polynucleotides.

Methods for measuring the binding of RGN peptides to target sequences are known in the art and include: chromatin immunoprecipitation assays, gel migration translocation assays, DNA pull-down assays, reporter assays, and microdisc capture and detection assays. Similarly, methods for measuring the cleavage or modification of target nucleic acid molecules including target sequences are known in the art and include in vitro or in vivo cleavage assays, wherein cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without the attachment of appropriate tracers (e.g., radioisotopes, fluorescent substances) to the target sequence to facilitate the detection of degradation products. Alternatively, nick-triggered exponential amplification reaction (NTEXPAR) assays can be used (see, for example, Zhang et al. (2016) Chem. Sci. 7:4951-4957). In vivo shear can be assessed using the Surveyor assay (Guschin et al. (2010) Methods Mol Biol 649:247-256).

In some implementations, the method involves using a single type of RGN, base editor, or PE that is complexed with more than one guide RNA (i.e., crRNA). The more than one guide RNA (i.e., crRNA) may target different regions of a single gene or may target multiple genes.

In embodiments where no donor polynucleotide is provided, double-strand breaks introduced by the RGN peptide can be repaired via non-homologous end-joining (NHEJ) repair. Due to the error-prone nature of NHEJ, repair of double-strand breaks can lead to modifications of the target sequence. As used herein, “modification” of a nucleic acid molecule refers to a change in the nucleotide sequence of the nucleic acid molecule, which can be the deletion, insertion, or substitution of one or more nucleotides, or a combination thereof. Modification of a target nucleic acid molecule, including the target sequence, can result in altered protein product expression or inactivation of the coding sequence.

In embodiments where a donor polynucleotide is present, during the repair of an introduced double-strand break, the donor sequence in the donor polynucleotide can be integrated into or exchanged with the target nucleotide sequence, resulting in the introduction of a foreign donor sequence. Therefore, the donor polynucleotide includes the donor sequence to be introduced into the target sequence of interest. In some embodiments, the donor sequence alters the original target nucleotide sequence so that the newly integrated donor sequence is not recognized and cleaved by the RGN. In some embodiments, the donor polynucleotide is single-stranded DNA. Integration of the donor sequence can be enhanced by including flanking sequences within the donor polynucleotide; these flanking sequences, referred to herein as "homologous arms," have substantial sequence identity with the sequences flanking the target nucleotide sequence, thereby allowing for homology-guided repair. In some embodiments, the homology arm has a length of at least 30 base pairs, at least 35 base pairs, at least 40 base pairs, at least 45 base pairs, at least 50 base pairs, at least 55 base pairs, at least 60 base pairs, at least 65 base pairs, at least 70 base pairs, at least 75 base pairs, at least 80 base pairs, at least 85 base pairs, at least 90 base pairs, at least 95 base pairs, at least 100 base pairs, and up to 2000 base pairs or more, and has at least 90%, at least 95%, or higher sequence homology with the corresponding sequence within its target nucleotide sequence. Unbound by any single theory, the distance between the splice site and the PAM site of type V RGN could potentially allow for several re-cuttings after non-homologous end-joint (NHEJ) but before the PAM/septum binding is completely disrupted. This could allow for more opportunities for homology-dependent repair when donor polynucleotides are introduced as part of gene editing.

In those embodiments in which RGN peptides introduce double-stranded cross-cutting, the donor polynucleotide may include a donor sequence flanked by compatible protrusions, allowing the donor sequence to be directly linked to the cleaved target nucleotide sequence containing the protrusions during double-stranded cut repair via a non-homologous repair process.

In embodiments where the method involves the use of an RGN (i.e., an RGN that can only cleave a single strand of a double-stranded polynucleotide), the method may include introducing two RGN cleavage enzymes that target the same or overlapping target sequences and cleave different strands of the polynucleotide. For example, an RGN cleavage enzyme that cleaves only the positive (+) strand of a double-stranded polynucleotide may be introduced together with a second RGN cleavage enzyme that cleaves only the negative (-) strand of the double-stranded polynucleotide.

In some embodiments, a method is provided for binding to and detecting a target nucleotide sequence, wherein the method includes introducing at least one guide RNA (i.e., crRNA) or a polynucleotide encoding it and at least one RGN polypeptide or a polynucleotide encoding it into a cell, organelle, or embryo; expressing the guide RNA (i.e., crRNA) and/or the RGN polypeptide (if the encoding sequence is introduced), wherein the RGN polypeptide is a non-nuclease-active RGN and further includes a detectable tracer, and the method further includes detecting the detectable tracer. The detectable tracer may be fused with RGN to form a fusion protein (e.g., fluorescent protein), or may be a small molecule ligated to or incorporated into the RGN polypeptide that can be detected visually or by other means.

This document also provides methods for regulating the expression of a target gene of interest, including a target sequence, or a gene regulated by a target sequence. The methods include introducing at least one guide RNA (i.e., crRNA) or a polynucleotide encoding it and at least one RGN polypeptide or a polynucleotide encoding it into a cell, organelle, or embryo; expressing the guide RNA (i.e., crRNA) and/or the RGN polypeptide (if the encoding sequence is introduced), wherein the RGN polypeptide is a nuclease-free RGN. In some embodiments of these embodiments, the nuclease-free RGN is a fusion protein including expression regulator domains (i.e., epigenetic modification domains, transcription activation domains, or transcription repression domains) as described herein.

This disclosure also provides methods for binding to and/or modifying a target nucleic acid molecule of interest that includes a target sequence. The method comprises delivering a system to a cell, organelle, or embryo containing the target sequence, the system comprising: at least one guide RNA (i.e., crRNA) or a polynucleotide encoding it; and at least one fusion polypeptide comprising the RGN of this disclosure and a base-editing polypeptide (e.g., cytosine deaminerase or adenine deaminerase) or a polynucleotide encoding the fusion polypeptide. The method comprises delivering a system to a cell, organelle, or embryo containing the target sequence, the system comprising: at least one guide RNA or a polynucleotide encoding it; and at least one fusion polypeptide comprising the RGN of this disclosure and a polymerase-editing polypeptide (e.g., DNA polymerase or reverse transcriptase) or a polynucleotide encoding the fusion polypeptide.

In some embodiments of the fusion polypeptide, including RGN and base-editing polypeptides, the binding of the fusion polypeptide to the target sequence results in the modification of one or more nucleotides adjacent to the target sequence. The nucleotides adjacent to the target sequence and modified by the deaminase can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs counting from the 5' or 3' end of the target sequence.

In some embodiments employing the PE or PE system disclosed herein, the binding of the PE or PE system results in the modification of one or more nucleotides within or adjacent to the target sequence using DNA synthesis templates of varying lengths, such that the editing window of the polymerase editor can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs, counting from the 5' or 3' end of the target sequence.

To minimize the removal of edits by polymerase editor insertions caused by DNA mismatch repair mechanisms, one or more components of the mismatch repair system can be inactivated, for example, using a dominant-negative version. A non-limiting example of a component of the mismatch repair system that can be inactivated or has its activity reduced is MLH1. The dominant-negative MLH1 or the polynucleotide encoding it can be introduced into the cell simultaneously, before, or after the introduction of the PE system. A non-limiting example of dominant-negative MLH1 is the sequence shown in SEQ ID NO: 2688. In some embodiments, the DNA construct encoding the PE includes a dominant-negative MLH1 coding sequence at its N-terminus or C-terminus, and the dominant-negative MLH1 coding sequence can be linked to the PE coding sequence via a peptide conjugate and/or NLS.

Those skilled in the art will understand that any of the methods disclosed herein can be used to target a single target sequence or multiple target sequences. Therefore, the methods include the combined use of a single RGN peptide with multiple different guide RNAs (i.e., crRNAs) to target multiple different sequences within a single gene and/or multiple genes. This document also covers methods in which multiple different guide RNAs (i.e., crRNAs) are introduced in combination with multiple different RGN peptides. These guide RNAs (i.e., crRNAs) and guide RNA/RGN peptide systems can target multiple different sequences within a single gene and/or multiple genes.

In some embodiments, the guide RNA (i.e., crRNA) and guide RNA/RGN polypeptide system can use a single polynucleotide encoding a crRNA tandem array to target multiple different sequences within a single gene and/or multiple genes. The crRNA tandem array is transcribed into a single transcript and then processed into multiple single guide RNAs (i.e., crRNAs) by the V-H, V-I, or V-J type RGN system disclosed herein, which can then target multiple different sequences.

This document also provides a method for improving the efficiency of cleaving and/or modifying nucleic acid molecules including target sequences. In some embodiments, the method includes delivering an RGN system, base editing system, PE system, or RNP complex described herein to a target sequence or a cell including a target sequence. The RGN system, base editing system, PE system, or RNP complex includes a variant RGN peptide described herein, such that the cleavage or modification of the target nucleic acid molecule occurs with higher efficiency compared to methods involving delivering a reference RGN system, base editing system, PE system, or RNP complex to the same target nucleic acid molecule. In some embodiments, the reference RGN system, base editing system, PE system, or RNP complex does not include a variant RGN peptide described herein. In some embodiments, the reference RGN system, base editing system, PE system, or RNP complex includes those having, for example, SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081 RGN polypeptides with amino acid sequences represented by any one of the following: 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687. In some embodiments, the reference RGN system, base editing system, PE system, or RNP complex includes a variant RGN polypeptide that differs from a variant RGN polypeptide of the RGN system, base editing system, PE system, or RNP complex being tested for cleavage and/or modification of the nucleic acid molecule. In some embodiments, the reference RGN system, base editing system, PE system, or RNP complex includes SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 96 1, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1 Variant RGN peptides of any of the following: 108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687, which lack the corresponding mutations of variant RGN peptides of the RGN system, base editing system, PE system, or RNP complexes that are being tested for cleavage and/or modification of nucleic acid molecules. In some embodiments, the reference RGN is a different variant of the RGN. In some embodiments, the reference RGN system, base editing system, PE system, or RNP complex includes a different variant of the RGN polypeptide that shares the same parent RGN polypeptide with a variant RGN polypeptide of the RGN system, base editing system, PE system, or RNP complex that is being tested for cleavage and/or modification of nucleic acid molecules.

In some embodiments, the efficiency of cutting and/or modifying the target sequence is increased by 2 to 100 times, or 2 to 80 times, or 2 to 5 times. In some embodiments, the efficiency of cutting and/or modifying the target sequence is increased by 2, 2.5, 3, 3.5, 4, 4.5, 5, or higher. In some embodiments, the efficiency of cutting and/or modifying the target sequence is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, or higher. In some embodiments, gene editing efficiency is measured by next-generation sequencing, by degradation-tracked insertion or deletion (TIDE) analysis, flow cytometry, or a combination thereof. In some embodiments, the efficiency of cleavage and/or modification of the target sequence is assessed by measuring the percentage change in the expression of the target sequence or the polypeptide encoded by the target sequence in cells containing the target sequence. In some embodiments, performance is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof. In some embodiments, the target sequence encodes a protein expressed on the cell surface, and when measured by flow cytometry, the efficiency of cleavage and/or modification of the target sequence is assessed by measuring the percentage of cells with a reduced protein expression on the cell surface. X. Target polynucleotide

This disclosure provides methods for modifying target polynucleotides, including target sequences, or for modifying the expression of target polynucleotides in eukaryotic cells, wherein the modification can be in vivo, in vitro, or extracellular. In some embodiments, the method includes sampling cells or cell populations from human or non-human animals or plants (including microalgae); and modifying cells or multiple cells. Culture can occur at any stage in vitro. It is even possible to reintroduce cells or multiple cells into non-human animals or plants (including microalgae).

Using natural variation, plant breeders combine most of the useful genes to obtain desired qualities, such as yield, quality, uniformity, tolerance, and resistance to pests. These desired qualities also include growth, day length preference, temperature requirements, onset date of flowering or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, and tolerance to various environmental factors, including drought, heat, humidity, cold, wind, and unfavorable soil conditions including high salinity. The sources of these useful genes include natural or exotic varieties, heirloom varieties, closely related wild plants, and induced mutations, such as those induced by mutagens on plant material. This invention provides plant breeders with new tools for inducing mutations. Therefore, those skilled in the art can analyze genomes to find the source of useful genes and use the present invention in varieties with desired characteristics or traits to induce the increase of useful genes more precisely than previous mutagens, thereby accelerating and improving plant breeding programs.

The target polynucleotide (PMN) for RGN, base editing, or PE systems can be any polynucleotide, whether endogenous or exogenous in eukaryotic cells. For example, a target PMN can be a polynucleotide present in the nucleus of a eukaryotic cell. The target PMN can be a sequence that cognizes a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). Without being bound by theory, the non-target strand of the target sequence is adjacent to the PAM at its 3' end and is recognized by the RGN, base editing, or PE system. The precise sequence and length requirements of the PAM vary depending on the RGN used, but a PAM typically contains 2–5 nucleotides adjacent to the anterior septum (i.e., the target sequence).

The target polynucleotides of the RGN, base editing, or PE systems disclosed herein may include a wide range of disease-associated genes and polynucleotides, as well as genes and polynucleotides associated with signaling biochemical pathways. Examples of target polynucleotides include sequences associated with signaling biochemical pathways, such as genes or polynucleotides associated with signaling biochemical pathways. Examples of target polynucleotides include disease-associated genes or polynucleotides. "Disease-associated" genes or polynucleotides refer to any gene or polynucleotide that produces transcription or translation products at an abnormal level or in an abnormal form in cells obtained from diseased tissue compared to non-disease-controlled tissues or cells. This can be a gene that has been altered to an abnormally high level of expression; it can be a gene that has been altered to an abnormally low level of expression, wherein the altered expression is associated with the occurrence and/or progression of disease. Disease-associated genes also refer to genes with one or more mutations or variants that are directly responsible for the disease (e.g., causal mutations) or are in chain disequilibrium with one or more genes that are responsible for the disease (e.g., causal mutations). The transcribed or translated products can be known or unknown and can be at normal or abnormal levels. In some embodiments, the disease can be an animal disease. In some embodiments, the disease can be a bird disease. In some embodiments, the disease can be a mammal disease. In some embodiments, the disease can be a human disease. Non-restricted examples of disease-related genes and polynucleotides in humans are available from the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University (Baltimore, MD) and the National Center for Biotechnology Information at the National Library of Medicine (Bethesda, MD), both of which are accessible via the Global Information Network.

The method includes contacting a target nucleic acid molecule including a target sequence with the RGN of the present disclosure, wherein the target nucleic acid molecule is contacted with the RGN in an effective amount and under conditions suitable for editing the target sequence (e.g., excision of a portion of the target polynucleotide, splicing, insertion of a donor polynucleotide, modification of expression, base editing, polymerase editing). In some embodiments, the method includes contacting the target nucleic acid molecule including the target sequence with (a) the RGN of the present disclosure; and with (b) a gRNA (i.e., crRNA) that targets the target sequence with the RGN of (a); wherein the target nucleic acid molecule is contacted with the RGN and the gRNA (i.e., crRNA) in an effective amount and under conditions suitable for editing the target sequence. In some embodiments, the target sequence includes a sequence associated with a disease or condition, and editing the target sequence results in a sequence unrelated to the disease or condition. In some implementations, the target sequence is located in a pair of genes in the crop, where a specific pair of genes is associated with traits that result in lower agronomic value in the plant. Editing the target sequence results in a pair of genes associated with traits that increase the agronomic value of the plant.

Fusion proteins comprising the RGN peptide disclosed herein and base-editing peptides (such as deaminers as described herein) can be generated. In some embodiments, the method includes contacting a DNA molecule with (a) a fusion protein comprising a variant of the RGN peptide disclosed herein and a base-editing peptide such as a deaminer; and (b) contacting a gRNA targeting a target nucleotide sequence of the DNA molecule with the fusion protein of (a); wherein the DNA molecule contacts the fusion protein and gRNA in an effective amount and under conditions suitable for nucleobase deamination. In some embodiments, the target DNA sequence includes a sequence associated with a disease or condition, and wherein nucleobase deamination results in a sequence unrelated to the disease or condition. In some implementations, the target DNA sequence is located in the paired genes of the crop, where a specific paired gene for the trait of interest results in a plant with lower agronomic value. Nucleobase deamination results in paired genes that improve plant traits and increase the plant's agronomic value.

Fusion proteins comprising the RGN polypeptide and polymerase-editing polypeptide (such as DNA polymerase or reverse transcriptase as described herein) can be generated. In some embodiments, the method includes contacting a DNA molecule with (a) a fusion protein comprising a variant RGN polypeptide and a polymerase-editing polypeptide such as reverse transcriptase, as disclosed herein; and with (b) a PEgRNA that targets the fusion protein of (a) to a target nucleotide sequence of the DNA molecule; wherein the DNA molecule is contacted with the fusion protein and PEgRNA in an effective amount and under conditions suitable for editing the target nucleotide sequence. In some embodiments, the target DNA sequence includes a sequence associated with a disease or condition, and wherein editing the target DNA sequence results in a sequence associated with the disease or condition. In some implementations, the target DNA sequence is located in the paired genes of a crop, where a specific paired gene for the trait of interest results in a plant with lower agronomical value. Editing the target DNA sequence results in paired genes that improve plant traits and increase the plant's agronomic value.

In some embodiments, the target DNA sequence includes a T→C or A→G point mutation associated with a disease or condition, wherein deamination of the C or G base of the mutant results in a sequence unrelated to the disease or condition. In some embodiments, deamination corrects point mutations in sequences associated with the disease or condition.

In some embodiments, a disease- or symptom-associated sequence is written into a protein, and the edit target sequence introduces a termination codon into the disease- or symptom-associated sequence, causing truncation of the written protein. In some embodiments, the exposure occurs in an individual susceptible to or diagnosed with the disease or symptom. In some embodiments, the disease or symptom is associated with point mutations or single-base mutations in the genome. In some embodiments, the disease is a genetic disease, cancer, metabolic disease, or lysosomal storage disease. XI. Cells including polynucleotide gene modifications.

This document provides information on cells and organisms that have been modified with target nucleic acid molecules using RGNs and/or guide RNAs (i.e., crRNAs), RGN systems, base editing systems, PE systems, or RNP complex-mediated processes as described herein. In some embodiments, the RGN is capable of recognizing and binding to the target sequence at its 5' PAM. In some embodiments, the RGNs disclosed herein are active without the need for tracrRNA. In some embodiments, the RGNs disclosed herein comprise V-H, V-I, or V-J type RGNs. In some embodiments, the RGN comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1 The amino acid sequence or its active variant or fragment represented by any one of 082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687. In some embodiments, the guide RNA comprises a crRNA duplicant or an active variant or fragment thereof having a nucleotide sequence as shown in any of SEQ ID NOs: 5 to 8, 1617 to 1620, 1631 and 1957 to 1983. In some embodiments, the guide RNA (i.e., crRNA) comprises a nucleotide sequence as shown in any of SEQ ID NOs: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658 or an active variant or fragment thereof. In some embodiments, the guide RNA (i.e., crRNA) comprises a nucleotide sequence or an active variant or fragment thereof as shown in any of SEQ ID NO: 39 to 49, 51, 53, 54, 58, 61, 62, 64, 66, 68, 69, 78, 80, 81, 83, 84, 86, 88, 91, 96, 98, 99, 101, 109 to 128, 130, 132, 133, 135, 137 to 139, 147 to 153, 159, 163, 165, 168, 170, 173, 175, 176, 178, 179 and 182.

The modified cells can be eukaryotic (e.g., mammalian, plant, insect, avian cells) or prokaryotic. Prokaryotic cells can originate from species including, but not limited to, archaea and bacteria (e.g., Bacillus, Klebsiella sp., Streptococcus, Rhizobium, Escherichia sp., Pseudomonas, Salmonella, Shigella, Vibrio, Yersinia, Mycobacterium, Agrobacterium, Lactobacillus).

Eukaryotic cells may include cells derived from animals (e.g., mammals, humans, insects, fish, birds, and reptiles), plants, fungi, amoebae, algae, and yeasts. In some embodiments, cells modified by the methods disclosed in this invention include hematopoietic cells, such as, but not limited to, cells of the immune system including, but not limited to, B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells. In some embodiments, cells modified by the methods disclosed in this invention include primary cells. In some implementations, primary cells include primary T cells.

The methods described herein can be used to modify any plant species, including but not limited to monocots and dicots. Examples of plants of interest include, but are not limited to: corn, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and rapeseed, Brassica sp., alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, alligator, fig, guava, mango, olive, papaya, cashew, macadamia nut, almond, oats, vegetables, ornamental plants, and conifers.

Vegetables include, but are not limited to: tomatoes, lettuce, mung beans, lima beans, peas, and members of the genus *Curcumis* such as cucumbers, cantaloupes, and honeydew melons. Ornamental plants include, but are not limited to: azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, Christmas flowers, and chrysanthemums. In certain embodiments, the plants of this invention are crops (e.g., corn, sorghum, wheat, sunflowers, tomatoes, cruciferous plants, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

Organelles and embryos including at least one target sequence are also provided, the target sequence having been modified by processes such as RGN, base editor, PE and/or guide RNA (i.e., crRNA) as described herein. The genetically modified cells, organisms, organelles and embryos may be hetero-conjugated or homo-conjugated to the modified target sequence.

Chromosomal modifications in cells, organisms, organelles, or embryos can result in altered (updated or downdated) expression of protein products or integrated sequences. In those embodiments where chromosomal modifications result in gene inactivation or nonfunctional protein product expression, the modified cell, organism, organelle, or embryo is referred to as "knockout." Knockout phenotypes can result from deletion mutations (i.e., the deletion of at least one nucleotide), insertion mutations (i.e., the insertion of at least one nucleotide), or meaningless mutations (i.e., the substitution of at least one nucleotide, resulting in the introduction of a termination codon). In some embodiments, chromosomal modifications result in updating of protein products that are missing or reduced due to mutations in one or more target sequences.

Alternatively, chromosomal modifications of cells, organisms, organelles, or embryos can generate "knock-in," which is caused by chromosomal integration of the nucleotide sequence of a coding protein. In some of these embodiments, the coding sequence is integrated into the chromosome, causing the chromosomal sequence encoding the wild-type protein to be inactivated, but the exogenously introduced protein is expressed.

In some embodiments, the RGN system disclosed herein comprises a fusion of RGN with a base-editing polypeptide (e.g., a deaminase) or a fusion of RGN with a polymerase-editing polypeptide (e.g., DNA polymerase or RT), and mutations (one or more) introduced by these RGN systems result in the production of variant protein products. The expressed variant protein products may have at least one amino acid substitution and/or at least one amino acid addition or deletion. When compared to wild-type protein, the variant protein product encoded by the altered chromosomal sequence may exhibit modified characteristics or activities, including but not limited to altered enzyme activity or substrate specificity. In some embodiments, chromosomal modification may lead to altered protein expression patterns. As a non-limiting example, chromosomal alterations in regulatory regions controlling protein product expression can lead to overexpression, downexpression, or alteration of tissue or temporary expression patterns of the protein product. In some implementations, the reduction or elimination of gene expression is caused by one or more mutations introduced by these RGN systems.

Modified cells can be introduced into an organism. In the case of autologous cell transplantation, these cells may originate from the same organism (e.g., a human), where the cells are modified in vitro. Alternatively, in the case of allogeneic cell transplantation, the cells originate from another organism within the same species (e.g., another human). The modified cells can then be grown into organisms, such as plants, according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same or different modified plants, resulting in hybrids with the genetic modification. This disclosure provides genetically modified seeds. Progeny, variants, and mutants of regenerated plants are also included within the scope of this disclosure, provided that these portions include gene modifications. Further, processed plant products or byproducts retaining gene modifications are provided, including, for example, soybean meal. XII. Pharmaceutical Compositions

The pharmaceutical composition disclosed herein may include: the RGN peptide described herein or its active variants and fragments, and polynucleotides encoding them; the gRNA (i.e., crRNA) described herein or its active variants and fragments, or polynucleotides encoding them; the RGN, base editor, and PE system described herein that includes the RGN peptide and/or gRNA (i.e., crRNA); or cells that include the RGN peptide or polynucleotides encoding RGN, gRNA (i.e., crRNA), or polynucleotides encoding gRNA (i.e., crRNA), or any of the RGN, base editor, and PE system; and pharmaceutically acceptable carriers.

Pharmaceutical compositions are compositions used to prevent, reduce, cure, or treat a symptom or disease. Compositions include active ingredients (i.e., RGN peptides, polynucleotides encoding RGN, gRNA (i.e., crRNA), polynucleotides encoding gRNA (i.e., crRNA), RGN systems, base editors, PE, or cells including any of these) and pharmaceutically acceptable carriers.

As used herein, "pharmaceutically acceptable carrier" refers to a material that does not cause significant irritation to a organism and does not eliminate the activity and properties of the active ingredient (i.e., RGN peptides, polynucleotides encoding RGN, gRNA (i.e., crRNA), polynucleotides encoding gRNA (i.e., crRNA), RGN systems, base editors, PE, or cells including any of these). The carrier must have sufficiently high purity and sufficiently low toxicity to be suitable for administration to the individual being treated. The carrier may be inert or otherwise possess medicinal benefits. In some embodiments, pharmaceutically acceptable carriers include one or more compatible solid or liquid fillers, thinners, or encapsulating substances suitable for administration to humans or other vertebrates. In some embodiments, pharmaceutically acceptable carriers are not naturally occurring. In some implementation methods, pharmaceutically acceptable carriers and active ingredients have not been found in nature.

The pharmaceutical composition used in the methods disclosed in this invention can be formulated with suitable carriers, excipients, and other pharmaceutical preparations that provide suitable transfer, delivery, tolerability, etc. Many suitable formulations are known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st edition, 2005). Suitable formulations include powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (e.g., LIPOFECTIN vesicles), lipid nanoparticles, DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Pharmaceutical formulations for oral or non-oral use can be formulated into dosage forms suitable for the appropriate unit dose of the active ingredient. For example, these unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc.

This disclosure provides pharmaceutical compositions including lipid-based formulations containing an active ingredient (i.e., an RGN peptide, a polynucleotide encoding an RGN, gRNA (i.e., crRNA), a polynucleotide encoding gRNA (i.e., crRNA), an RGN system, a base editor, a PE, or a cell including any of these). Lipid-based formulations may contain liposomes. Lipid-based formulations may contain lipid nanoparticles (LNPs). In some embodiments, the active ingredient is encapsulated in and/or disposed on the surface of the lipid particles. In some embodiments, the active ingredient is covalently attached to the lipid particles. In some embodiments, the active ingredient is non-covalently attached to the lipid particles. Covalent attachment involves sharing electrons in chemical bonds. Non-covalent interactions include dispersed electromagnetic interactions, such as hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds.

In some embodiments, the active ingredient is encapsulated in liposomes. The term "encapsulate" means to enclose, surround, or encase. When it comes to the formulation of the compounds disclosed herein, encapsulation can be substantial, complete, or partial. The term "substantially encapsulated" or "substantially encapsulated" means that more than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more of the pharmaceutical composition or active ingredient of this disclosure may be encapsulated, surrounded, or encased in a delivery medium (e.g., liposomes or LNPs). The term "partially encapsulated" or "partially encapsulated" means that less than 50%, 40%, 30%, 20%, 10%, or less of the pharmaceutical composition or active ingredient of this disclosure may be encapsulated, surrounded, or encased in a delivery medium. Encapsulation can be determined by measuring the escape or activity of the pharmaceutical composition or active ingredient of this disclosure using fluorescence and/or electron microscopy. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or more of the pharmaceutical composition or active ingredient of this disclosure are encapsulated in a delivery agent (e.g., liposomes or LNPs).

Liposomes are spherical vesicle structures composed of a single or multiple lipid bilayer surrounding an internal aqueous compartment and a relatively opaque outer lipophilic phospholipid bilayer. Liposomes have attracted considerable attention as drug delivery carriers because they are biocompatible, non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from plasma enzyme degradation, and transport their payload across biological membranes and the blood-brain barrier (BBB) (see, for example, Spuch and Navarro (2011) Journal of Drug Delivery 2011).

Liposomes can be made from several different types of lipids (e.g., ionizable lipids, structured lipids, helper lipids, and pegylated lipids); however, phospholipids are most commonly used to produce liposomes as drug carriers. Although liposome formation is spontaneous when lipid membranes are mixed with aqueous solutions, it can be accelerated by applying forces in an oscillating manner using homogenizers, ultrasound, or extrusion devices (see, for example, Spuch and Navarro (2011) Journal of Drug Delivery 2011).

Traditional liposome formulations are primarily composed of natural phospholipids and phospholipids such as 1,2-distearyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, lecithin choline, and monosialotetrahexosylganglioside. In some embodiments, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) increases the stability of the liposomes.

Additives can be added to liposomes to modify their structure and properties. In some embodiments, cholesterol and/or sphingomyelin can be added to the liposome mixture to stabilize the liposome structure and prevent leakage of contents within the liposomes. In some embodiments, adding cholesterol to conventional liposome formulations reduces the rapid release of encapsulated active ingredients (i.e., RGN peptides, polynucleotides encoding RGN, gRNA (i.e., crRNA), polynucleotides encoding gRNA (i.e., crRNA), the RGN system, or cells containing any of these) into the plasma. In some embodiments, liposomes are prepared from hydrogenated lecithin acetylcholine or lecithin acetylcholine, cholesterol, and didicetyl phosphate. In some embodiments, the average liposome vesicle size is adjusted to approximately 50 or 100 nm. In some embodiments, Trojan horse liposomes (also known as molecular Trojan horses or polyethylene glycol-modified immunoliposomes) can be used in pharmaceutical compositions for delivering the active ingredient across the BBB (described on the Global Information Network at cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long). Unbound by any theory, neutral lipid particles with specific antibodies bound to their surface are believed to allow passage across the bbb via endocytosis. In some implementations, pharmaceutical compositions including Trojan horse liposomes can be used to deliver the active ingredient (i.e., RGN peptides, polynucleotides encoding RGN, gRNA (i.e., crRNA), polynucleotides encoding gRNA (i.e., crRNA), the RGN system, or cells including any of these) to the brain via intravascular injection.

In some embodiments, liposomes include stable nucleic acid-lipid particles (SNALP) (see, for example, Morrissey et al. (2005) Nature Biotechnology 23(8):1002-1007; Zimmerman et al. (2006) Nature 441:111-114). SNALPs comprise a mixture of cationic lipids and fusogenic lipids and are coated with polyethylene glycol (PEG) to allow for cellular uptake and endosome release of the active ingredient. In some embodiments, SNALPs are a class of LNPs and include ionizable lipids that are cationic at low pH (e.g., DLinDMA, COATSOME® SS-OC), neutral cofactor lipids (e.g., DSPC), cholesterol, and diffusible polyethylene glycol (PEG)-lipids (e.g., Brij S100). In some embodiments, the SNALP formulation contains the following lipids: 3- N- (β-methoxypoly(ethylene glycol)2000)carbamoyl-1,2-dimyrestyloxy-propylamine (PEG-CDMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA); 1,2-distearate-sn-glycero-3-phosphate choline (DSPC); and cholesterol. In some embodiments, SNALP contains synthetic cholesterol, dipalmitoylphosphatidylcholine (DOPC), PEG-cDMA, and DLinDMA (see, for example, Geisbert et al. (2010) Lancet 375:1896-1905). In some embodiments, SNALP contains synthetic cholesterol, DSPC, PEG-cDMA, and DLinDMA (see, for example, Judge et al. (2009) J. Clin. Invest. 119:661-673). In some embodiments, the SNALP formulation contains COATSOME® SS-OC, DSPC, Brij S100, and cholesterol. In some embodiments, the SNALP formulation contains ionizable lipids, DSPC, cholesterol, and PEG lipids. In some embodiments, the SNALP formulation includes the ionizable lipids and/or PEG lipids disclosed in WO2022173531 or WO2022173531, each of which is incorporated herein by reference in its entirety.

In some implementations, the size of SNALP liposomes is approximately 80–100 nm. SNALP has been used as an efficient delivery molecule for highly vascularized HepG2-derived hepatocellular carcinomas (see, for example, Li et al. (2012) Gene Therapy 19:775–780).

Unbound by any single theory, during SNALP formulation, ionizable lipids are used to condense the lipids with the active ingredient (e.g., nucleic acid molecules) during particle formation. When positively charged under progressively acidic endosomal conditions, the ionizable lipids mediate the fusion of SNALP with the endosomal membrane, enabling the release of the active ingredient into the cytoplasm. During formulation, PEG-LIPID stabilizes the particles and reduces aggregation, subsequently providing a neutral, hydrophilic exterior to improve pharmacokinetic properties. In some implementations, SNALP liposomes are prepared by blending DLinDMA and PEG-cDMA with DSPC, cholesterol, and active ingredients using a lipid:active ingredient ratio of 25:1 and a cholesterol:DLinDMA:DSPC:PEG-cDMA molar ratio of 48:40:10:2.

In some embodiments, the pharmaceutical composition of this disclosure includes LNPs. In some embodiments, lipids may be formulated with the active ingredient of this disclosure to form LNPs. An LNP comprises a plurality of lipid molecules physically bound together by intermolecular forces. In some embodiments, an LNP comprises a liposome. In some embodiments, an LNP differs from a liposome in that it does not have a continuous lipid bilayer. In some embodiments, an LNP comprises a solid particle having a mixture of solid and liquid lipids. In some embodiments, an LNP comprises dendrimer lipid nanoparticles (DLNPs), SNALPs, and lipid-like nanoparticles (LLNPs). Generally, "nanoparticles" refers to any particle with a diameter less than 1000 nanometers (nm). In some embodiments, nanoparticles have a diameter of 500 nm or less. In some embodiments, the nanoparticles have a diameter between 25 nm and 200 nm, or 100 nm or less. In some embodiments, the nanoparticles have a diameter between 35 nm and 60 nm. In some embodiments, the LNP comprises lipid particles with a size between about 1 and about 100 nm.

LNPs comprise four components: ionizable cationized lipids, fused amphoteric phospholipids, cholesterol, and PEGylated lipids. In some embodiments, the ionizable cationized lipid component complexes with negatively charged polynucleotides and enhances endosome escape. In some embodiments, the phospholipid component plays a role in modifying the lipid bilayer structure. In some embodiments, the cholesterol component helps stabilize LNPs. In some embodiments, the PEGylated lipid component reduces LNP aggregation and nonspecific uptake.

In some embodiments, LNP includes ionizable lipids that are cationic at low pH (e.g., DLinDMA, COATSOME® SS-OC), neutral co-lipids (e.g., DSPC), cholesterol, and diffuse polyethylene glycol (PEG)-lipids (e.g., Brij S100). In some embodiments, LNP comprises the following lipids: 3- N- (β-methoxypoly(ethylene glycol)2000)carbamoyl-1,2-dimyrestyloxy-propylamine (PEG-CDMA); 1,2-dilinoleyloxy- N,N- dimethyl-3-aminopropane (DLinDMA); 1,2-distearate-sn-glycero-3-phosphate choline (DSPC); and cholesterol. In some embodiments, LNP comprises synthetic cholesterol, dipalmitoylphosphatidylcholine (DOPC), PEG-cDMA, and DLinDMA (see, for example, Geisbert et al. (2010) Lancet 375:1896-1905). In some embodiments, LNP comprises synthetic cholesterol, DSPC, PEG-cDMA, and DLinDMA (see, for example, Judge et al. (2009) J. Clin. Invest. 119:661-673).

Useful ionizable cationic lipids in LNPs include: COATSOME® SS-OC; 1,2-diimino-3-dimethylammonium-propane (DLinDAP); DLinDMA; 1,2-dilinoleyloxy-keto- N ,N- dimethyl-3-aminopropane (DlinK-DMA); 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DlinKC2-DMA); 5A2-SC8 (Zhou et al. (2016) Proc. Natl. Acad. Sci. USA). 113:520-525); C12-200 (Love et al. (2010) Proc.Natl.Acad.Sci. USA 107:1864-1869); 246C10 (Kim et al. (2021) Sci Adv 7(9):EABF4398); ckK-E12 (Fenton et al. (2016) Advanced Materials 28(15):2939-2943); 1,2-distearyloxy- N , N -dimethyl-3-aminopropane (DSDMA); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA); 1,2-dilinathyloxy- N,N 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA); and dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA); Jayaraman et al. (2012) Angew Chem Int Ed Engl. 51(34):8529-8533. Cationic lipids are further described in international references WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, and W. The following patents are published: O2010080724, WO201021865, WO2022173531, WO2022150485 and WO2008103276, U.S. Patents 7,893,302 and 7,404,969 and U.S. Patent Application No. US20100036115, each of which is incorporated herein by reference in its entirety.

The zwitterionic phospholipids that are useful for LNP include: DSPC, DOPE, and DOPC. PEG lipids useful for LNP include: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG); (3-o-[2 ^'' -(methoxypolyethylene glycol 2000)succinoyl]-1,2-dimyristoyl-sn-diol (PEG-S-DMG); R-3-[(ω-methoxy-poly ⁽ ethylene glycol) 2000)aminomethoxypropyl-3-amine (R-3-[(ω-methoxy-poly(ethylene glycol) 2000)]-1,2-dimyristoyl-sn-glycol) [carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG); and C16 PEG-ceramide (C16 PEG-ceramide). In some embodiments, the LNP contains a 50:10:38.5:1.5 molar ratio of DLinkC2-DMA or C12-200:DSPC:cholesterol:PEG-DMG (see, for example, Basha et al. (2011) Molecular Therapy 19(12):2186-2200). In some embodiments, the LNP contains a 26.5:20:52:1.5 ionizable lipid:DOPE:cholesterol:PEG lipid (see, for example, Han et al. (2022) Sci Adv 8(3):eabj6901; Kim et al. (2021) Sci Adv 8(3):eabj6901). 7(9):eabf4398). PEG lipids are further described in WO2012099755, WO2022173531 and WO2022150485, each of which is incorporated herein by reference in its entirety. In some embodiments, the proportion of PEG in the LNP formulation may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulation.

In some embodiments, the LNP formulation includes COATSOME® SS-OC, DSPC, Brij S100, and cholesterol. In some embodiments, the LNP formulation includes ionizable lipids, DSPC, cholesterol, and PEG lipids. In some embodiments, the LNP formulation includes ionizable lipids and/or PEG lipids disclosed in WO2022173531 or WO2022150485, each of which is incorporated herein by reference in its entirety. In some embodiments, the LNP formulation comprises the ionizable lipids and/or PEG lipids, DSPC, and cholesterol disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety). In some embodiments, the LNP formulation comprises any of CAT1 to CAT35 and any of CHM-001 to CHM-016 disclosed in WO2022173531 or WO2022150485, each of which is incorporated herein by reference in its entirety. In some embodiments, the LNP formulation comprises any of CAT1 to CAT35 and any of CHM-001 to CHM-016 disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety), DSPC, and cholesterol.

In some embodiments, the LNP disclosed herein comprises 44 to 60 mol% cationic lipids, 19 to 25 mol% auxiliary lipids, 25 to 33 mol% structural lipids, and 0.2 to 0.8 mol% PEG lipids (including end points). In some embodiments, the LNP disclosed herein comprises 44 to 54 mol% cationic lipids, 19 to 25 mol% auxiliary lipids, 24 to 32 mol% structural lipids, and 1.2 to 1.8 mol% PEG lipids (including end points). In some embodiments, the LNP disclosed herein comprises 44 to 54 mol% cationic lipids, 8 to 14 mol% auxiliary lipids, 35 to 43 mol% structural lipids, and 1.2 to 1.8 mol% PEG lipids (including end points). In some embodiments, the LNP disclosed herein comprises 45 to 55 mol% cationic lipids, 5 to 9 mol% auxiliary lipids, 36 to 44 mol% structural lipids, and 2.5 to 3.5 mol% PEG lipids (including end points).

In some embodiments, the LNP disclosed herein comprises 49 mol% cationic lipids, 22 mol% auxiliary lipids, 28.5% structural lipids, and 0.05 mol% PEG lipids. In some embodiments, the LNP disclosed herein comprises 49 mol% SS-OC, 22 mol% DSPC, 28.5 mol% cholesterol, and 0.05 mol% Brij S100.

In some embodiments, the LNP disclosed herein comprises 50 mol% cationic lipids, 7 mol% auxiliary lipids, 40% structured lipids, and 3 mol% PEG lipids. In some embodiments, the LNP disclosed herein comprises 50 mol% ionizable lipids, 7 mol% DSPC, 40 mol% cholesterol, and 0.05 mol% PEG lipids. In some embodiments, the LNP of this disclosure includes 50 mol% of any one of CAT1 to CAT35 disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety), 7 mol% of DSPC, 40 mol% of cholesterol, and 0.05 mol% of any one of CHM-001 to CHM-016 disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety). In some embodiments, the LNP of this disclosure includes 50 mol% of CAT7 disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety), 7 mol% of DSPC, 40 mol% of cholesterol, and 0.05 mol% of CHM-006 disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety).

In some embodiments, the size of the LNP is approximately 80 to 100 nm. In other embodiments, the size of the LNP is approximately 80 nm, approximately 81 nm, approximately 82 nm, approximately 83 nm, approximately 84 nm, approximately 85 nm, approximately 86 nm, approximately 87 nm, approximately 88 nm, approximately 89 nm, approximately 90 nm, approximately 91 nm, approximately 92 nm, approximately 93 nm, approximately 94 nm, approximately 95 nm, approximately 96 nm, approximately 97 nm, approximately 98 nm, approximately 99 nm, or approximately 100 nm. The size of individual LNPs within a LNP group can vary by approximately ±5 nm.

In some paradigms, the charge of LNPs is considered. Cationic lipids can bind to negatively charged lipids to induce non-bilayer structures that promote intracellular delivery. Because charged LNPs are rapidly cleared from circulation after intravenous injection, ionizable cationic lipids with pKa values below 7 have been developed (see, for example, Basha et al. (2011) Molecular Therapy 19(12):2186-2200). Negatively charged polymers (e.g., polynucleotides) can be loaded into LNPs at low pH values (e.g., pH 4), where the ionizable lipids exhibit a positive charge. However, at physiological pH values, LNPs exhibit a low surface charge suitable for longer circulation times.

The preparation of LNPs and the encapsulation of the active ingredient are described in, for example, Basha et al. (2011) Molecular Therapy 19(12):1286-2200; Han et al. (2022) Sci Adv 8(3): eabj6901; Kim et al. (2021) Sci Adv 7(9): eabf4398; Finn et al. (2018) Cell Reports 22:2227–2235; Wei et al. (2020) Nature Communications 11:3232; WO2011127255; and WO2008103276. The lipids are commercially available (e.g., from Tekmira Pharmaceuticals, Vancouver, Canada; Avanti Polar Lipids, Inc., Alabaster, AL) or synthetic (e.g., Kim et al. (2021) Sci Adv 7(9): eabf4398). The synthesis of cationic lipids is also described in International Publications Nos. WO2012040184, 2011153120, 2011149733, 2011090965, 2011043913, 2011022460, 2012061259, 2012054365, 2012044638, 2010080724 and WO201021865. Cholesterol is commercially available (e.g., from Sigma-Aldrich, St. Louis, MO).

In some embodiments, encapsulation can be performed by dissolving a lipid mixture comprising cationic lipids (e.g., DLin-DMA): phospholipids (e.g., DSPC, DOPE): cholesterol: PEG lipids (e.g., in a 40:10:40:10 molar ratio) in ethanol. The active ingredient (e.g., a polynucleotide comprising or containing code-disclosed content such as guide RNA or RGN) can be dissolved in an acidic buffer (e.g., citrate, acetate) at pH 3 or 4. In some embodiments, the lipid solution and the active ingredient solution can be mixed using a microfluidic system (Chen et al. (2012) J.Amer.Chem.Soc.134:6948-6951; e.g., NanoAssemblr from Precision NanoSystems) or by dropwise addition of the lipid solution to the active ingredient solution. Ethanol removal and neutralization of the formulation buffer can be performed by dialyzing with phosphate-buffered saline (PBS) for, for example, 16 hours or overnight using a dialysis cartridge (e.g., Life Technologies' 3500 molecular weight cutoff cartridge). The size, polydispersity index (PDI), and zeta potential of the LNP can be assessed using dynamic light scattering. The encapsulation efficiency of active ingredients such as RNA can be determined by assays such as the Quant-iT™ RiboGreen (Thermo Fisher) assay. In some embodiments where the encapsulated active ingredient is a polynucleotide, the polynucleotide can be extracted from the eluted nanoparticles and quantified at 260 nm. The pKa of the LNP can be assessed using the 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) assay (Zhang et al. (2011) Langmuir 27(5):1907-1914). In some implementations, the final lipid:active ingredient weight ratio includes 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1 and 5:1.

In some embodiments of pharmaceutical compositions comprising ribonucleoprotein (RNP) complexes (i.e., RGN and guide RNA) encapsulated in LNPs, the inclusion of additional permanent cationic lipids (e.g., 1,2-dioleyl-3-trimethylammonium-propane (DOTAP)) allows for the formation of RNP-encapsulated LNPs by mixing an ethanolic solution of the lipid with a physiological pH RNP solution (e.g., PBS buffer; Wei et al. (2020) Nature Communications 11:32 32). In some embodiments, the permanent cationic lipids are included in the LNPs at 10 to 20 mole% of the total lipids. In some embodiments, the LNP formulations described herein may additionally include permeability enhancer molecules. The non-restricted permeability enhancer molecule is described in US2005/0222064.

In some administration methods, LNP components are biodegradable because they do not accumulate to cytotoxic levels in vivo at therapeutically effective doses. LNP formulations can be modified by replacing cationized lipids with biodegradable cationized lipids called rapidly eliminated lipid nanoparticles (reLNPs). In some administration methods, in rats, from doses of 1 mg/kg to 10 mg/kg, the rapid metabolism of rapidly eliminated lipids has increased LNP tolerability and therapeutic index by an order of magnitude. Incorporating enzymatically degradable ester linkages can improve the degradation and metabolic profile of the cationized components while maintaining the activity of the reLNP formulation. Ester linkages can be internally located within the lipid chain or terminally located at the ends of the lipid chain. Internal ester linkages can replace any carbon in the lipid chain.

In some embodiments, the LNP composition does not induce an innate immune response at therapeutic dose levels that would lead to substantial side effects. In some embodiments, the LNP composition described herein does not induce toxicity at therapeutic dose levels.

In some embodiments, the active ingredient (i.e., RGN peptides, polynucleotides encoding RGN, gRNA (i.e., crRNA), polynucleotides encoding gRNA (i.e., crRNA), RGN, a base editor or PE system, or a cell including any of these) is formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLNs) may be spherical with an average diameter between 10 and 1000 nm. SLNs have a solid lipid core acceptor that can solubilize lipophilic molecules and can be stabilized with surfactants and/or emulsifiers. In some embodiments, the lipid nanoparticles may be self-assembled lipid-polymer nanoparticles (see, for example, Zhang et al. (2008) ACS Nano 2(8):1696-1702). In some embodiments, lipid-based formulations comprising an active ingredient (i.e., an RGN peptide, a polynucleotide encoding an RGN, gRNA (i.e., crRNA), a polynucleotide encoding gRNA (i.e., crRNA), an RGN system, or a cell including any of these) may be formulated for controlled release and/or targeted delivery. As used herein, “controlled release” refers to a release profile of a drug combination or compound that conforms to a specific release pattern to achieve a therapeutic effect.

In some embodiments, lipid-based formulations containing active ingredients (i.e., RGN peptides, polynucleotides encoding RGN, gRNA (i.e., crRNA), polynucleotides encoding gRNA (i.e., crRNA), RGN systems, or cells including any of these) include at least one controlled release coating. Controlled-release coatings include: OPADRY® (Colorcon Inc., Harleysville, PA); polyvinylpyrrolidone/vinyl acetate copolymer; polyvinylpyrrolidone; hydroxypropyl methylcellulose; hydroxypropyl cellulose; hydroxyethyl cellulose; EUDRAGIT RL® (Evonik, Essen, Germany); EUDRAGIT RS® (Evonik, Essen, Germany); and cellulose derivatives such as ethylcellulose aqueous dispersion (AQUACOAT® and SURELEASE®, Colorcon Inc., Harleysville, PA)). In some embodiments, controlled release and/or targeted delivery of the formulation may include at least one biodegradable polyester, which may contain a polycationic sidechain. The biodegradable polyester includes poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the biodegradable polyester may contain a PEG adjuvant to form a polyethylene glycol polymer.

In some implementations, LNP modulators can be prepared such that they are passively or actively directed to different types of cells in the body, including hepatocytes, immune cells, tumor cells, endothelial cells, antigen-presenting cells, and leukocytes (Akinc et al. (2010) Mol Ther. 18: 1357-1364; Song et al. (2005) Nat Biotechnol. 23:709-717; Judge et al. (2009) J Clin Invest. 119:661-673; Kaufmann et al. (2010) Microvasc Res 80:286-293; Santel et al. (2006) Gene Ther 13:1222-1234; Santel et al. (2006) Gene Ther 13:1360-1370; Gutbier et al. (2010) Pulm Pharmacol. Ther. 23:334-344; Basha et al. (2011) Mol. Ther. 19:2186-2200; Fenske and Cullis (2008) Expert Opin Drug Deliv. 5:25-44; Peer et al. (2008) Science 319:627-630; Peer and Lieberman (2011) Gene Ther. 18:1127-1133; all of them are incorporated herein by reference in their entirety. An example of a formulation that passively targets hepatocytes includes lipid nanoparticle formulations based on Dlin-DMA, Dlin-KC2-DMA, and MC3, which have been shown to bind to apolipoprotein E and promote their in vivo binding and uptake into hepatocytes (Akinc et al. (2010) Mol Ther. 18:1357-1364).

LNP modulators can also be selectively targeted by expressing different ligands on their surface, such as folic acid, transferrin, N-acetylgalactosamine (GalNAc), and antibodies (Kolhatkar et al. (2011) Curr Drug Discov Technol. 8:197-206; Musacchio and Torchilin (2011) Front Biosci. 16:1388-1412; Yu et al. (2010) Mol Membr Biol. 27:286-298; Patil et al. (2008) Crit Rev Ther Drug Carrier Syst. 25:1-61; Benoit et al. (2011) Biomacromolecules. 12:2708-2714; Zhao et al. (2008) Expert Opin Drug Deliv.). 5:309-319; Akinc et al. (2010) Mol Ther. 18: 1357-1364; Srinivasan et al. (2012) Methods Mol Biol. 820:105-116; Ben-Arie et al. (2012) Methods Mol Biol. 757:497-507; Peer, D (2010) J of controlled release 148(1):63-68; Peer et al. (2007) Proc Natl Acad Sci USA. 104:4095-4100; Kim et al. (2011) Methods Mol Biol. 721:339-353; Subramanya et al. (2010) Mol Ther. 18:2028-2037; Song et al. (2005) Nat Biotechnol. 23:709-717; Peer et al. (2008) Science 319:627-630; Peer and Lieberman (2011) Gene Ther. 18:1127-1133; all of them are incorporated herein by reference in their entirety.

In some embodiments, the active ingredient (i.e., RGN peptide, RGN-coding polynucleotide, gRNA (i.e., crRNA), gRNA-coding polynucleotide (i.e., crRNA), RGN system or cell including any of these) may be encapsulated within the LNP, and then the LNP may be encapsulated within polymers, polymer receptors, hydrogels and/or surgical sealants described herein and/or known in the art. In some implementations, the polymer, hydrogel, or surgical sealant includes: poly(lactic-co-glycolic acid, PLGA); ethylene vinyl acetate (EVAc); poloxamer; GELSITE® (NanoTherapeutics, Inc., Alachua, FL); HYLENEX® (Halozyme Therapeutics, San Diego, CA); surgical sealants such as fibroblast polymer (Ethicon Inc., Cornelia, GA); and TISSELL® (Baxter International, Inc., Deerfield, IL); PEG-based sealants; and COSEAL® (Baxter International, Inc., Deerfield, IL).

LNP and LNP formulations are further described in, for example, U.S. Patents No. 7,982,027; No. 7,799,565; No. 8,058,069; No. 8,283,333; No. 7,901,708; No. 7,745,651; No. 7,803,397; No. 8,101,741; No. 8,188,263; No. 7,915,399; No. 8,236,943 and No. 7,838,658; European Patent No. 1766035; No. 1519714; No. 1781593 and No. 1664316.

In embodiments in which cells modified with or incorporated herein by means of the RGN polypeptide, RGN-coding polynucleotide, gRNA (i.e., crRNA), gRNA (i.e., crRNA)-coding polynucleotide, or RGN, base editor, or PE system, or cells modified therewith, are delivered to an individual, the cells are delivered as a suspension together with a pharmaceutically acceptable carrier. Those skilled in the art will recognize that a pharmaceutically acceptable carrier used in a cellular composition will not contain a quantity of buffer, compound, cryopreservative, preservative, or other reagent that substantially interferes with the viability of the cells to be delivered to the individual. Formulations containing cells may include, for example, osmotic pressure buffers that allow cell membrane integrity to be maintained, and optionally, nutrients that maintain cell viability or enhance implantation upon administration. Such formulations and suspensions are known to those skilled in the art and/or can be adapted for use with the cells disclosed herein using routine experiments.

Cellular components may also be emulsified or presented as liposome components, provided that the emulsification process does not adversely affect cell viability. Cells and any other active ingredients may be mixed with pharmaceutically acceptable excipients compatible with the active ingredients in amounts suitable for use in the treatment methods disclosed herein.

Additives contained in cellular components may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of peptides) that form with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, tartaric acid, phenylethanolic acid, etc.). Salts formed with free carboxyl groups may also be derived from inorganic bases (e.g., hydroxides of sodium, potassium, ammonium, calcium, or iron) and organic bases (e.g., isopropylamine, trimethylamine, 2-ethylamineethanol, histidine, procaine, etc.).

Physiologically tolerable and pharmaceutically acceptable carriers are known in the art. Exemplary liquid carriers are sterile aqueous solutions containing no materials other than the active ingredient and water, or buffers such as sodium phosphate at physiological pH, physiological saline, or both (e.g., phosphate-buffered saline). Furthermore, aqueous carriers may contain one or more buffered salts and salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol, and other solutes. Liquid compositions may also contain a liquid phase other than water and exclude water. Examples of such added liquid phases are glycerol, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in cellular compositions effective in the treatment of a particular disorder or condition may depend on the nature of the disorder or condition and may be determined by standard clinical techniques.

The RGN polypeptide, RGN-coding polynucleotide, gRNA (i.e., crRNA), gRNA-coding polynucleotide (i.e., crRNA) disclosed in this invention, RGN system, base editor, PE, or cells including any of these can be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the specific administration method and dosage form. In some embodiments, these pharmaceutical compositions are formulated to achieve a physiologically compatible pH, and depending on the formulation and route of administration, the pH ranges from about 3 to about 11, or from about pH 3 to about pH 7. In some embodiments, the pH can be adjusted to a range of about pH 5.0 to about pH 8. In some embodiments, the composition may include at least one compound in a therapeutically effective amount as described herein, together with one or more pharmaceutically acceptable excipients. In some embodiments, the composition includes a combination of compounds described herein, or a combination of reagents that are useful in treating or preventing bacterial growth (e.g., but not limited to, antibacterial or antimicrobial agents), or that contain the contents of this disclosure.

Suitable excipients include, for example, carrier molecules, which include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymerized amino acids, amino acid copolymers, and inactive viral particles. Other exemplary excipients may include antioxidants (e.g., but not limited to, ascorbic acid), chelating agents (e.g., but not limited to, EDTA), carbohydrates (e.g., but not limited to, dextrin, hydroxyl cellulose, and hydroxyl methyl cellulose), stearic acid, liquids (e.g., but not limited to, oils, water, brine, glycerol, and ethanol), humectants or emulsifiers, pH buffers, and so on.

In some embodiments, the formulation is provided in single-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored under freeze-dried conditions, requiring the addition of a sterile liquid carrier, such as saline solution, water for injection, semi-liquid foam, or gel, immediately prior to use. Immediate-injection solutions and suspensions can be prepared from the aforementioned types of sterile powders, granules, and tablets. In some embodiments, the active ingredient is dissolved in a buffer solution, which is frozen in single-dose or multi-dose containers and subsequently thawed for injection or refrigerated/stabilized until use.

One or more therapeutic agents may be included in a controlled-release system. To prolong the action of a drug, it is often desirable to slow its absorption by subcutaneous, intrathecal, or intramuscular injection. This can be accomplished by using a liquid suspension of a water-poorly soluble crystalline or amorphous material. The rate of drug absorption then depends on its dissolution rate, which in turn depends on crystal size and crystal form. Alternatively, delayed absorption of non-oral drug forms is achieved by dissolving or suspending the drug in an oil medium. In some implementations, the use of long-term sustained-release implants is particularly suitable for the treatment of chronic conditions. Long-term sustained-release implants are well known to those skilled in the art. XIII. Treatment Methods

Methods for treating diseases in individuals requiring treatment are provided herein. These methods include administering to the individual requiring treatment an effective amount of: the RGN polypeptide described herein or an active variant or fragment thereof, or a polynucleotide encoding such a polypeptide; the gRNA (i.e., crRNA) described herein or a polynucleotide encoding such crRNA; the RGN system described herein; or cells modified by or comprising any of these components.

In some embodiments, treatment includes in vivo gene editing by administering the RGN polypeptide, gRNA (i.e., crRNA), or RGN system, base editor, PE, or coding RGN polypeptide, gRNA (i.e., crRNA), or RGN system, base editor, PE (one or more) polynucleotides. In some embodiments, treatment includes in vitro gene editing, wherein cells are in vitro gene-modified with the RGN polypeptide, gRNA (i.e., crRNA), or RGN system, base editor, PE, or coding RGN polypeptide, gRNA (i.e., crRNA), or RGN system, base editor, PE (one or more) polynucleotides, and then the modified cells are administered to an individual. In some embodiments, the genetically modified cells originate from the individual to whom the modified cells are subsequently administered, and the transplanted cells are referred to herein as autologous. In some embodiments, the genetically modified cells originate from a different individual (i.e., a donor) of the same species as the individual to whom the modified cells are administered (i.e., the recipient), and the transplanted cells are referred herein as allogeneic. In some examples described herein, the cells may be amplified in a culture prior to administration to the individual requiring treatment.

In some embodiments, the disease to be treated with the composition disclosed in this invention is a disease that can be treated with immunotherapy (e.g., with chimeric antigen receptor (CAR) T cells). Such diseases include, but are not limited to, cancer.

In some embodiments, the diseases to be treated by the compositions disclosed herein are associated with gene defects (e.g., lysosomal storage disorders or metabolic diseases such as type 1 diabetes). Therefore, in some embodiments, the compositions disclosed herein are used to edit genomic sequences that cause diseases or disorders or symptoms of such diseases or disorders. For example, deamination of target nucleosides leads to the correction of gene defects, thereby restoring the lost function of gene products.

In some embodiments, the disease to be treated with the composition disclosed herein is associated with a causal mutation. As used herein, "causal mutation" refers to a specific nucleotide, multiple nucleotides, or nucleotide sequence in the genome that contributes to the severity of a disease or disorder in an individual. Correction of the causal mutation results in an improvement in at least one symptom caused by the disease or disorder. In some embodiments, the causal mutation is adjacent to a PAM site identified by the RGN disclosed herein. The causal mutation can be corrected using the RGN disclosed herein, a fusion polypeptide comprising the RGN disclosed herein and an editing peptide (i.e., a base editor), or a fusion polypeptide comprising the RGN disclosed herein and an editing polymerase (i.e., a polymerase editor). Non-limiting examples of diseases associated with causal mutations include cystic fibrosis, Hurler syndrome, Friedreich’s Ataxia, Huntington’s Disease, and sickle cell disease. Additional non-limiting examples of disease-associated genes and mutations are available from the McCausfield-Nathan Institute for Genetic Medicine at Johns Hopkins University (Baltimore, MD) and the National Center for Biotechnology Information at the National Library of Medicine (Bethesda, MD), accessible via the Global Information Network.

In some embodiments, the methods provided herein are used to introduce inactivation site mutations into genes or their pairs that encode gene products associated with a disease or symptom. For example, in some embodiments, this document provides a method for introducing inactivation site mutations into oncogenes using RGN fusion peptides (e.g., in the treatment of proliferative diseases). In some embodiments, the inactivation mutation may generate a premature stop codon in the coding sequence, resulting in the expression of a truncated gene product, for example, a truncated protein lacking the function of the full-length protein. In some embodiments, the methods provided herein aim to restore the function of dysfunctional genes via genome editing. RGN fusion proteins can be validated for efficacy in in vitro gene editing-based human therapies, for example, by correcting disease-related mutations in human cell cultures. Those skilled in the art will understand that RGN fusion peptides, including RGN and a base-editing peptide, or RGN fusion peptides including RGN and a polymerase-editing peptide, can be used to correct single-point mutations.

In some embodiments, methods for treating diseases in individuals requiring treatment include generating induced pluripotent stem cells (iPSCs); or isolating mesenchymal stem cells from an individual; contacting the iPSCs or mesenchymal stem cells with any of the RGN peptide, RGN, base editor, or PE system disclosed herein, components including the RGN peptide, RGN, base editor, or PE system, or pharmaceutical components to genetically modify target nucleic acid molecules within the cells; differentiating the modified iPSCs or modified mesenchymal stem cells into genetically modified mature cells or their precursors; and administering the genetically modified mature cells or their precursors into an individual. In some implementations, iPSCs or mesenchymal stem cells are autologous or allogeneic cells. In some implementations, iPSCs or mesenchymal stem cells are obtained from a donor whose human leukocyte antigen (HLA) is a perfect match for the individual. In some implementations, myeloablative therapy is administered to the individual before the modified cells are given.

Any method known in the art for generating patient-specific iPS cells may be used, including but not limited to the methods described in Takahashi and Yamanaka, Cell 126(4):663-76, 2006. For example, the generation steps may include: a) isolating somatic cells from an individual, such as skin cells or fibroblasts; and b) introducing a set of pluripotency-associated genes into somatic cells to induce the cells to become pluripotency-associated stem cells. This group of potential-associated genes may be one or more selected from a group consisting of: OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT, and LIN28. Mesenchymal stem cells can be isolated using any method known in the art, such as from the patient's bone marrow or peripheral blood. For example, bone marrow aspirate can be collected into a syringe with heparin. Cells can be washed and centrifuged on Percoll. Cells can be cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS) (Pittinger M F, Mackay A M, Beck S C et al., Science 1999; 284:143-147).

The genetically modified cells used in this disclosure and delivered to an individual include both autologous and allogeneic cells. Allogeneic cells refer to cells derived from one or more donors (i.e., from which one or more individuals obtained genetically modified cells). Autologous cells refer to cells derived from the individual receiving the treatment (i.e., the recipient of the genetically modified cells). Due to the risk of transplant rejection, efforts are made to optimize the major histocompatibility complex (MHC)/human leukocyte antigen (HLA) match between the donor and recipient tissues. HLA is present on the cell surface and helps the body distinguish between its own cells and those of other cells, enabling the body to attack foreign entities such as bacteria and viruses. HLA typing of donor and recipient tissues involves determining the genotypes of six HLA antigens or paired genes between one or more donors and recipients to assess the degree of matching among the six HLAs. HLA paired genes typically refer to two each at loci HLA-A, HLA-B, and HLA-DR, or one each at loci HLA-A, HLA-B, and HLA-C, and one each at loci HLA-DRB1, HLA-DQB1, and HLA-DPB1 (see, for example, Kawase et al., 2007, Blood 110:2235-2241). In some implementations, matching four of the six HLAs between one or more donors and recipients is sufficient to administer cells obtained from the donor to the recipient. In some implementations, a match of 5 out of 6 HLA types between one or more donors and recipients is sufficient to administer cells obtained from one or more donors to the recipient. In other implementations, a match of 6 out of 6 HLA types between one or more donors and recipients is used to administer cells obtained from one or more donors to the recipient. Generally, a match of 4, 5, or 6 out of 6 HLA types is the standard in clinical care. When all 6 HLA types match between one or more donors and recipients, the match is called a perfect match.

As used herein, the terms "treatment," "treating," "palliating," or "ameliorating" are used interchangeably. These terms refer to a means of obtaining a beneficial or desired outcome, which includes, but is not limited to, therapeutic benefits and/or preventive benefits. A therapeutic benefit means any treatment-related improvement or effect on one or more diseases, conditions, or symptoms under treatment. For preventive benefits, the composition may be administered to an individual at risk of developing a particular disease, condition, or symptom, or to an individual reporting one or more physical symptoms of a disease, even if the disease, condition, or symptom may not yet be present. In some implementations, treatment may be administered after the onset of one or more symptoms and/or after a disease has been diagnosed. In certain implementation methods, treatment can be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of disease. For instance, susceptible individuals can be treated before symptoms appear (e.g., based on symptom history and/or genetic or other susceptibility factors). Treatment can also continue after symptoms have subsided to prevent or delay their prevention or recurrence.

The term "effective dose" or "therapeutic effective dose" refers to a dosage amount sufficient to achieve a beneficial or desired outcome. Therapeutic effective doses can vary depending on one or more of the following: the individual being treated and the nature of the disease, the individual's weight and age, the severity of the disease, the method of administration, etc., which can be easily determined by someone of ordinary knowledge in the art. Specific dosages can vary depending on one or more of the following: the specific drug selected, the administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, and the delivery system used.

The term "delivery" refers to the placement of an active ingredient into an individual by means or pathway that causes the introduced active ingredient to be at least locally located at a desired site (e.g., a site of injury or repair), thereby producing (one or more) desired effects. In some embodiments, this disclosure provides methods including delivering any of the RGN peptides, nucleic acid molecules, systems, ribonucleoprotein complexes, vectors, pharmaceutical compositions, and/or gRNA (i.e., crRNA) described herein. In some embodiments, this disclosure further provides cells produced by such methods and organisms (e.g., animals or plants) including or derived from such cells. In some embodiments, RGN peptides and/or nucleic acid molecules, as described herein, combined (and optionally complexed) with guide RNA, are delivered to cells.

In those implementations in which cells are delivered, cells can be delivered via any appropriate pathway leading to a desired address in an individual, at least a portion of which is implanted and remains viable. The survival time of cells after delivery to an individual can range from a few hours (e.g., twenty-four hours) to a few days, to several years, or even the patient's lifespan, i.e., long-term implantation.

In some implementations, delivery includes delivery via viral transport. Viral vectors, including those encoding RGN peptides, ribonucleoprotein complexes, or vectors of nucleic acids disclosed herein, can be delivered directly to patients (i.e., in vivo) or used to treat cells in vitro, and modified cells can be selectively delivered to patients (i.e., ex vivo). Traditional virus-based systems may include, but are not limited to, retrotransfer viruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible using retrotransfer virus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgenic gene. Lentiviral vectors are retrotransfer virus vectors capable of transducing or infecting non-dividing cells and typically producing high viral potency. In applications where short-term expression is preferred, adenovirus-based systems may be used. Adenovirus-based vectors can achieve very high transduction efficiency in many cell types without requiring cell division.

In administration methods including delivery via AAV carriers, the therapeutically effective dose in humans can be in the range of about 20 to about 50 ml of saline solution containing about 1× ^10¹⁰ to about 1× ^10⁵⁰ functional AAVs/ml. The dose can be adjusted to balance the therapeutic benefits with any side effects. In some administration methods, the AAV dose can be about 1× ^10⁵ to 1× ^10⁵⁰ genomic AAVs, about 1× ^10⁸ to 1× ^10²⁰ genomic AAVs, about 1× ^10¹⁰ to about 1× ^10¹⁶ genomic AAVs, or about 1× ^10¹¹ to about 1× ^10¹⁶ genomic AAVs. The human dose can be about 1× ^10¹³ genomic AAVs. These concentrations can be delivered in carrier solutions ranging from about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml. Other effective doses can be readily determined by those skilled in the art through experiments establishing dose-response curves. See, for example, U.S. Patent No. 8,404,658B2.

In some embodiments, delivery includes delivery via other non-viral methods of nucleic acid delivery. In the absence of limitation, exemplary non-viral delivery methods include RNP complexes, liposome transfection, nuclear transfection, microinjection, gene guns, virions, liposomes, LNPs, immunoliposomes, polycationic or liposome-nucleic acid conjugates, naked DNA, artificial viral particles, and agent-enhanced uptake of DNA. Liposome transfection is described in, for example, U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and the liposome transfection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Suitable for the effective receptor recognition of polynucleotides in liposome-transfected cationic and neutral lipids, including those in Feigner's WO1991/17424 and WO1991/16024. Delivery may be to cells (e.g., in vitro or ex vivo) or to the target tissue (e.g., in vivo). In some embodiments, the administration of the pharmaceutical composition disclosed herein comprises an intravenous injection of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg/day or more of the active ingredient in the pharmaceutical composition comprising liposomes or LNPs daily. In some embodiments, the administration of the pharmaceutical composition comprising liposomes or LNPs comprises a dose of about 0.01 to 1 mg per kg of body weight. In some implementations, a pharmaceutical composition including liposomes or LNPs is administered at a dose of approximately 1 to 10 mg per kg of body weight.

Suitable routes of administration for the pharmaceutical composition described herein include, but are not limited to: external, subcutaneous, transdermal, intradermal, intralesional, intra-articular, intraperitoneal, intrabladder, transmucosal, gum, intradental, intraosseous, transtympanic, intra-organ, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periorbital, intratumoral, intracerebral, and intraventricular administration.

In some embodiments, the pharmaceutical composition described herein is delivered to an individual by injection, inhalation (e.g., aerosol), via catheter, via suppository, or via implantation, the implant being a porous, non-porous, or gel-like material comprising a membrane (e.g., a silicone rubber membrane or fiber). In some embodiments, the pharmaceutical composition is formulated for delivery to an individual, for example, for gene editing.

In some embodiments, the pharmaceutical composition is formulated according to standard procedures for intravenous or subcutaneous administration to an individual (e.g., a person). In other embodiments, the pharmaceutical composition for injection is a solution in a sterile isotonic aqueous buffer. Where necessary, the pharmaceutical composition may also contain a solubilizing agent and a local anesthetic (e.g., lidocaine) to reduce pain at the injection site. Generally, the composition is provided alone or in combination in a sealed container (e.g., an ampoule or sachet) indicating the amount of active agent, for example as a dry refrigerated powder or anhydrous concentrate. Where the pharmaceutical composition is to be administered by perfusion, it may be dispensed using a perfusion bottle containing sterile pharmaceutical-grade water or saline solution. In cases where the pharmaceutical composition is administered by injection, ampoules containing sterile water for injection or saline solution can be provided, allowing the composition to be mixed before administration.

In some implementations, the pharmaceutical composition may be contained within lipid particles or vesicles (e.g., liposomes or microcrystals), which are also suitable for parenteral administration.

Although the description of pharmaceutical compositions provided herein is primarily directed at those suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all kinds of animals or organisms.

As used herein, the term "individual" refers to any single entity to which one wishes to diagnose, treat, or cure. In some embodiments, the individual is an animal. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

The efficacy of a treatment can be assessed by a skilled clinician. However, a treatment is considered “effective” if any or all of the signs or symptoms of a disease or disorder are altered in a beneficial manner (e.g., reduced by at least 10%), or if the symptoms or markers of another clinically accepted disease are improved or ameliorated. Efficacy can also be measured by the absence of a deterioration, as assessed by inpatient evaluation, or by the cessation or at least slowing of disease progression without medical intervention. Methods for measuring these indicators are known to those of ordinary knowledge in the art. Treatment includes: (1) suppressing the disease, e.g., halting or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing symptom resolution; and (3) preventing or reducing the likelihood of symptom progression.

The articles “a” and “an” are used in this text to refer to one or more (i.e., at least one) grammatical objects. As an example, “polypeptide” means one or more polypeptides.

All publications and patent applications mentioned in this specification indicate a level of expertise among those who are familiar with the subject matter of this disclosure. All publications and patent applications are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated herein by reference.

Although the foregoing invention has been described in considerable detail with illustrations and examples for the purpose of clarity, it will be apparent that certain modifications and alterations may be made within the scope of the appended embodiments. Non-restrictive embodiments include:

1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding the RGN polypeptide, the RGN polypeptide comprising, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, The amino acid sequence represented by any one of 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity.

2. The nucleic acid molecule as described in embodiment 1, wherein the RGN polypeptide comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, The amino acid sequence represented by any one of 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 95% sequence identity.

3. A nucleic acid molecule as described in embodiment 1 or 2, wherein the RGN polypeptide comprises, as in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687.

4. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 1.

5. The nucleic acid molecule of embodiment 4, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are positively charged amino acid residues.

6. The nucleic acid molecule of embodiment 4 or 5, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are arginine (R).

7. The nucleic acid molecule of embodiment 6, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of the amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of the RGN polypeptide are R.

8. A nucleic acid molecule as described in embodiment 6 or 7, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 450; (b) an amino acid sequence as shown in SEQ ID NO: 463; (c) an amino acid sequence as shown in SEQ ID NO: 471; (d) an amino acid sequence as shown in SEQ ID NO: 481; (e) an amino acid sequence as shown in SEQ ID NO: 484; (f) an amino acid sequence as shown in SEQ ID NO: 485; (g) an amino acid sequence as shown in SEQ ID NO: 486; (h) an amino acid sequence as shown in SEQ ID NO: 500; (i) an amino acid sequence as shown in SEQ ID NO: 502; (j) an amino acid sequence as shown in SEQ ID NO: 505; (k) an amino acid sequence as shown in SEQ ID NO: 508; (l) an amino acid sequence as shown in SEQ ID NO: 505; (d) an amino acid sequence as shown in SEQ ID NO: 508; (e) an amino acid sequence as shown in SEQ ID NO: 450; (f) an amino acid sequence as shown in SEQ ID NO: 463; (c) an amino acid sequence as shown in SEQ ID NO: 471; (d) an amino acid sequence as shown in SEQ ID NO: 481; (e) an amino acid sequence as shown in SEQ ID NO: 484; (f) an amino acid sequence as shown in SEQ ID NO: 485; (g) an amino acid sequence as shown in SEQ ID NO: 486; (h) an amino acid sequence as shown in SEQ ID NO: 500; (i) an amino acid sequence as shown in SEQ ID NO: 502; (j) an amino acid sequence as shown in SEQ ID NO: 505; (k) an amino The amino acid sequence shown in SEQ ID NO: 539; (m) the amino acid sequence shown in SEQ ID NO: 602; (n) the amino acid sequence shown in SEQ ID NO: 707; (o) the amino acid sequence shown in SEQ ID NO: 708; and (p) the amino acid sequence shown in SEQ ID NO: 717.

9. A nucleic acid molecule as described in Embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 1, and wherein at the following amino acid position combinations selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; (c) amino acid positions 530, 552, 677, and 801; (d) amino acid positions 530, 577, and 790; (e) amino acid positions 517, 530, 538, 552, and 677; (f) amino acid positions 530, 538, 677, and 801; (g) amino acid positions 538, 538, 677, and 801; 552, 677, and 801; (h) amino acid positions 517, 530, 538, and 552; (i) amino acid positions 517 and 530; (j) amino acid positions 530, 538, and 552; (k) amino acid positions 517, 530, 538, and 677; (l) amino acid positions 517, 552, 677, and 801; (m) amino acid positions 530, 677, and 801; (n) amino acid positions 517, 530, 677, and 801; (o) amino acid positions 517, 530, and 677; (p) amino acid positions 530, 538, 552, and 677; (q) amino acid positions 517, 552, and 677; and (r) amino acid position 517, 530, 552, 677 and 801.

10. A nucleic acid molecule as described in embodiment 9, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 1, and wherein the amino acid residue is arginine (R) at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide: (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; (c) amino acid positions 530, 552, 677, and 801; (d) amino acid positions 530, 577, and 790; (e) amino acid positions 517, 530, 538, 552, and 677; (f) amino acid positions 530, 538, 677, and 690. 801; (g) Amino acid positions 538, 552, 677 and 801; (h) Amino acid positions 517, 530, 538 and 552; (i) Amino acid positions 517 and 530; (j) Amino acid positions 530, 538 and 552; (k) Amino acid positions 517, 530, 538 and 677; (l) Amino acid positions 517, 552, 677 and 801; (m) Amino acid positions 530, 677 and 801; (n) Amino acid positions 517, 530, 677 and 801; (o) Amino acid positions 517, 530 and 677; (p) Amino acid positions 530, 538, 552 and 677; (q) Amino acid positions 517, 552 and 677; and (r) amino acid positions 517, 530, 552, 677 and 801.

11. The nucleic acid molecule of embodiment 9 or 10, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1072; (b) an amino acid sequence as shown in SEQ ID NO: 1080; (c) an amino acid sequence as shown in SEQ ID NO: 1082; (d) an amino acid sequence as shown in SEQ ID NO: 1105; (e) an amino acid sequence as shown in SEQ ID NO: 1084; (f) an amino acid sequence as shown in SEQ ID NO: 1081; (g) an amino acid sequence as shown in SEQ ID NO: 1083; (h) an amino acid sequence as shown in SEQ ID NO: 1069; (i) an amino acid sequence as shown in SEQ ID NO: 1034; (j) an amino acid sequence as shown in SEQ ID NO: 1059; (k) an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 1070; (l) the amino acid sequence shown in SEQ ID NO: 1078; (m) the amino acid sequence shown in SEQ ID NO: 1064; (n) the amino acid sequence shown in SEQ ID NO: 1074; (o) the amino acid sequence shown in SEQ ID NO: 1051; (p) the amino acid sequence shown in SEQ ID NO: 1079; (q) the amino acid sequence shown in SEQ ID NO: 1056; and (r) the amino acid sequence shown in SEQ ID NO: 1087.

12. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 1, and wherein the amino acid residue is arginine (R) at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide: (a) amino acid positions 517, 530, 552 and 677; (b) amino acid positions 530, 538, 552 and 801; and (c) amino acid positions 530, 552, 677 and 801.

13. The nucleic acid molecule of embodiment 12, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 1, and wherein at the following combinations of amino acid positions selected from the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; and (c) amino acid positions 530, 552, 677, and 801.

14. The nucleic acid molecule of embodiment 12 or 13, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1072; (b) an amino acid sequence as shown in SEQ ID NO: 1080; and (c) an amino acid sequence as shown in SEQ ID NO: 1082.

15. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of the amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4.

16. The nucleic acid molecule of embodiment 15, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are positively charged amino acid residues.

17. The nucleic acid molecule of embodiment 15 or 16, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are R.

18. The nucleic acid molecule of embodiment 17, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of the RGN polypeptide are R.

19. A nucleic acid molecule as described in embodiment 17 or 18, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 809; (b) an amino acid sequence as shown in SEQ ID NO: 810; (c) an amino acid sequence as shown in SEQ ID NO: 866; (d) an amino acid sequence as shown in SEQ ID NO: 898; (e) an amino acid sequence as shown in SEQ ID NO: 909; (f) an amino acid sequence as shown in SEQ ID NO: 912; (g) an amino acid sequence as shown in SEQ ID NO: 916; (h) an amino acid sequence as shown in SEQ ID NO: 960; (i) an amino acid sequence as shown in SEQ ID NO: 961; (j) an amino acid sequence as shown in SEQ ID NO: 963; (k) an amino acid sequence as shown in SEQ ID NO: 964; (v) an amino acid sequence as shown in SEQ ID NO: 965; (v) an amino acid sequence as shown in SEQ ID NO: 96 ... The amino acid sequence shown in SEQ ID NO: 966; (l) the amino acid sequence shown in SEQ ID NO: 970; (m) the amino acid sequence shown in SEQ ID NO: 975; (n) the amino acid sequence shown in SEQ ID NO: 1012; and (o) the amino acid sequence shown in SEQ ID NO: 1019.

20. A nucleic acid molecule as described in embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein the amino acid residue is arginine (R) at the following amino acid position combinations selected from the following amino acid position combinations of the RGN polypeptide: (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid positions 377, 625, and 691; (g) amino acid positions 377, 625, 698 and 747; (h) amino acid positions 377, 638, 691 and 698; (i) amino acid positions 377, 625 and 638; (j) amino acid positions 377, 638, 698 and 747; (k) amino acid positions 377, 625, 638, 691 and 698; (l) amino acid positions 625, 638 and 698; (m) amino acid positions 377, 625, 691 and 698; (n) amino acid positions 377, 638 and 698; (o) amino acid positions 625, 691, 698 and 747; (p) amino acid positions 638 and 698; and (q) amino acid positions 377 and 691.

21. The nucleic acid molecule of embodiment 20, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue is arginine (R) at the following amino acid position combinations selected from the following amino acid position combinations of the RGN polypeptide: (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid positions 377, 625, and 698. 691; (g) amino acid positions 377, 625, 698 and 747; (h) amino acid positions 377, 638, 691 and 698; (i) amino acid positions 377, 625 and 638; (j) amino acid positions 377, 638, 698 and 747; (k) amino acid positions 377, 625, 638, 691 and 698; (l) amino acid positions 625, 638 and 698; (m) amino acid positions 377, 625, 691 and 698; (n) amino acid positions 377, 638 and 698; (o) amino acid positions 625, 691, 698 and 747; (p) amino acid positions 638 and 698; and (q) amino acid positions 377 and 691.

22. A nucleic acid molecule as described in embodiment 20 or 21, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1123; (b) an amino acid sequence as shown in SEQ ID NO: 1158; (c) an amino acid sequence as shown in SEQ ID NO: 1159; (d) an amino acid sequence as shown in SEQ ID NO: 1148; (e) an amino acid sequence as shown in SEQ ID NO: 1160; (f) an amino acid sequence as shown in SEQ ID NO: 1122; (g) an amino acid sequence as shown in SEQ ID NO: 1146; (h) an amino acid sequence as shown in SEQ ID NO: 1147; (i) an amino acid sequence as shown in SEQ ID NO: 1121; (j) an amino acid sequence as shown in SEQ ID NO: 1149; (k) an amino acid sequence as shown in SEQ ID NO: 1149; (v ... The amino acid sequence shown in SEQ ID NO: 1156; (l) the amino acid sequence shown in SEQ ID NO: 1132; (m) the amino acid sequence shown in SEQ ID NO: 1144; (n) the amino acid sequence shown in SEQ ID NO: 1126; (o) the amino acid sequence shown in SEQ ID NO: 1154; (p) the amino acid sequence shown in SEQ ID NO: 1116; and (q) the amino acid sequence shown in SEQ ID NO: 1108.

23. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4.

24. The nucleic acid molecule of embodiment 23, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are positively charged amino acid residues.

25. The nucleic acid molecule of embodiment 23 or 24, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein the amino acid residue at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide is R or lysine (K).

26. The nucleic acid molecule of embodiment 25, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are R or K.

27. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein at the amino acid position combinations selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R) and/or lysine (K): (a) amino acid positions 736, 743, 752, 753, 755, 757, 758 and 760; (b) amino acid positions 740, 747, 751, 753, 755, 760, 764 and 766; (c) amino acid positions 685, 691 and 698; and (d) amino acid positions 685, 692, 695, 702 and 707.

28. The nucleic acid molecule of embodiment 27, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein at the amino acid positions selected from the following combinations of amino acid positions of the RGN polypeptide, the amino acid residue is arginine (R) and/or lysine (K): (a) amino acid positions 736, 743, 752, 753, 755, 757, 758, and 760; (b) amino acid positions 740, 747, 751, 753, 755, 760, 764, and 766; (c) amino acid positions 685, 691, and 698; and (d) amino acid positions 685, 692, 695, 702, and 707.

29. The nucleic acid molecule of embodiment 27 or 28, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1168; (b) an amino acid sequence as shown in SEQ ID NO: 1171; (c) an amino acid sequence as shown in SEQ ID NO: 1169; and (d) an amino acid sequence as shown in SEQ ID NO: 1170.

30. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein the RGN polypeptide comprises the deletion of amino acid residues corresponding to amino acid residues 751 to 769 of SEQ ID NO: 4.

31. The nucleic acid molecule of embodiment 30, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein the RGN polypeptide comprises the deletion of an amino acid residue corresponding to amino acid residues 751 to 769 of SEQ ID NO: 4.

32. The nucleic acid molecule as described in embodiment 30 or 31, wherein the RGN polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1173.

33. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more of the amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596 and 862 of the RGN polypeptide comprises substitution or insertion of one or more glycine (G) at the N-terminal position adjacent to the amino acid position, at the amino acid position, or at the C-terminal position adjacent to the amino acid position.

34. The nucleic acid molecule of embodiment 33, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more of the amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596, and 862 of the RGN polypeptide comprises one or more substitutions or insertions of G in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position.

35. The nucleic acid molecule as described in embodiment 33 or 34, wherein the substitution or insertion of one or more Gs comprises the substitution or insertion of one, two or three Gs.

36. The nucleic acid molecule of embodiment 33, wherein the substitution or insertion of one or more Gs is selected from: (a) inserting three Gs between amino acid positions 293 and 294; (b) inserting two Gs between amino acid positions 359 and 360; (c) substituting the G at amino acid position 361; (d) inserting one G between amino acid positions 402 and 403; (e) substituting the G at amino acid position 401; (f) substituting the G at amino acid position 596; and (g) substituting the G at amino acid position 862.

37. The nucleic acid molecule of embodiment 36, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) the amino acid sequence shown in SEQ ID NO: 1304; (b) the amino acid sequence shown in SEQ ID NO: 1322; (c) the amino acid sequence shown in SEQ ID NO: 1324; (d) the amino acid sequence shown in SEQ ID NO: 1328; (e) the amino acid sequence shown in SEQ ID NO: 1330; (f) the amino acid sequence shown in SEQ ID NO: 1361; and (g) the amino acid sequence shown in SEQ ID NO: 1400.

38. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more of the amino acid positions 566, 567, and 584 of the RGN polypeptide comprises one or more substitutions or insertions of G in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position.

39. The nucleic acid molecule of embodiment 38, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more of the amino acid positions 566, 567, and 584 of the RGN polypeptide comprises one or more substitutions or insertions of G in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position.

40. The nucleic acid molecule as described in embodiment 38 or 39, wherein the substitution or insertion of one or more Gs comprises the substitution or insertion of one, two or three Gs.

41. The nucleic acid molecule of embodiment 38, wherein the substitution or insertion of one or more Gs is selected from: (a) substitution of G at amino acid position 566 and substitution of G at amino acid position 567; and (b) substitution of G at amino acid position 584.

42. The nucleic acid molecule of embodiment 41, wherein the RGN polypeptide comprises an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1223; and (b) an amino acid sequence as shown in SEQ ID NO: 1228.

43. A nucleic acid molecule as described in any of embodiments 1 to 42, wherein the polynucleotide encoding the RGN polypeptide comprises mRNA, which includes a heterologous 5' untranslated region (UTR) and/or a heterologous 3' UTR.

44. A nucleic acid molecule as described in any of embodiments 1 to 43, wherein the polynucleotide encoding the RGN polypeptide is operatively linked to a promoter heterologous to the polynucleotide.

45. A nucleic acid molecule as described in any one of embodiments 1 to 44, wherein, when bound to guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA-guided sequence-specific manner, wherein the target sequence includes a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence.

46. The nucleic acid molecule of embodiment 45, wherein the target sequence is disposed adjacent to and at its 3' of the original spacer adjacent motif (PAM).

47. The nucleic acid molecule of embodiment 46, wherein a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 recognizes a PAM having a common nucleotide sequence shown in AYG, wherein the PAM is adjacent to and at its 5' of the target sequence; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 1933 or 1944 recognizes a PAM having a common nucleotide sequence shown in ATTN, wherein the PAM is adjacent to and at its 5' of the target sequence; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947 or 1949 recognizes a PAM having a common nucleotide sequence shown in VTTN, wherein the PAM is adjacent to and at its 5' of the target sequence; and d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1... 4) RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is located at its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1937 or 1950 identify PAMs having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1951 identify PAMs having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1953 identify PAMs having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'.

48. The nucleic acid molecule as described in any of embodiments 45 to 47, wherein the gRNA does not include trans-activated CRISPR RNA (tracrRNA).

49. A nucleic acid molecule as described in any of embodiments 45 to 48, wherein the gRNA has a total length of 35 to 250 nucleotides (nt), or 35 to 170 nt, or 35 to 125 nt, or 40 to 100 nt, or 40 to 70 nt, or 40 to 60 nt.

50. A nucleic acid molecule as described in any of embodiments 45 to 48, wherein the gRNA has a maximum length of 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt, 83 nt, or more. nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 160 nt, 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt, 180 nt, 190 nt, 200 nt, 205 nt, 210 nt, 215 nt, 220 nt, 225 nt, 230 nt, 235 nt, 240 nt, 245 nt or 250 nt total length.

51. A nucleic acid molecule as described in any of embodiments 45 to 48, wherein the gRNA comprises a crRNA backbone having a total length of up to 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt.

52. The nucleic acid molecule as described in any one of embodiments 1 to 51, wherein the nucleotide sequence encoding the RGN polypeptide includes any one of SEQ ID NO: 29 to 32 and 2659 to 2685.

53. A nucleic acid molecule as described in any of embodiments 45 to 52, wherein the gRNA contains at least one chemical modification.

54. The nucleic acid molecule of embodiment 53, wherein at least one chemical modification comprises: bridging nucleic acid (BNA) modification; 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluorine (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and thiophosphate (PS) modification.

55. The nucleic acid molecule as described in embodiment 54, wherein the BNA includes 2′,4′ BNA modification.

56. The nucleic acid molecule as described in embodiment 55, wherein the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification and S-restricted ethyl (cEt) modification.

57. The nucleic acid molecule as described in embodiment 56, wherein the 2′,4′ BNA is an LNA modification.

58. The nucleic acid molecule as described in embodiment 56, wherein the 2′,4′ BNA is cEt modified.

59. The nucleic acid molecule as described in embodiment 53 or 54, wherein at least one chemical modification includes BNA modification, 2'-O-Me modification or PS modification.

60. The nucleic acid molecule as described in any one of embodiments 45 to 59, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding.

61. The nucleic acid molecule of embodiment 60, wherein the RGN polypeptide is capable of producing double-strand breaks.

62. The nucleic acid molecule as described in any one of embodiments 1 to 59, wherein the RGN polypeptide is nuclease-inactivating.

63. The nucleic acid molecule of claim 62, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 408, 597, 678, 827, 896, 979, 2686, and 2687.

64. A nucleic acid molecule as described in any of embodiments 1 to 63, wherein the RGN polypeptide is operatively fused with a base-editing polypeptide.

65. The nucleic acid molecule as described in embodiment 64, wherein the base-editing polypeptide includes a deaminase.

66. The nucleic acid molecule as described in any of claims 1 to 63, wherein the RGN polypeptide is operatively fused with a polymerase-editing polypeptide.

67. The nucleic acid molecule as claimed in claim 66, wherein the polymerase editing polypeptide comprises DNA polymerase.

68. The nucleic acid molecule as claimed in claim 66, wherein the polymerase-editing polypeptide comprises a reverse transcriptase.

69. A nucleic acid molecule as described in any of embodiments 1 to 63, wherein the RGN polypeptide is operatively fused to an effector domain.

70. The nucleic acid molecule of embodiment 69, wherein the effector domain is operatively fused to the N-terminus or C-terminus of the RGN polypeptide.

71. The nucleic acid molecule as described in embodiment 69, wherein the effector domain is operatively fused to an internal address of the RGN polypeptide.

72. The nucleic acid molecule as described in any of embodiments 69 to 71, wherein the effector domain includes a splicing domain, a deaminase domain, or a phenoregulatory domain.

73. The nucleic acid molecule as described in embodiment 72, wherein the expression regulatory subdomain includes a transcription activation domain, a transcription repression domain, or an epigenetic modification domain.

74. A nucleic acid molecule as described in any of embodiments 1 to 73, wherein the RGN polypeptide includes one or more nuclear localization signals (NLS).

75. The nucleic acid molecule of embodiment 74, wherein one or more NLS comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any of SEQ ID NO: 33, 35, 407, and 1927.

76. A nucleic acid molecule as described in any of embodiments 1 to 75, wherein the nucleotide sequence encoding the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

77. The nucleic acid molecule as described in embodiment 76, wherein the eukaryotic cell is a mammalian cell.

78. A nucleic acid molecule as described in any of embodiments 1 to 77, wherein the nucleic acid molecule is an isolated nucleic acid molecule.

79. A vector comprising a nucleic acid molecule as described in any one of embodiments 1 to 78.

80. The vector as described in embodiment 79, wherein the vector further comprises at least one nucleotide sequence of a coding guide RNA (gRNA).

81. The vector as described in embodiment 79, wherein the vector further comprises at least two, at least three, at least four, at least five, at least six or more nucleotide sequences, each nucleotide sequence coding for guiding RNA.

82. A vector comprising a nucleic acid molecule as described in any one of embodiments 45 to 78, further comprising at least one nucleotide sequence encoding the gRNA.

83. The vector as described in embodiment 80 or 82, wherein the at least one nucleotide sequence encoding the gRNA comprises a nucleotide sequence encoding one or more gRNAs.

84. The vector as described in embodiment 83, wherein the nucleotide sequence encoding one or more gRNAs is operatively linked to a single promoter.

85. The vector as described in embodiment 80 or 82, wherein the at least one nucleotide sequence encoding the gRNA comprises two or more nucleotide sequences, each nucleotide sequence encoding the gRNA.

86. The vector as described in embodiment 85, wherein each nucleotide sequence encoding the gRNA is operatively linked to a promoter.

87. The vector as described in embodiments 84 or 86, wherein the promoter comprises the RNA polymerase III promoter.

88. The vector as described in embodiment 87, wherein the RNA polymerase III promoter comprises the human U6 promoter or the human 7SK promoter.

89. The vector as described in embodiment 88, wherein the human U6 promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1672.

90. The vector as described in embodiment 88, wherein the human 7SK promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1673.

91. The vector as described in any of embodiments 79 to 90, wherein the vector further comprises a promoter operatively linked to a nucleic acid molecule comprising a polynucleotide encoding an RGN polypeptide.

92. The vector as described in embodiment 91, wherein the promoter comprises the RNA polymerase II promoter.

93. The vector as described in embodiment 92, wherein the RNA polymerase II promoter comprises the CMV promoter or the CBh promoter.

94. The carrier as described in embodiment 93, wherein the CMV initiator further includes a CMV upstream enhancer.

95. The vector of embodiment 94, wherein the CMV initiator and the CMV upstream enhancer comprise a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1674.

96. The vector as described in embodiment 94, wherein the CBh promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1675.

97. The vector as described in any of embodiments 79 to 96, wherein the vector further comprises a nucleotide sequence of a coding peptide linker.

98. The carrier as described in embodiment 97, wherein the peptide linker comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with an amino acid sequence as shown in SEQ ID NO: 34 or 1928.

99. The carrier as described in any of embodiments 79 to 98, wherein the carrier further comprises a polyadenylated (polyA) tail.

100. The carrier as described in embodiment 99, wherein the polyA tail comprises an SV40 polyA tail.

101. The vector as described in embodiment 100, wherein the SV40 polyA tail comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1678.

102. The vector as described in any of embodiments 80 to 101, wherein the gRNA comprises at least one CRISPR RNA (crRNA) backbone (bb) and at least one spacer.

103. The vector as described in embodiment 102, wherein the gRNA comprises two crRNA bbs and a spacer, in the form of crRNA bb-spacer-crRNA bb from 5' to 3'.

104. The vector as described in embodiment 102, wherein the gRNA comprises three crRNA bb, a first spacer and a second spacer, in the form of crRNA bb-first spacer-crRNA bb-second spacer-crRNA bb from 5' to 3'.

105. The vector as described in embodiment 102, wherein the gRNA comprises a crRNA bb and a spacer, in the form of crRNA bb-spacer from 5' to 3'.

106. The vector as described in any of embodiments 102 to 104, wherein at least one crRNA bb comprises two or more crRNA bbs, and wherein the two or more crRNA bbs are distinct.

107. The vector as described in any of embodiments 102 to 104, wherein at least one crRNA bb comprises two or more crRNA bbs, and wherein the two or more crRNA bbs are identical.

108. The vector as described in any of embodiments 102 to 104, wherein at least one crRNA bb comprises two or more crRNA bbs, and wherein the two or more crRNA bbs are a mixture of some identical crRNA bbs and some different crRNA bbs.

109. The carrier as described in any of embodiments 102 to 108, wherein at least one septum comprises two or more septum, and wherein the two or more septum target different target sequences.

110. The carrier as described in any of embodiments 102 to 108, wherein at least one septum comprises two or more septum, and wherein the two or more septum target the same target sequence.

111. A vector of any of embodiments 80 to 110, wherein the gRNA is selected from the group consisting of: a) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a CRISPR RNA (crRNA) backbone comprising a nucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 1; b) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 2; c) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 7 or differing from SEQ ID NO: 8 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 2; 7. A nucleotide sequence having 1 to 5 different nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; and d) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence having 1 to 5 different nucleotides as shown in SEQ ID NO: 8 or having 1 to 5 different nucleotides from SEQ ID NO: 8, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence having 1 to 5 different nucleotides as shown in SEQ ID NO: 1617 or having 1 to 5 different nucleotides from SEQ ID NO: 1617, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence having 1 to 5 different nucleotides from SEQ ID NO: 1617. The following are considered RGN polypeptides: (a) nucleotide sequences shown in SEQ ID NO: 1618 or differing from SEQ ID NO: 1618 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (b) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence shown in SEQ ID NO: 1619 or differing from SEQ ID NO: 1619 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (c) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1620 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (d) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1620 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence having a crRNA backbone of SEQ ID NO: 4. The RNA includes a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1631 or differing from SEQ ID NO: 1631 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1; (j) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1957 or differing from SEQ ID NO: 1957 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1930; (k) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1958 or differing from SEQ ID NO: 1958 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1930; The amino acid sequence of SEQ ID NO: 1931 has an amino acid sequence with at least 90% sequence identity; (l) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1959 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1932; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1960 or differing from SEQ ID NO: 1960 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1933; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1961 or differing from SEQ ID NO: 1932 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1933; 1961 has a nucleotide sequence that is 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1962 or having 1 to 5 nucleotides different from SEQ ID NO: 1962, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935; (p) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1963 or having 1 to 5 nucleotides different from SEQ ID NO: 1963, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936; (q) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1963 or having 1 to 5 nucleotides different from SEQ ID NO: 1963; The RNA includes a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1964 or differing from SEQ ID NO: 1964 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1965 or differing from SEQ ID NO: 1965 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938; (s) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1966 or differing from SEQ ID NO: 1966 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938. The amino acid sequence of SEQ ID NO: 1939 has at least 90% sequence identity; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1967 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1968 or differing from SEQ ID NO: 1968 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1969 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941; 1969 has a nucleotide sequence that is 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942; (w) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1970 or having 1 to 5 nucleotides different from SEQ ID NO: 1970, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1971 or having 1 to 5 nucleotides different from SEQ ID NO: 1971, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944; (y) gRNA, including CRISPR RNA, CRISPR... The RNA includes a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1972 or differing from SEQ ID NO: 1972 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1945; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1973 or differing from SEQ ID NO: 1973 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1974 or differing from SEQ ID NO: 1974 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; The amino acid sequence of SEQ ID NO: 1947 has at least 90% sequence identity; (bb) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1975 or differing from SEQ ID NO: 1975 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1976 or differing from SEQ ID NO: 1976 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1977 or differing from SEQ ID NO: 1949 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; 1977 contains nucleotide sequences that are 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1978 or having 1 to 5 nucleotides different from SEQ ID NO: 1978, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951; (ff) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1979 or having 1 to 5 nucleotides different from SEQ ID NO: 1979, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1979 or having 1 to 5 nucleotides different from SEQ ID NO: 1979; The RNA includes a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1980 or differing from SEQ ID NO: 1980 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1981 or differing from SEQ ID NO: 1981 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954; (iii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1982 or differing from SEQ ID NO: 1982 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954. The amino acid sequence of 1955 has an amino acid sequence with at least 90% sequence identity; and (jj)gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1983 or differing from SEQ ID NO: 1983 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1956.

112. The vector of embodiment 111, wherein the gRNA is selected from the group consisting of: a) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a CRISPR RNA (crRNA) backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; The amino acid sequence shown in SEQ ID NO: 3 has an amino acid sequence with at least 90% sequence identity; and d) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 8 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1617 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1618 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 4 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 4 has at least 90% sequence identity; (g) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1619 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1620 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (j) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 5 nucleotides different from that of SEQ ID NO: 1957, wherein the RGN polypeptide includes an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1930; (k) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 5 nucleotides different from that of SEQ ID NO: 1958, wherein the RGN polypeptide includes an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 5 nucleotides different from that of SEQ ID NO: 1959, wherein the RGN polypeptide includes an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1931. The amino acid sequence of SEQ ID NO: 1932 has at least 90% sequence identity; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1960 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934; The amino acid sequence of SEQ ID NO: 1935 has at least 90% sequence identity; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936; (q) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937. The amino acid sequence of SEQ ID NO: 1938 has at least 90% sequence identity; (s) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1939; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1939; The amino acid sequence of SEQ ID NO: 1941 has at least 90% sequence identity; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943. The amino acid sequence of SEQ ID NO: 1944 has at least 90% sequence identity; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1945; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; The amino acid sequence of SEQ ID NO: 1947 has at least 90% sequence identity; (bb) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1950 has at least 90% sequence identity; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951; (ff) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; The amino acid sequence of SEQ ID NO: 1953 has at least 90% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954; (iii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955; The amino acid sequence of 1956 has an amino acid sequence with at least 90% sequence identity.

113. The vector of embodiment 111, wherein the gRNA is selected from the group consisting of: a) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a CRISPR RNA (crRNA) backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; The amino acid sequence shown in SEQ ID NO: 3 has an amino acid sequence with at least 90% sequence identity; and d) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 8 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1617 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1618 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 4 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 4 has at least 90% sequence identity; (g) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1619 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1620 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (j) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 4 nucleotides different from that of SEQ ID NO: 1957, wherein the RGN polypeptide includes an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1930; (k) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 4 nucleotides different from that of SEQ ID NO: 1958, wherein the RGN polypeptide includes an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 4 nucleotides different from that of SEQ ID NO: 1959, wherein the RGN polypeptide includes an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1931. The amino acid sequence of SEQ ID NO: 1932 has at least 90% sequence identity; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1960 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934. The amino acid sequence of SEQ ID NO: 1935 has at least 90% sequence identity; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936; (q) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937. The amino acid sequence of SEQ ID NO: 1938 has at least 90% sequence identity; (s) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1939; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1939; The amino acid sequence of SEQ ID NO: 1941 has at least 90% sequence identity; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943. The amino acid sequence of SEQ ID NO: 1944 has at least 90% sequence identity; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1945; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; The amino acid sequence of SEQ ID NO: 1947 has at least 90% sequence identity; (bb) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1950 has at least 90% sequence identity; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951; (ff) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; The amino acid sequence of SEQ ID NO: 1953 has at least 90% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954; (iii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955. The amino acid sequence of 1956 has an amino acid sequence with at least 90% sequence identity.

114. The vector of embodiment 111, wherein the gRNA is selected from the group consisting of: a) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a CRISPR RNA (crRNA) backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; The amino acid sequence shown in SEQ ID NO: 3 has an amino acid sequence with at least 95% sequence identity; and d) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 8 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1617 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1618 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 4 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 4 has at least 95% sequence identity; (g) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1619 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1620 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (j) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 3 nucleotides different from that of SEQ ID NO: 1957, wherein the RGN polypeptide includes an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 1930; (k) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 3 nucleotides different from that of SEQ ID NO: 1958, wherein the RGN polypeptide includes an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that is 3 nucleotides different from that of SEQ ID NO: 1959, wherein the RGN polypeptide includes an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 1931. The amino acid sequence of SEQ ID NO: 1932 has at least 95% sequence identity; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1960 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934. The amino acid sequence of SEQ ID NO: 1935 has at least 95% sequence identity; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936; (q) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937. The amino acid sequence of SEQ ID NO: 1938 has at least 95% sequence identity; (s) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1939; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1939; The amino acid sequence of SEQ ID NO: 1941 has at least 95% sequence identity; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943. The amino acid sequence of SEQ ID NO: 1944 has at least 95% sequence identity; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1945; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946; The amino acid sequence of SEQ ID NO: 1947 has at least 95% sequence identity; (bb) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1948; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1950 has at least 95% sequence identity; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1951; (ff) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; The amino acid sequence of SEQ ID NO: 1953 has at least 95% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1981 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954; (iii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1982 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1983 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1955 by 3 nucleotides; The amino acid sequence of 1956 has an amino acid sequence with at least 95% sequence identity.

115. The vector of embodiment 111, wherein the gRNA is selected from the group consisting of: a) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a CRISPR RNA (crRNA) backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; The amino acid sequence shown in SEQ ID NO: 3 has an amino acid sequence with at least 95% sequence identity; and d) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 8 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1617 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1618 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 4 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 4 has at least 95% sequence identity; (g) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1619 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1620 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (j) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1957 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1930; (k) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1958 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1959 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931. The amino acid sequence of SEQ ID NO: 1932 has at least 95% sequence identity; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1960 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934; The amino acid sequence of SEQ ID NO: 1935 has at least 95% sequence identity; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936; (q) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937. The amino acid sequence of SEQ ID NO: 1938 has at least 95% sequence identity; (s) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1939; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1939; The amino acid sequence of SEQ ID NO: 1941 has at least 95% sequence identity; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943. The amino acid sequence of SEQ ID NO: 1944 has at least 95% sequence identity; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1945; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946; The amino acid sequence of SEQ ID NO: 1947 has at least 95% sequence identity; (bb) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1975 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1948; (cc) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1976 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1977 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1950 has at least 95% sequence identity; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1951; (ff) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; The amino acid sequence of SEQ ID NO: 1953 has at least 95% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1981 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1982 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1983 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1955 by 2 nucleotides; The amino acid sequence of 1956 has an amino acid sequence with at least 95% sequence identity.

116. The vector of embodiment 111, wherein the gRNA is selected from the group consisting of: a) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a CRISPR RNA (crRNA) backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; The amino acid sequence shown in SEQ ID NO: 3 has an amino acid sequence with at least 95% sequence identity; and d) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 8 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1617 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1618 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 4 ... The amino acid sequence of SEQ ID NO: 4 has at least 95% sequence identity; (g) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1619 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1620 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (j) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1957 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1930; (k) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1958 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1959 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931; The amino acid sequence of SEQ ID NO: 1932 has at least 95% sequence identity; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1960 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1961 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1962 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1932 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1933 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1934 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1932 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 193 ... The amino acid sequence of SEQ ID NO: 1935 has at least 95% sequence identity; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1963 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936; (q) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1964 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1965 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1937 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1935 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1936 ...5 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1935 by one nucleotide The amino acid sequence of SEQ ID NO: 1938 has at least 95% sequence identity; (s) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1966 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1939; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1967 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1968 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1939 ... The amino acid sequence of SEQ ID NO: 1941 has at least 95% sequence identity; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1969 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1970 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1971 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1941 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1942 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1943 ... The amino acid sequence of SEQ ID NO: 1944 has at least 95% sequence identity; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1972 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1945; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1973 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1974 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1944 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1945 ... The amino acid sequence of SEQ ID NO: 1947 has at least 95% sequence identity; (bb) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1948; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1950 has at least 95% sequence identity; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1951; (ff) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; The amino acid sequence of SEQ ID NO: 1953 has at least 95% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1981 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954; (iii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1982 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1983 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1955 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1954 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 195 ... The amino acid sequence of 1956 has an amino acid sequence with at least 95% sequence identity.

117. The vector of embodiment 111, wherein the gRNA is selected from the group consisting of: a) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 5, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1; b) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 6, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2; c) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 7, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3; and d) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 8, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 8. (e) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1617, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1618, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; (g) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1619, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1620, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1620, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; RNA, CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1631, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1; (j) gRNA, comprising CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1957, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1930; (k) gRNA, comprising CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1958, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1931; (l) gRNA, comprising CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1959, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1932; (m) gRNA, comprising CRISPR RNA, CRISPR... The RNA includes a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1960, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1933; (n) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1961, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1934; (o) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1962, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1935; (p) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1963, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1936; (q) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1963, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1936; The RNA includes a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1964, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1937; (r) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1965, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1938; (s) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1966, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1939; (t) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1967, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1940; (u) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1967, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1940; The RNA includes a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1968, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1941; (v) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1969, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1942; (w) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1970, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1943; (x) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1971, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1944; (y) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1971, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1944; The RNA includes a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1972, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1945; (z) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1973, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1946; (aa) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1974, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1947; (bb) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1975, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1948; (cc) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1975, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1948; The RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1976, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1949; (dd)gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1977, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1950; (ee)gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1978, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1951; (ff)gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1979, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1952; (gg)gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1979, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1952; The RNA includes a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1980, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1953; (hh) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1981, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1954; (ii) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1982, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1955; and (jj) gRNA, comprising CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1983, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1956.

118. The vector as described in any one of embodiments 79 to 117, wherein the gRNA comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequences of any one of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658.

119. The vector as described in embodiment 118, wherein the gRNA comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the nucleotide sequences of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671, and 2126 to 2658.

120. The vector as described in embodiment 118 or 119, wherein the gRNA comprises the nucleotide sequence of any one of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658.

121. The vector as described in any of embodiments 79 to 120, wherein the vector is an adeno-associated virus (AAV) vector.

122. The vector as described in embodiment 121, wherein the vector comprises a nucleotide sequence as shown in any of SEQ ID NO: 1679 to 1683.

123. A cell comprising a nucleic acid molecule as described in any of embodiments 1 to 78 or a vector as described in any of embodiments 79 to 122.

124. The cell as described in embodiment 123, wherein the cell is a prokaryotic cell.

125. The cell as described in embodiment 123, wherein the cell is a eukaryotic cell.

126. The cell as described in embodiment 125, wherein the eukaryotic cell is a mammalian cell.

127. The cell as described in embodiment 126, wherein the mammalian cell is a human cell.

128. The cell as described in embodiment 127, wherein the human cell is an immune cell.

129. The cell as described in embodiment 128, wherein the immune cell is a stem cell.

130. The cell as described in embodiment 129, wherein the stem cell is an induced pluripotent stem cell.

131. The cell as described in embodiment 125, wherein the eukaryotic cell is an insect or bird cell.

132. The cell as described in embodiment 125, wherein the eukaryotic cell is a fungal cell.

133. The cell as described in embodiment 125, wherein the eukaryotic cell is a plant cell.

134. A plant or part of a plant comprising cells as described in embodiment 133.

135. A method for producing an RGN polypeptide, comprising culturing cells as described in embodiment 123 under conditions in which the RGN polypeptide is expressed.

136. A method for manufacturing an RGN polypeptide, comprising: introducing a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide into a cell, the RNA-guided nuclease (RGN) polypeptide comprising, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1 The amino acid sequence represented by any one of 087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity; and the cells are cultured under conditions in which the RGN polypeptide is expressed.

137. The method of embodiment 136, wherein the RGN polypeptide comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 10 The amino acid sequence represented by any one of 87, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

138. The method as described in embodiments 136 or 137, wherein the RGN polypeptide comprises, as in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687.

139. The method of any of embodiments 136 to 138, wherein the heterologous nucleic acid molecule comprising the nucleotide sequence of the coding RNA guiding nuclease (RGN) polypeptide comprises mRNA having a heterologous 5' untranslated region (UTR) and/or a heterologous 3' UTR.

140. The method of any one of embodiments 136 to 139, wherein when bound to guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA guide sequence specific manner, wherein the target sequence includes a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence.

141. The method of any of embodiments 136 to 140 further includes purifying the RGN polypeptide.

142. The method of any of embodiments 136 to 141, wherein the cell further expresses one or more guide RNAs capable of binding to the RGN polypeptide to form an RGN ribonucleoprotein complex.

143. The method of embodiment 142 further includes purifying the RGN ribonucleoprotein complex.

144. An RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, The amino acid sequence represented by any one of 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity.

145. The RGN polypeptide as described in embodiment 144, wherein the RGN polypeptide comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, The amino acid sequence represented by any one of 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 95% sequence identity.

146. The RGN polypeptide as described in embodiment 144 or 145, wherein the RGN polypeptide comprises as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687.

147. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein the amino acid residues at one or more of the amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 1.

148. The RGN polypeptide of embodiment 147, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are positively charged amino acid residues.

149. The RGN polypeptide of embodiment 147 or 148, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are arginine (R).

150. The RGN polypeptide of embodiment 149, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of the RGN polypeptide are R.

151. The RGN polypeptide as described in embodiments 149 or 150, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 450; (b) an amino acid sequence as shown in SEQ ID NO: 463; (c) an amino acid sequence as shown in SEQ ID NO: 471; (d) an amino acid sequence as shown in SEQ ID NO: 481; (e) an amino acid sequence as shown in SEQ ID NO: 484; (f) an amino acid sequence as shown in SEQ ID NO: 485; (g) an amino acid sequence as shown in SEQ ID NO: 486; (h) an amino acid sequence as shown in SEQ ID NO: 500; (i) an amino acid sequence as shown in SEQ ID NO: 502; (j) an amino acid sequence as shown in SEQ ID NO: 505; (k) an amino acid sequence as shown in SEQ ID NO: 505; The amino acid sequence shown in SEQ ID NO: 508; (l) the amino acid sequence shown in SEQ ID NO: 539; (m) the amino acid sequence shown in SEQ ID NO: 602; (n) the amino acid sequence shown in SEQ ID NO: 707; (o) the amino acid sequence shown in SEQ ID NO: 708; and (p) the amino acid sequence shown in SEQ ID NO: 717.

152. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein the amino acid residue is arginine (R) at the following combinations of amino acid positions selected from the following combinations of amino acid positions of the RGN polypeptide: (a) amino acid positions 517, 530, 552 and 677; (b) amino acid positions 530, 538, 552 and 801; (c) amino acid positions 530, 552 (d) Amino acid positions 530, 577, and 790; (e) Amino acid positions 517, 530, 538, 552, and 677; (f) Amino acid positions 530, 538, 677, and 801; (g) Amino acid positions 538, 552, 677, and 801; (h) Amino acid positions 517, 530, 538, and 552; (i) Amino acid position 517 and 530; (j) amino acid positions 530, 538 and 552; (k) amino acid positions 517, 530, 538 and 677; (l) amino acid positions 517, 552, 677 and 801; (m) amino acid positions 530, 677 and 801; (n) amino acid positions 517, 530, 677 and 801; (o) amino acid positions 517, 530 and 677; (p) amino acid positions 530, 538, 552 and 677; (q) amino acid positions 517, 552 and 677; and (r) amino acid positions 517, 530, 552, 677 and 801.

153. The RGN polypeptide of embodiment 152, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein the amino acid residue is arginine (R) at the following combinations of amino acid positions selected from the following combinations of amino acid positions of the RGN polypeptide: (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; (c) Amino acid positions 530, 552, 677 and 801; (d) Amino acid positions 530, 577 and 790; (e) Amino acid positions 517, 530, 538, 552 and 677; (f) Amino acid positions 530, 538, 677 and 801; (g) Amino acid positions 538, 552, 677 and 801; (h) Amino acid positions 517, 530, 538 and 552; (i) Amino acid position 517 (j) Amino acid positions 530, 538 and 552; (k) Amino acid positions 517, 530, 538 and 677; (l) Amino acid positions 517, 552, 677 and 801; (m) Amino acid positions 530, 677 and 801; (n) Amino acid positions 517, 530, 677 and 801; (o) Amino acid positions 517, 530 and 677; (p) Amino acid positions 530, 538, 552 and 677; (q) Amino acid positions 517, 552 and 677; and (r) Amino acid positions 517, 530, 552, 677 and 801.

154. The RGN polypeptide of embodiment 152 or 153, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1072; (b) an amino acid sequence as shown in SEQ ID NO: 1080; (c) an amino acid sequence as shown in SEQ ID NO: 1082; (d) an amino acid sequence as shown in SEQ ID NO: 1105; (e) an amino acid sequence as shown in SEQ ID NO: 1084; (f) an amino acid sequence as shown in SEQ ID NO: 1081; (g) an amino acid sequence as shown in SEQ ID NO: 1083; (h) an amino acid sequence as shown in SEQ ID NO: 1069; (i) an amino acid sequence as shown in SEQ ID NO: 1034; (j) an amino acid sequence as shown in SEQ ID NO: 1059; (k) an amino acid sequence as shown in SEQ ID NO: 1082; (v) an amino acid sequence as shown in SEQ ID NO: 1083; (v) an amino acid sequence as shown in SEQ ID NO: 1069; (vi) an amino acid sequence as shown in SEQ ID NO: 1034; (j) an amino acid sequence as shown in SEQ ID NO: 1059; (k) an amino acid sequence as shown in SEQ ID NO: 1083; (v) an amino acid sequence as shown in SEQ ID NO: 1084; (v) an amino acid sequence as shown in SEQ ID NO: 1085 ... The amino acid sequence shown in NO: 1070; (l) the amino acid sequence shown in SEQ ID NO: 1078; (m) the amino acid sequence shown in SEQ ID NO: 1064; (n) the amino acid sequence shown in SEQ ID NO: 1074; (o) the amino acid sequence shown in SEQ ID NO: 1051; (p) the amino acid sequence shown in SEQ ID NO: 1079; (q) the amino acid sequence shown in SEQ ID NO: 1056; and (r) the amino acid sequence shown in SEQ ID NO: 1087.

155. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4.

156. The RGN polypeptide of embodiment 155, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are positively charged amino acid residues.

157. The RGN polypeptide as described in embodiment 155 or 156, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of the RGN polypeptide are R.

158. The RGN polypeptide of embodiment 157, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of the RGN polypeptide are R.

159. The RGN polypeptide of embodiment 157 or 158, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 809; (b) an amino acid sequence as shown in SEQ ID NO: 810; (c) an amino acid sequence as shown in SEQ ID NO: 866; (d) an amino acid sequence as shown in SEQ ID NO: 898; (e) an amino acid sequence as shown in SEQ ID NO: 909; (f) an amino acid sequence as shown in SEQ ID NO: 912; (g) an amino acid sequence as shown in SEQ ID NO: 916; (h) an amino acid sequence as shown in SEQ ID NO: 960; (i) an amino acid sequence as shown in SEQ ID NO: 961; (j) an amino acid sequence as shown in SEQ ID NO: 963; (k) an amino acid sequence as shown in SEQ ID NO: 964; (v) an amino acid sequence as shown in SEQ ID NO: 965; (v) an amino acid sequence as shown in SEQ ID NO: 96 ... The amino acid sequence shown in SEQ ID NO: 966; (l) the amino acid sequence shown in SEQ ID NO: 970; (m) the amino acid sequence shown in SEQ ID NO: 975; (n) the amino acid sequence shown in SEQ ID NO: 1012; and (o) the amino acid sequence shown in SEQ ID NO: 1019.

160. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue is arginine (R) at the following amino acid position combinations selected from the following amino acid position combinations of the RGN polypeptide: (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid position 377, 625 and 691; (g) amino acid positions 377, 625, 698 and 747; (h) amino acid positions 377, 638, 691 and 698; (i) amino acid positions 377, 625 and 638; (j) amino acid positions 377, 638, 698 and 747; (k) amino acid positions 377, 625, 638, 691 and 698; (l) amino acid positions 625, 638 and 698; (m) amino acid positions 377, 625, 691 and 698; (n) amino acid positions 377, 638 and 698; (o) amino acid positions 625, 691, 698 and 747; (p) amino acid positions 638 and 698; and (q) amino acid positions 377 and 691.

161. The RGN polypeptide of embodiment 160, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein at the following combinations of amino acid positions selected from the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino group (g) Amino acid positions 377, 625, and 691; (h) Amino acid positions 377, 638, 691, and 698; (i) Amino acid positions 377, 625, and 638; (j) Amino acid positions 377, 638, 698, and 747; (k) Amino acid positions 377, 625, 638, 691, and 698; (l) Amino acid positions 625, 638, and 698; (m) Amino acid positions 377, 625, 691, and 698; (n) Amino acid positions 377, 638, and 698; (o) Amino acid positions 625, 691, 698, and 747; (p) Amino acid position 638 and 698; and (q) amino acid positions 377 and 691.

162. The RGN polypeptide as described in embodiments 160 or 161, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 1123; (b) an amino acid sequence as shown in SEQ ID NO: 1158; (c) an amino acid sequence as shown in SEQ ID NO: 1159; (d) an amino acid sequence as shown in SEQ ID NO: 1148; (e) an amino acid sequence as shown in SEQ ID NO: 1160; (f) an amino acid sequence as shown in SEQ ID NO: 1122; (g) an amino acid sequence as shown in SEQ ID NO: 1146; (h) an amino acid sequence as shown in SEQ ID NO: 1147; (i) an amino acid sequence as shown in SEQ ID NO: 1121; (j) an amino acid sequence as shown in SEQ ID NO: 1149; (k) an amino acid sequence as shown in SEQ ID NO: 1148; (v) an amino acid sequence as shown in SEQ ID NO: 1149 ... The amino acid sequence shown in NO: 1156; (l) the amino acid sequence shown in SEQ ID NO: 1132; (m) the amino acid sequence shown in SEQ ID NO: 1144; (n) the amino acid sequence shown in SEQ ID NO: 1126; (o) the amino acid sequence shown in SEQ ID NO: 1154; (p) the amino acid sequence shown in SEQ ID NO: 1116; and (q) the amino acid sequence shown in SEQ ID NO: 1108.

163. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4.

164. The RGN polypeptide of embodiment 163, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are positively charged amino acid residues.

165. The RGN polypeptide of embodiment 163 or 164, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein the amino acid residue at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide is R or lysine (K).

166. The RGN polypeptide of embodiment 165, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide is R or K.

167. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein at the amino acid position combinations selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R) and/or lysine (K): (a) amino acid positions 736, 743, 752, 753, 755, 757, 758 and 760; (b) amino acid positions 740, 747, 751, 753, 755, 760, 764 and 766; (c) amino acid positions 685, 691 and 698; and (d) amino acid positions 685, 692, 695, 702 and 707.

168. The RGN polypeptide of embodiment 167, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein at the amino acid positions selected from the following combinations of amino acid positions of the RGN polypeptide, the amino acid residue is arginine (R) and/or lysine (K): (a) amino acid positions 736, 743, 752, 753, 755, 757, 758, and 760; (b) amino acid positions 740, 747, 751, 753, 755, 760, 764, and 766; (c) amino acid positions 685, 691, and 698; and (d) amino acid positions 685, 692, 695, 702, and 707.

169. The RGN polypeptide of embodiment 167 or 168, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1168; (b) an amino acid sequence as shown in SEQ ID NO: 1171; (c) an amino acid sequence as shown in SEQ ID NO: 1169; and (d) an amino acid sequence as shown in SEQ ID NO: 1170.

170. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the RGN polypeptide comprises the deletion of amino acid residues corresponding to amino acid residues 751 to 769 of SEQ ID NO: 4.

171. The RGN polypeptide of embodiment 170, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the RGN polypeptide comprises the deletion of amino acid residues corresponding to amino acid residues 751 to 769 of SEQ ID NO: 4.

172. The RGN polypeptide as described in embodiment 170 or 171, wherein the RGN polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1173.

173. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more of the amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596 and 862 of the RGN polypeptide comprises substitution or insertion of one or more glycine (G) in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position.

174. The RGN polypeptide of embodiment 173, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more of the amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596, and 862 of the RGN polypeptide comprises one or more substitutions or insertions of G in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position.

175. The RGN polypeptide as described in embodiments 173 or 174, wherein the substitution or insertion of one or more Gs comprises the substitution or insertion of one, two, or three Gs.

176. The RGN polypeptide of embodiment 173, wherein the substitution or insertion of one or more Gs is selected from: (a) the insertion of three Gs between amino acid positions 293 and 294; (b) the insertion of two Gs between amino acid positions 359 and 360; (c) the substitution of G at amino acid position 361; (d) the insertion of one G between amino acid positions 402 and 403; (e) the substitution of G at amino acid position 401; (f) the substitution of G at amino acid position 596; and (g) the substitution of G at amino acid position 862.

177. The RGN polypeptide of embodiment 176, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) the amino acid sequence shown in SEQ ID NO: 1304; (b) the amino acid sequence shown in SEQ ID NO: 1322; (c) the amino acid sequence shown in SEQ ID NO: 1324; (d) the amino acid sequence shown in SEQ ID NO: 1328; (e) the amino acid sequence shown in SEQ ID NO: 1330; (f) the amino acid sequence shown in SEQ ID NO: 1361; and (g) the amino acid sequence shown in SEQ ID NO: 1400.

178. The RGN polypeptide of embodiment 144, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more of the amino acid positions 566, 567, and 584 of the RGN polypeptide comprises one or more substitutions or insertions of G in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position.

179. The RGN polypeptide of embodiment 178, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more of the amino acid positions 566, 567, and 584 of the RGN polypeptide comprises one or more substitutions or insertions of G in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position.

180. The RGN polypeptide as described in embodiments 178 or 179, wherein the substitution or insertion of one or more Gs comprises the substitution or insertion of one, two, or three Gs.

181. The RGN polypeptide as described in embodiment 178, wherein the substitution or insertion of one or more Gs is selected from: (a) substitution of G at amino acid position 566 and substitution of G at amino acid position 567; and (b) substitution of G at amino acid position 584.

182. The RGN polypeptide of embodiment 181, wherein the RGN polypeptide comprises an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1223; and (b) an amino acid sequence as shown in SEQ ID NO: 1228.

183. The RGN polypeptide as described in any of embodiments 144 to 182, wherein the RGN polypeptide is operatively fused with a heterologous polypeptide.

184. The RGN polypeptide as described in embodiment 183, wherein the heterologous polypeptide includes a base-editing polypeptide, a polymerase-editing polypeptide, an effector domain, an expression regulator, a detectable tracer, or a purified tracer.

185. The RGN polypeptide as described in embodiment 184, wherein the heterologous polypeptide includes a base-editing polypeptide.

186. The RGN polypeptide as described in embodiment 185, wherein the base-editing polypeptide includes a deaminerase.

187. The RGN polypeptide as described in embodiment 184, wherein the polymerase editing polypeptide comprises DNA polymerase.

188. The RGN polypeptide as described in embodiment 184, wherein the polymerase-editing polypeptide comprises reverse transcriptase.

189. The RGN polypeptide as described in embodiment 184, wherein the heterologous polypeptide includes an effector domain.

190. The RGN polypeptide as described in embodiment 189, wherein the effector domain is operatively fused at the N-terminus or C-terminus of the RGN polypeptide.

191. The RGN polypeptide as described in embodiment 189, wherein the effector subdomain is operatively fused to an internal address of the RGN polypeptide.

192. The RGN polypeptide as described in any of embodiments 189 to 191, wherein the effector domain includes a cleavage domain, a deaminase domain, or a performance regulation domain.

193. The RGN polypeptide as described in embodiment 192, wherein the expression regulatory domain includes a transcriptional activation domain, a transcriptional repression domain, or an epigenetic modification domain.

194. The RGN polypeptide as described in any one of embodiments 144 to 193, wherein, when bound to guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA guide sequence-specific manner, wherein the target sequence includes a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence.

195. The RGN polypeptide of embodiment 194, wherein the target sequence is disposed adjacent to and disposed at the 3' of the original spacer adjacent motif (PAM).

196. The RGN polypeptide of embodiment 195, wherein a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 recognizes a PAM having a common nucleotide sequence shown in AYG, wherein the PAM is adjacent to and at its 5' of the target sequence; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 1933 or 1944 recognizes a PAM having a common nucleotide sequence shown in ATTN, wherein the PAM is adjacent to and at its 5' of the target sequence; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947 or 1949 recognizes a PAM having a common nucleotide sequence shown in VTTN, wherein the PAM is adjacent to and at its 5' of the target sequence; and d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4) RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is located at its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1937 or 1950 identify PAMs having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1951 identify PAMs having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1953 identify PAMs having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'.

197. The RGN polypeptide as described in any one of embodiments 194 to 196, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding.

198. The RGN polypeptide as described in embodiment 197, wherein the cleavage of the RGN polypeptide results in double strand breakage.

199. The RGN polypeptide as described in any one of embodiments 144 to 196, wherein the RGN polypeptide is nuclease-inactivating.

200. The RGN polypeptide of embodiment 199, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 408, 597, 678, 827, 896, 979, 2686, and 2687.

201. The RGN polypeptide as described in embodiments 199 or 200, wherein the RGN polypeptide is operatively fused with a base-editing polypeptide.

202. The RGN polypeptide as described in embodiment 1201, wherein the base-editing polypeptide includes a deaminerase.

203. The RGN polypeptide as described in embodiments 199 or 200, wherein the RGN polypeptide is operatively fused with a polymerase-editing polypeptide.

204. The RGN polypeptide as described in embodiment 203, wherein the polymerase editing polypeptide comprises DNA polymerase.

205. The RGN polypeptide as described in embodiment 203, wherein the polymerase editing polypeptide comprises reverse transcriptase.

206. The RGN polypeptide as described in any of embodiments 144 to 205, wherein the RGN polypeptide includes one or more nuclear localization signals (NLS).

207. The RGN polypeptide of embodiment 206, wherein one or more NLS comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any of SEQ ID NO: 33, 35, 407, and 1927.

208. The RGN polypeptide as described in any of embodiments 144 to 207, wherein the RGN polypeptide is an isolated RGN polypeptide.

209. A ribonucleoprotein (RNP) complex comprising an RGN polypeptide as described in any one of embodiments 144 to 208 and a guide RNA bound to the RGN polypeptide.

210. The RNP complex as described in embodiment 209, wherein the guide RNA and the RGN polypeptide have not been found to be complexed with each other in nature.

211. The RNP complex as described in embodiments 209 or 210, wherein the guiding RNA comprises a spacer hybridized with a eukaryotic target sequence.

212. The RNP complex as described in embodiment 211, wherein the eukaryotic target sequence comprises the mammalian target sequence.

213. The RNP complex as described in any of embodiments 209 to 212, wherein the guiding RNA does not include tracrRNA.

214. An RNP complex as described in any of embodiments 209 to 213, wherein the guiding RNA has a total length of 35 to 250 nucleotides (nt), or 35 to 170 nt, or 35 to 125 nt, or 40 to 100 nt, or 40 to 70 nt, or 40 to 60 nt.

215. An RNP complex as described in any one of embodiments 209 to 213, wherein the guiding RNA has a maximum of 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt nt, 83 nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 160 nt, 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt, 180 nt, 190 nt, 200 nt, 205 nt, 210 nt, 215 nt, 220 nt, 225 nt, 230 nt, 235 nt, 240 nt, 245 nt or 250 nt total length.

216. An RNP complex as described in any of embodiments 209 to 213, wherein the guiding RNA comprises a crRNA backbone having a total length of up to 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt.

217. The RNP complex as described in any of embodiments 209 to 216, wherein the guiding RNA comprises at least one chemical modification.

218. The RNP complex as described in embodiment 217, wherein the at least one chemical modification comprises: bridging nucleic acid (BNA) modification; 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluorine (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and thiophosphate (PS) modification.

219. The RNP complex as described in embodiment 218, wherein the BNA comprises 2′,4′ BNA modification.

220. The RNP complex as described in embodiment 219, wherein the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA ^NC [N-Me] modification, 2′-O,4′-C-ethylene bridging nucleic acid (2′,4′-ENA) modification and S-restricted ethyl (cEt) modification.

221. The RNP complex as described in embodiment 220, wherein the 2′,4′ BNA is an LNA modification.

222. The RNP complex as described in embodiment 220, wherein the 2′,4′ BNA is cEt modified.

223. The RNP complex as described in embodiments 217 or 218, wherein at least one chemical modification includes BNA modification, 2'-O-Me modification or PS modification.

224. A guide RNA (gRNA) comprising CRISPR RNA (crRNA), wherein the crRNA comprises a crRNA backbone and spacers, wherein the crRNA backbone is selected from the group consisting of: a) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides; b) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides; c) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 7 or differing from SEQ ID NO: 7 by 1 to 5 nucleotides; d) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 8 or differing from SEQ ID NO: 8 by 1 to 5 nucleotides; (e) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1617 or differing from SEQ ID NO: 1617 by 1 to 5 nucleotides; 1617 has a nucleotide sequence that differs by 1 to 5 nucleotides; (f) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1618 or differing from SEQ ID NO: 1618 by 1 to 5 nucleotides; (g) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1619 or differing from SEQ ID NO: 1619 by 1 to 5 nucleotides; (h) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1620 by 1 to 5 nucleotides; (i) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1631 or differing from SEQ ID NO: 1631 by 1 to 5 nucleotides; (j) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1957 or differing from SEQ ID NO: 1957 by 1 to 5 nucleotides; (k) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1617 or differing from SEQ ID NO: 1957 by 1 to 5 nucleotides; The following are considered as follows: (1) a nucleotide sequence as shown in SEQ ID NO: 1958 or differing from SEQ ID NO: 1958 by 1 to 5 nucleotides; (l) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1959 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides; (m) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1960 or differing from SEQ ID NO: 1960 by 1 to 5 nucleotides; (n) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1961 or differing from SEQ ID NO: 1961 by 1 to 5 nucleotides; (o) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1962 or differing from SEQ ID NO: 1962 by 1 to 5 nucleotides; (p) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1963 or differing from SEQ ID NO: 1958 by 1 to 5 nucleotides; 1963 has a nucleotide sequence that differs by 1 to 5 nucleotides; (q) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1964 or differing from SEQ ID NO: 1964 by 1 to 5 nucleotides; (r) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1965 or differing from SEQ ID NO: 1965 by 1 to 5 nucleotides; (s) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1966 or differing from SEQ ID NO: 1966 by 1 to 5 nucleotides; (t) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1967 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides; (u) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1968 or differing from SEQ ID NO: 1968 by 1 to 5 nucleotides; (v) crRNA backbone, including a nucleotide sequence as shown in SEQ ID NO: 1963; (w) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1970 or differing from SEQ ID NO: 1970 by 1 to 5 nucleotides; (x) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1971 or differing from SEQ ID NO: 1971 by 1 to 5 nucleotides; (y) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1972 or differing from SEQ ID NO: 1972 by 1 to 5 nucleotides; (z) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1973 or differing from SEQ ID NO: 1973 by 1 to 5 nucleotides; (aa) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1974 or differing from SEQ ID NO: 1975 by 1 to 5 nucleotides; 1974 has nucleotide sequences that differ by 1 to 5 nucleotides; (bb) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1975 or differing from SEQ ID NO: 1975 by 1 to 5 nucleotides; (cc) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1976 or differing from SEQ ID NO: 1976 by 1 to 5 nucleotides; (dd) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1977 or differing from SEQ ID NO: 1977 by 1 to 5 nucleotides; (ee) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1978 or differing from SEQ ID NO: 1978 by 1 to 5 nucleotides; (ff) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1979 or differing from SEQ ID NO: 1979 by 1 to 5 nucleotides; (gg) crRNA backbone, including nucleotide sequences as shown in SEQ ID NO: 1979 by 1 to 5 nucleotides; The following are included: (i) a nucleotide sequence as shown in SEQ ID NO: 1980 or differing from SEQ ID NO: 1980 by 1 to 5 nucleotides; (ii) a crRNA backbone as shown in SEQ ID NO: 1981 or differing from SEQ ID NO: 1981 by 1 to 5 nucleotides; (ii) a crRNA backbone as shown in SEQ ID NO: 1982 or differing from SEQ ID NO: 1982 by 1 to 5 nucleotides; and (jj) a crRNA backbone as shown in SEQ ID NO: 1983 or differing from SEQ ID NO: 1983 by 1 to 5 nucleotides.

225. The gRNA of embodiment 224, wherein the crRNA backbone is selected from the group consisting of: (a) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 5 nucleotides; (b) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 5 nucleotides; (c) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 5 nucleotides; (d) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 8 by 5 nucleotides; (e) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1617 by 5 nucleotides; (f) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1618 by 5 nucleotides; (g) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by 5 nucleotides; (h) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 5 nucleotides; (v ... SEQ ID NO: 1620 has a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1957; (i) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1958; (l) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1959; (m) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1960; (n) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1961; (o) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1962; (p) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1962. 1963 has a nucleotide sequence that differs from SEQ ID NO: 1964 by 5 nucleotides; (q) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 5 nucleotides; (r) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 5 nucleotides; (s) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1966 by 5 nucleotides; (t) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1967 by 5 nucleotides; (u) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1968 by 5 nucleotides; (v) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1969 by 5 nucleotides; (w) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1970 by 5 nucleotides; (x) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1964 by 5 nucleotides; 1971 has a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides; (y) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides; (z) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1973 by 5 nucleotides; (aa) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1974 by 5 nucleotides; (bb) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1975 by 5 nucleotides; (cc) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1976 by 5 nucleotides; (dd) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1977 by 5 nucleotides; (ee) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1978 by 5 nucleotides; (ff) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides; 1979 has a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1980; (gg) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1981; (ii) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1982; and (jj) crRNA backbone, including a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1983.

226. The gRNA of embodiment 224, wherein the crRNA backbone is selected from the group consisting of: (a) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 4 nucleotides; (b) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 4 nucleotides; (c) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 4 nucleotides; (d) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 8 by 4 nucleotides; (e) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1617 by 4 nucleotides; (f) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1618 by 4 nucleotides; (g) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by 4 nucleotides; (h) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by 4 nucleotides; and (v ... SEQ ID NO: 1620 has a nucleotide sequence that differs from SEQ ID NO: 1957 by 4 nucleotides; (i) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1958 by 4 nucleotides; (j) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1959 by 4 nucleotides; (m) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1960 by 4 nucleotides; (n) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1961 by 4 nucleotides; (o) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1962 by 4 nucleotides; (p) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1962 by 4 nucleotides; 1963 has a nucleotide sequence that differs from SEQ ID NO: 1964 by 4 nucleotides; (q) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 4 nucleotides; (r) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 4 nucleotides; (s) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1966 by 4 nucleotides; (t) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1967 by 4 nucleotides; (u) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1968 by 4 nucleotides; (v) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1969 by 4 nucleotides; (w) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1970 by 4 nucleotides; (x) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1964 by 4 nucleotides; 1971 has a nucleotide sequence that differs from SEQ ID NO: 1972 by 4 nucleotides; (y) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1973 by 4 nucleotides; (z) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1973 by 4 nucleotides; (aa) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1974 by 4 nucleotides; (bb) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1975 by 4 nucleotides; (cc) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1976 by 4 nucleotides; (dd) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1977 by 4 nucleotides; (ee) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1978 by 4 nucleotides; (ff) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1972 by 4 nucleotides; 1979 has a nucleotide sequence that is 4 nucleotides different from SEQ ID NO: 1980; (gg)crRNA backbone, including a nucleotide sequence that is 4 nucleotides different from SEQ ID NO: 1981; (ii)crRNA backbone, including a nucleotide sequence that is 4 nucleotides different from SEQ ID NO: 1982; and (jj)crRNA backbone, including a nucleotide sequence that is 4 nucleotides different from SEQ ID NO: 1983.

227. The gRNA of embodiment 224, wherein the crRNA backbone is selected from the group consisting of: a) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 3 nucleotides; b) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 3 nucleotides; c) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 3 nucleotides; d) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 8 by 3 nucleotides; (e) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1617 by 3 nucleotides; (f) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1618 by 3 nucleotides; (g) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by 3 nucleotides; (h) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 3 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 3 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 3 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 8 by 3 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 9 ... SEQ ID NO: 1620 has a nucleotide sequence that differs from SEQ ID NO: 1957 by 3 nucleotides; (i) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1958 by 3 nucleotides; (j) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1959 by 3 nucleotides; (m) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1960 by 3 nucleotides; (n) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1961 by 3 nucleotides; (o) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1962 by 3 nucleotides; (p) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1962 by 3 nucleotides; 1963 has a nucleotide sequence that differs from SEQ ID NO: 1964 by 3 nucleotides; (q) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 3 nucleotides; (s) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1966 by 3 nucleotides; (t) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1967 by 3 nucleotides; (u) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1968 by 3 nucleotides; (v) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1969 by 3 nucleotides; (w) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1970 by 3 nucleotides; (x) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1964 by 3 nucleotides; 1971 has a nucleotide sequence that differs from SEQ ID NO: 1972 by 3 nucleotides; (y) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1973 by 3 nucleotides; (z) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1973 by 3 nucleotides; (aa) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1974 by 3 nucleotides; (bb) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1975 by 3 nucleotides; (cc) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1976 by 3 nucleotides; (dd) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1977 by 3 nucleotides; (ee) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1978 by 3 nucleotides; (ff) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1972 by 3 nucleotides; 1979 has a nucleotide sequence that is 3 nucleotides different from SEQ ID NO: 1980; (gg) crRNA backbone, including a nucleotide sequence that is 3 nucleotides different from SEQ ID NO: 1981; (ii) crRNA backbone, including a nucleotide sequence that is 3 nucleotides different from SEQ ID NO: 1982; and (jj) crRNA backbone, including a nucleotide sequence that is 3 nucleotides different from SEQ ID NO: 1983.

228. The gRNA of embodiment 224, wherein the crRNA backbone is selected from the group consisting of: (a) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 2 nucleotides; (b) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 2 nucleotides; (c) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 2 nucleotides; (d) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 8 by 2 nucleotides; (e) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1617 by 2 nucleotides; (f) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1618 by 2 nucleotides; (g) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by 2 nucleotides; (h) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 2 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 2 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 2 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 8 by 2 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 9 ... SEQ ID NO: 1620 has a nucleotide sequence that differs from SEQ ID NO: 1957 by 2 nucleotides; (i) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1958 by 2 nucleotides; (j) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1959 by 2 nucleotides; (m) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1960 by 2 nucleotides; (n) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1961 by 2 nucleotides; (o) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1962 by 2 nucleotides; (p) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1962 by 2 nucleotides; 1963 has a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides; (q) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides; (r) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides; (s) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1966 by 2 nucleotides; (t) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1967 by 2 nucleotides; (u) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1968 by 2 nucleotides; (v) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1969 by 2 nucleotides; (w) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1970 by 2 nucleotides; (x) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides; 1971 has a nucleotide sequence that differs from SEQ ID NO: 1972 by 2 nucleotides; (z) crRNA backbone, including SEQ ID NO: 1973 by 2 nucleotides; (aa) crRNA backbone, including SEQ ID NO: 1974 by 2 nucleotides; (bb) crRNA backbone, including SEQ ID NO: 1975 by 2 nucleotides; (cc) crRNA backbone, including SEQ ID NO: 1976 by 2 nucleotides; (dd) crRNA backbone, including SEQ ID NO: 1977 by 2 nucleotides; (ee) crRNA backbone, including SEQ ID NO: 1978 by 2 nucleotides; (ff) crRNA backbone, including SEQ ID NO: 1979 has a nucleotide sequence that differs from SEQ ID NO: 1980 by 2 nucleotides; (gg)crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1981 by 2 nucleotides; (ii)crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1982 by 2 nucleotides; and (jj)crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1983 by 2 nucleotides.

229. The gRNA of embodiment 224, wherein the crRNA backbone is selected from the group consisting of: (a) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by one nucleotide; (b) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by one nucleotide; (c) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by one nucleotide; (d) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 8 by one nucleotide; (e) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1617 by one nucleotide; (f) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1618 by one nucleotide; (g) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by one nucleotide; (h) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by one nucleotide; (c) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by one nucleotide; (d) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by one nucleotide; (e) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 8 by one nucleotide; (f) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1618 by one nucleotide; (g) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by one nucleotide; (h) a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by one nucleotide; (g ... SEQ ID NO: 1620 has a nucleotide sequence that differs from SEQ ID NO: 1957 by one nucleotide; (i) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1958 by one nucleotide; (j) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1959 by one nucleotide; (m) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1960 by one nucleotide; (n) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1961 by one nucleotide; (o) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1962 by one nucleotide; (p) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1962 by one nucleotide; 1963 has a nucleotide sequence that differs from SEQ ID NO: 1964 by 1 nucleotide; (q) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 1 nucleotide; (r) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1965 by 1 nucleotide; (s) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1966 by 1 nucleotide; (t) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1967 by 1 nucleotide; (u) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1968 by 1 nucleotide; (v) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1969 by 1 nucleotide; (w) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1970 by 1 nucleotide; (x) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1964 by 1 nucleotide; 1971 has a nucleotide sequence that differs from SEQ ID NO: 1972 by 1 nucleotide; (y) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1972 by 1 nucleotide; (z) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1973 by 1 nucleotide; (aa) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1974 by 1 nucleotide; (bb) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1975 by 1 nucleotide; (cc) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1976 by 1 nucleotide; (dd) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1977 by 1 nucleotide; (ee) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1978 by 1 nucleotide; (ff) crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1972 by 1 nucleotide; 1979 has a nucleotide sequence that is 1 nucleotide different from SEQ ID NO: 1980; (gg) crRNA backbone, including a nucleotide sequence that is 1 nucleotide different from SEQ ID NO: 1981; (ii) crRNA backbone, including a nucleotide sequence that is 1 nucleotide different from SEQ ID NO: 1982; and (jj) crRNA backbone, including a nucleotide sequence that is 1 nucleotide different from SEQ ID NO: 1983.

230. The gRNA of embodiment 224, wherein the crRNA backbone is selected from the group consisting of: (a) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 5; (b) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 6; (c) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 7; (d) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 8; (e) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1617; (f) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1618; (g) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1619; (h) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1620; (i) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1620; The nucleotide sequence shown in SEQ ID NO: 1931; (j) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1957; (k) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1958; (l) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1959; (m) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1960; (n) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1961; (o) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1962; (p) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1963; (q) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1964; (r) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1964; The nucleotide sequence shown in SEQ ID NO: 1965; (s) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1966; (t) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1967; (u) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1968; (v) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1969; (w) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1970; (x) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1971; (y) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1972; (z) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1973; (aa) crRNA main chain, including the ...t) crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 196 The nucleotide sequence shown in SEQ ID NO: 1974; (bb)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1975; (cc)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1976; (dd)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1977; (ee)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1978; (ff)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1979; (gg)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1980; (hh)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1981; (ii)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1982; and (jj)crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1983.

231. The gRNA as described in any of embodiments 224 to 230, wherein the gRNA does not include tracrRNA.

232. The gRNA as described in any of embodiments 224 to 230, wherein the gRNA is composed of cRNA.

233. The gRNA as described in any of embodiments 224 to 232, wherein the gRNA has a total length of 35 to 250 nucleotides (nt), or 35 to 170 nt, or 35 to 125 nt, or 40 to 100 nt, or 40 to 70 nt, or 40 to 60 nt.

234. The gRNA as described in any one of embodiments 224 to 232, wherein the gRNA has at most 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt, 83 nt nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 160 nt, 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt, 180 nt, 190 nt, 200 nt, 205 nt, 210 nt, 215 nt, 220 nt, 225 nt, 230 nt, 235 nt, 240 nt, 245 nt or 250 nt total length.

235. The gRNA as described in any of embodiments 224 to 232, wherein the gRNA has a crRNA backbone having a total length of up to 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt or 40 nt.

236. The gRNA as described in any one of embodiments 224 to 235, wherein the spacer comprises a nucleotide sequence as shown in any one of SEQ ID NO: 294 to 396, 1789 to 1796, 1798 to 1880, 1890 to 1893 and 2082 to 2125.

237. The gRNA as described in any one of embodiments 224 to 236, wherein the crRNA comprises the nucleotide sequence shown in any one of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658.

238. The gRNA as described in any of embodiments 224 to 237, wherein the gRNA includes at least one chemical modification.

239. The gRNA as described in embodiment 238, wherein at least one chemical modification comprises: bridging nucleic acid (BNA) modification; 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluorine (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and thiophosphate (PS) modification.

240. The gRNA as described in embodiment 239, wherein the BNA includes 2′,4′ BNA modification.

241. The gRNA as described in embodiment 240, wherein the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA ^NC [N-Me] modification, 2′-O,4′-C-ethylene bridging nucleic acid (2′,4′-ENA) modification and S-restricted ethyl (cEt) modification.

242. The gRNA as described in embodiment 241, wherein 2′,4′ BNA is LNA modified.

243. The gRNA as described in embodiment 241, wherein the 2′,4′ BNA is cEt modified.

244. The gRNA as described in embodiments 238 or 239, wherein at least one chemical modification includes BNA modification, 2'-O-Me modification or PS modification.

245. The gRNA as described in any of embodiments 224 to 244, wherein when the gRNA binds to an RNA-guided nuclease (RGN) polypeptide, the gRNA is capable of hybridizing with a target strand of a target sequence in a target nucleic acid molecule in a sequence-specific manner via a spacer of the crRNA.

246. The gRNA of embodiment 245, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides; b) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides; c) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 7 or differing from SEQ ID NO: 7 by 1 to 5 nucleotides; d) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 3 or differing from SEQ ID NO: 7 by 1 to 5 nucleotides; The amino acid sequence shown in SEQ ID NO: 4 has an amino acid sequence with at least 90% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 8 or differs from SEQ ID NO: 8 by 1 to 5 nucleotides; (e) an RGN polypeptide comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1617 or differs from SEQ ID NO: 1617 by 1 to 5 nucleotides; (f) an RGN polypeptide comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1618 or differs from SEQ ID NO: 1618 by 1 to 5 nucleotides; (g) an RGN polypeptide comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1618 or differs from SEQ ID NO: 1618 by 1 to 5 nucleotides; (i) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1619 or having 1 to 5 nucleotide differences from SEQ ID NO: 1619; (h) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1620 or having 1 to 5 nucleotide differences from SEQ ID NO: 1620; (i) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1631 or having 1 to 5 nucleotide differences from SEQ ID NO: 1631; (j) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1930, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1957 or having 1 to 5 nucleotide differences from SEQ ID NO: 1630. (k) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1958 or having 1 to 5 nucleotide differences from SEQ ID NO: 1958; (l) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1959 or having 1 to 5 nucleotide differences from SEQ ID NO: 1959; (m) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1960 or having 1 to 5 nucleotide differences from SEQ ID NO: 1959. (n) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1961 or having 1 to 5 nucleotide differences from SEQ ID NO: 1961; (o) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1962 or having 1 to 5 nucleotide differences from SEQ ID NO: 1962; (p) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1963 or having 1 to 5 nucleotide differences from SEQ ID NO: 1962. 1963 has a nucleotide sequence that differs from SEQ ID NO: 1964 by 1 to 5 nucleotides; (q) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1964 or having 1 to 5 nucleotide differences from SEQ ID NO: 1964; (r) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1965 or having 1 to 5 nucleotide differences from SEQ ID NO: 1965; (s) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1939, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1966 or having 1 to 5 nucleotide differences from SEQ ID NO: 1965. 1966 has a nucleotide sequence that differs from SEQ ID NO: 1967 by 1 to 5 nucleotides; (t) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1967 or having 1 to 5 nucleotide differences from SEQ ID NO: 1967; (u) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1968 or having 1 to 5 nucleotide differences from SEQ ID NO: 1968; (v) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1969 or having 1 to 5 nucleotide differences from SEQ ID NO: 1968. (w) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1970 or having 1 to 5 nucleotide differences from SEQ ID NO: 1970; (x) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1971 or having 1 to 5 nucleotide differences from SEQ ID NO: 1971; (y) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1945, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1972 or having 1 to 5 nucleotide differences from SEQ ID NO: 1971. (z) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1973 or having 1 to 5 nucleotide differences from SEQ ID NO: 1973; (aa) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1974 or having 1 to 5 nucleotide differences from SEQ ID NO: 1974; (bb) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1975 or having 1 to 5 nucleotide differences from SEQ ID NO: 1974. 1975 has a nucleotide sequence that differs from SEQ ID NO: 1976 by 1 to 5 nucleotides; (cc) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1976 or having 1 to 5 nucleotide differences from SEQ ID NO: 1976; (dd) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1977 or having 1 to 5 nucleotide differences from SEQ ID NO: 1977; (ee) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1978 or having 1 to 5 nucleotide differences from SEQ ID NO: 1977. 1978 has a nucleotide sequence that differs from SEQ ID NO: 1979 by 1 to 5 nucleotides; (ff) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1979 or having 1 to 5 nucleotide differences from SEQ ID NO: 1979; (gg) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1980 or having 1 to 5 nucleotide differences from SEQ ID NO: 1980; (hh) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1981 or having 1 to 5 nucleotide differences from SEQ ID NO: 1979. (ii) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1982 or having 1 to 5 nucleotide differences from SEQ ID NO: 1982; and (jj) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1956, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1983 or having 1 to 5 nucleotide differences from SEQ ID NO: 1983.

247. The gRNA of embodiment 246, wherein the RGN is selected from the group consisting of: a) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by 5 nucleotides; b) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 6 by 5 nucleotides; c) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 7 by 5 nucleotides; d) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by 5 nucleotides; (e) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 5 nucleotide differences from SEQ ID NO: 1617; (f) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 5 nucleotide differences from SEQ ID NO: 1618; (g) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 5 nucleotide differences from SEQ ID NO: 1619; (h) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 5 nucleotide differences from SEQ ID NO: 1619; 1620 has a nucleotide sequence that is 5 nucleotides different; (i) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1; (j) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1930, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1957; (k) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1958; (l) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1932; (m) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein the crRNA backbone comprises a nucleotide sequence having at least 5 nucleotide differences from the amino acid sequence of SEQ ID NO: 1960; (n) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence having at least 5 nucleotide differences from the amino acid sequence of SEQ ID NO: 1961; (o) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence having at least 5 nucleotide differences from the amino acid sequence of SEQ ID NO: 1962; (p) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933; NO: 1963 has a nucleotide sequence that is 5 nucleotides different; (q) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1964; (r) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1965; (s) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1939, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1966; (t) RGN polypeptide, comprising an amino acid sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1964; The following amino acid sequences are included: (u) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1967 by 5 nucleotides; (v) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1968 by 5 nucleotides; (v) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1969 by 5 nucleotides; (w) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1970 by 5 nucleotides; (x) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1970 by 5 nucleotides; NO: 1944 has an amino acid sequence with at least 90% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1971 by 5 nucleotides; (y) RGN polypeptide, comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1945, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides; (z) RGN polypeptide, comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1946, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1973 by 5 nucleotides; (aa) RGN polypeptide, comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1947, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1944 by 5 nucleotides; SEQ ID NO: 1974 has a nucleotide sequence that is 5 nucleotides different; (bb) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1975; (cc) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1976; (dd) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1977; (ee) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1977; NO: 1978 has a nucleotide sequence that is 5 nucleotides different; (ff) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1979; (gg) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1980; (hh) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein the crRNA backbone comprises a nucleotide sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1981; (ii) RGN polypeptide, comprising an amino acid sequence that is 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1978; The amino acid sequence of 1955 has an amino acid sequence with at least 90% sequence identity, wherein the crRNA backbone includes a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1982; and the (jj)RGN polypeptide includes an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1956, wherein the crRNA backbone includes a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1983.

248. The gRNA of embodiment 246, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by 4 nucleotides; b) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 6 by 4 nucleotides; c) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 7 by 4 nucleotides; d) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by 4 nucleotides; (e) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having at least 4 nucleotide differences from SEQ ID NO: 1617; (f) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having at least 4 nucleotide differences from SEQ ID NO: 1618; (g) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having at least 4 nucleotide differences from SEQ ID NO: 1619; (h) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having at least 4 nucleotide differences from SEQ ID NO: 1619; 1620 has a nucleotide sequence that is 4 nucleotides different; (i) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1; (j) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1930, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1957; (k) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1958; (l) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1932; SEQ ID NO: 1959 has a nucleotide sequence that is 4 nucleotides different; (m) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1960; (n) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1961; (o) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1962; (p) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1959; NO: 1963 has a nucleotide sequence that is 4 nucleotides different; (q) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different with SEQ ID NO: 1964; (r) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different with SEQ ID NO: 1965; (s) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1939, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different with SEQ ID NO: 1966; (t) RGN polypeptide, comprising an amino acid sequence that is 4 nucleotides different with SEQ ID NO: 1964; The following amino acid sequences are included: (u) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1967 by 4 nucleotides; (v) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1969 by 4 nucleotides; (w) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1970 by 4 nucleotides; and (x) an RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1970 by 4 nucleotides. NO: 1944 has an amino acid sequence with at least 90% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1971 by 4 nucleotides; (y) RGN polypeptide, comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1945, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1972 by 4 nucleotides; (z) RGN polypeptide, comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1946, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1973 by 4 nucleotides; (aa) RGN polypeptide, comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1947, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1944 by 4 nucleotides; SEQ ID NO: 1974 has a nucleotide sequence that is 4 nucleotides different; (bb) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1975; (cc) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1976; (dd) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1977; (ee) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1977; NO: 1978 has a nucleotide sequence that is 4 nucleotides different; (ff) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1979; (gg) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1980; (hh) RGN polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein the crRNA backbone comprises a nucleotide sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1981; (ii) RGN polypeptide, comprising an amino acid sequence that is 4 nucleotides different from the amino acid sequence of SEQ ID NO: 1978; The amino acid sequence of 1955 has an amino acid sequence with at least 90% sequence identity, wherein the crRNA backbone includes a nucleotide sequence that is 4 nucleotides different from SEQ ID NO: 1982; and the (jj)RGN polypeptide includes an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1956, wherein the crRNA backbone includes a nucleotide sequence that is 4 nucleotides different from SEQ ID NO: 1983.

249. The gRNA of embodiment 246, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by 3 nucleotides; b) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 6 by 3 nucleotides; c) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 7 by 3 nucleotides; d) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by 3 nucleotides; (e) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 3 different nucleotides from SEQ ID NO: 1617; (f) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 3 different nucleotides from SEQ ID NO: 1618; (g) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 3 different nucleotides from SEQ ID NO: 1619; (h) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 3 different nucleotides from SEQ ID NO: 1619. 1620 has a nucleotide sequence that is 3 nucleotides different; (i) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence that is 3 nucleotides different from the amino acid sequence of SEQ ID NO: 1; (j) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1930, wherein the crRNA backbone comprises a nucleotide sequence that is 3 nucleotides different from the amino acid sequence of SEQ ID NO: 1957; (k) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein the crRNA backbone comprises a nucleotide sequence that is 3 nucleotides different from the amino acid sequence of SEQ ID NO: 1958; (l) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein the crRNA backbone comprises a nucleotide sequence that is 3 nucleotides different from the amino acid sequence of SEQ ID NO: 1932; (m) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1960; (n) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1961; (o) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1962; (p) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide ...3, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935 NO: 1963 has a nucleotide sequence that differs by 3 nucleotides; (q) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein the crRNA backbone comprises a nucleotide sequence that differs by 3 nucleotides with the amino acid sequence of SEQ ID NO: 1964; (r) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein the crRNA backbone comprises a nucleotide sequence that differs by 3 nucleotides with the amino acid sequence of SEQ ID NO: 1965; (s) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1939, wherein the crRNA backbone comprises a nucleotide sequence that differs by 3 nucleotides with the amino acid sequence of SEQ ID NO: 1966; (t) RGN polypeptide, comprising an amino acid sequence that differs by 3 nucleotides with the amino acid sequence of SEQ ID NO: 1964; The following amino acid sequences are included: (u) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1967 by 3 nucleotides; (v) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1968 by 3 nucleotides; (v) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1969 by 3 nucleotides; (w) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1970 by 3 nucleotides; (x) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1970 by 3 nucleotides; and (x) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1967 ... NO: 1944 has an amino acid sequence with at least 95% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1971 by 3 nucleotides; (y) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1945, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1972 by 3 nucleotides; (z) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1946, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1973 by 3 nucleotides; (aa) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1947, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1944 by 3 nucleotides; SEQ ID NO: 1974 has a nucleotide sequence that is 3 nucleotides different; (bb) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein the crRNA backbone comprises a nucleotide sequence that is 3 nucleotides different from the amino acid sequence of SEQ ID NO: 1975; (cc) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein the crRNA backbone comprises a nucleotide sequence that is 3 nucleotides different from the amino acid sequence of SEQ ID NO: 1976; (dd) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein the crRNA backbone comprises a nucleotide sequence that is 3 nucleotides different from the amino acid sequence of SEQ ID NO: 1977; (ee) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1951, wherein the crRNA backbone comprises a nucleotide sequence that is 3 nucleotides different from the amino acid sequence of SEQ ID NO: 1977; NO: 1978 has a nucleotide sequence that differs by 3 nucleotides; (ff) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein the crRNA backbone comprises a nucleotide sequence that differs by 3 nucleotides with the amino acid sequence of SEQ ID NO: 1979; (gg) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein the crRNA backbone comprises a nucleotide sequence that differs by 3 nucleotides with the amino acid sequence of SEQ ID NO: 1980; (hh) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein the crRNA backbone comprises a nucleotide sequence that differs by 3 nucleotides with the amino acid sequence of SEQ ID NO: 1981; (ii) RGN polypeptide, comprising an amino acid sequence that differs by 3 nucleotides with the amino acid sequence of SEQ ID NO: 1978; The amino acid sequence of 1955 has an amino acid sequence with at least 95% sequence identity, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1982 by 3 nucleotides; and the (jj)RGN polypeptide includes an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1956, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1983 by 3 nucleotides.

250. The gRNA of embodiment 246, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by 2 nucleotides; b) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 6 by 2 nucleotides; c) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 7 by 2 nucleotides; d) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by 2 nucleotides; (e) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 2 nucleotide differences from SEQ ID NO: 1617; (f) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 2 nucleotide differences from SEQ ID NO: 1618; (g) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 2 nucleotide differences from SEQ ID NO: 1619; (h) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence having 2 nucleotide differences from SEQ ID NO: 1619; 1620 has a nucleotide sequence that differs from SEQ ID NO: 1 by 2 nucleotides; (i) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1 by 2 nucleotides; (j) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1930, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1957 by 2 nucleotides; (k) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1958 by 2 nucleotides; (l) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1932 by 2 nucleotides; (m) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein the crRNA backbone comprises a nucleotide sequence having at least 2 nucleotide differences with SEQ ID NO: 1960; (n) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence having 2 nucleotide differences with SEQ ID NO: 1961; (o) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence having 2 nucleotide differences with SEQ ID NO: 1962; (p) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933; NO: 1963 has a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides; (q) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides; (r) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides; (s) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1939, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1966 by 2 nucleotides; (t) RGN polypeptide, comprising an amino acid sequence that differs from SEQ ID NO: 1964 by 2 nucleotides; The following amino acid sequences are included: (u) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1967 by 2 nucleotides; (v) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1968 by 2 nucleotides; (v) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1969 by 2 nucleotides; (w) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1970 by 2 nucleotides; and (x) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1970 by 2 nucleotides; and (x) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1967 ... NO: 1944 has an amino acid sequence with at least 95% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1971 by 2 nucleotides; (y) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1945, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1972 by 2 nucleotides; (z) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1946, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1973 by 2 nucleotides; (aa) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1947, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1944 by 2 nucleotides; SEQ ID NO: 1974 has a nucleotide sequence that differs by 2 nucleotides; (bb) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein the crRNA backbone comprises a nucleotide sequence that differs by 2 nucleotides with the amino acid sequence of SEQ ID NO: 1975; (cc) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein the crRNA backbone comprises a nucleotide sequence that differs by 2 nucleotides with the amino acid sequence of SEQ ID NO: 1976; (dd) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein the crRNA backbone comprises a nucleotide sequence that differs by 2 nucleotides with the amino acid sequence of SEQ ID NO: 1977; (ee) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1951, wherein the crRNA backbone comprises a nucleotide sequence that differs by 2 nucleotides with the amino acid sequence of SEQ ID NO: 1977; NO: 1978 has a nucleotide sequence that differs from SEQ ID NO: 1979 by 2 nucleotides; (ff) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1979 by 2 nucleotides; (gg) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1980 by 2 nucleotides; (hh) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1981 by 2 nucleotides; (ii) RGN polypeptide, comprising an amino acid sequence that differs from SEQ ID NO: 1979 by 2 nucleotides; The amino acid sequence of 1955 has an amino acid sequence with at least 95% sequence identity, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1982 by 2 nucleotides; and the (jj)RGN polypeptide includes an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1956, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1983 by 2 nucleotides.

251. The gRNA of embodiment 246, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by one nucleotide; b) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 6 by one nucleotide; c) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 7 by one nucleotide; d) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing from SEQ ID NO: 5 by one nucleotide; (e) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide ... 1620 has a nucleotide sequence that differs by one nucleotide; (i) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 1631; (j) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1930, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 1957; (k) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 1958; (l) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 1932; (m) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1960; (n) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1961; (o) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1962; (p) An RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide ...3, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein the crRNA backbone comprises a nucleotide sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935 NO: 1963 has a nucleotide sequence that differs by one nucleotide; (q) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1964; (r) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1965; (s) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1939, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1966; (t) RGN polypeptide, comprising an amino acid sequence that differs by one nucleotide with SEQ ID NO: 1964; The following amino acid sequences are included: (u) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1967 by one nucleotide; (v) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1968 by one nucleotide; (v) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1969 by one nucleotide; (w) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1970 by one nucleotide; (x) an RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1970 by one nucleotide; NO: 1944 has an amino acid sequence with at least 95% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1971 by one nucleotide; (y) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1945, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1972 by one nucleotide; (z) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1946, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1973 by one nucleotide; (aa) RGN polypeptide, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1947, wherein the crRNA backbone comprises a nucleotide sequence that differs from SEQ ID NO: 1944 by one nucleotide; SEQ ID NO: 1974 has a nucleotide sequence that differs by one nucleotide; (bb) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1975; (cc) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1976; (dd) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1977; (ee) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1951, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1977; NO: 1978 has a nucleotide sequence that differs by one nucleotide; (ff) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1979; (gg) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1980; (hh) RGN polypeptide, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein the crRNA backbone comprises a nucleotide sequence that differs by one nucleotide with SEQ ID NO: 1981; (ii) RGN polypeptide, comprising an amino acid sequence that differs by one nucleotide with the amino acid sequence of SEQ ID NO: 1978; The amino acid sequence of 1955 has an amino acid sequence with at least 95% sequence identity, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1982 by one nucleotide; and the (jj)RGN polypeptide includes an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1956, wherein the crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 1983 by one nucleotide.

252. The gRNA of embodiment 246, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 5; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 6; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 7; d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 8; (e) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1617; (f) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1617; The following are examples of amino acid sequences: (g) an RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA main chain includes the nucleotide sequence shown in SEQ ID NO: 1618; (h) an RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA main chain includes the nucleotide sequence shown in SEQ ID NO: 1619; (h) an RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA main chain includes the nucleotide sequence shown in SEQ ID NO: 1620; (i) an RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA main chain includes the nucleotide sequence shown in SEQ ID NO: 1631; (j) an RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 1930, wherein the crRNA main chain includes the nucleotide sequence shown in SEQ ID NO: 1957; (k) an RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 1931, wherein the crRNA main chain includes the nucleotide sequence shown in SEQ ID NO: 1957. The following are listed: (a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1932, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1959; (b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1933, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1960; (c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1934, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1961; (d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1935, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1962; (e) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1936, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1959; (f) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1936, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1959; (g) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1933, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1960; (n) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1934, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1961; (o) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1935, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1962; (p) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1936, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1959; (m) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1933, wherein The following are listed: (q) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1937, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1964; (r) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1938, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1965; (s) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1939, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1966; (t) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1940, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1967; (u) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1941, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1967; (v) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1942, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1969; (w) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1943, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1970; (x) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1944, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1971; (y) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1945, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1972; (z) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1946, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1969; The following are listed: (aa) RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 1947, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1974; (bb) RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 1948, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1975; (cc) RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 1949, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1976; (dd) RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 1950, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1977; (ee) RGN polypeptide, comprising the amino acid sequence shown in SEQ ID NO: 1951, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1977; The following are listed: (i) the nucleotide sequence shown in SEQ ID NO: 1978; (ii) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1952, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1979; (f) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1953, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1980; (hh) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1954, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1981; (ii) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1955, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1982; and (jj) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1956, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1983.

253. The gRNA as described in any of embodiments 245 to 252, wherein the target sequence is disposed adjacent to and at its 3' of the protoseptum adjacent motif (PAM).

254. The gRNA of embodiment 253, wherein a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 recognizes a PAM having a common nucleotide sequence shown in AYG, wherein the PAM is adjacent to and at its 5' of the target sequence; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 1933 or 1944 recognizes a PAM having a common nucleotide sequence shown in ATTN, wherein the PAM is adjacent to and at its 5' of the target sequence; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947 or 1949 recognizes a PAM having a common nucleotide sequence shown in VTTN, wherein the PAM is adjacent to and at its 5' of the target sequence; and d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4) RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is located at its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1937 or 1950 identify PAMs having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1951 identify PAMs having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) RGN polypeptides comprising amino acid sequences as shown in SEQ ID NO: 1953 identify PAMs having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'.

255. The gRNA as described in any one of embodiments 224 to 254, wherein the gRNA is an isolated gRNA.

256. A nucleic acid molecule comprising at least one polynucleotide encoding a gRNA as described in any one of embodiments 224 to 255.

257. The nucleic acid molecule as described in embodiment 256, wherein the nucleic acid molecule comprises at least two, at least three, at least four, at least five, at least six or more polynucleotides encoding gRNA as described in any one of embodiments 224 to 255.

258. A nucleic acid molecule comprising a polynucleotide encoding a gRNA as described in any one of embodiments 245 to 255.

259. The nucleic acid molecule as described in embodiment 258, wherein the nucleic acid molecule comprises at least two, at least three, at least four, at least five, at least six or more polynucleotides encoding gRNA as described in any one of embodiments 245 to 255.

260. The nucleic acid molecule as described in any one of embodiments 256 to 259, wherein the nucleic acid molecule is an isolated nucleic acid molecule.

261. A vector comprising a nucleic acid molecule as described in any one of embodiments 256 to 260.

262. The vector as described in embodiment 261, wherein the polynucleotide encoding at least one gRNA comprises a polynucleotide encoding one or more gRNAs.

263. The vector as described in embodiment 262, wherein a polynucleotide of the write-code one or more gRNAs is operatively linked to a single promoter.

264. The vector as described in embodiment 261, wherein at least one polynucleotide of the writing gRNA comprises two or more polynucleotides, each polynucleotide writing gRNA.

265. The vector as described in embodiment 264, wherein each polynucleotide encoding the gRNA is operatively linked to a promoter.

266. The vector as described in embodiments 263 or 265, wherein the promoter comprises the RNA polymerase III promoter.

267. The vector as described in embodiment 266, wherein the RNA polymerase III promoter comprises the human U6 promoter or the human 7SK promoter.

268. The vector as described in embodiment 267, wherein the human U6 promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1672.

269. The vector as described in embodiment 267, wherein the human 7SK promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1673.

270. The vector as described in any of embodiments 261 to 269, wherein the vector further comprises a polynucleotide encoding an RGN polypeptide.

271. The carrier as described in embodiment 270, wherein the carrier further comprises a promoter operatively linked to a polynucleotide encoding the RGN polypeptide.

272. The vector as described in embodiment 271, wherein the promoter comprises the RNA polymerase II promoter.

273. The vector as described in embodiment 272, wherein the RNA polymerase II promoter comprises the CMV promoter or the CBh promoter.

274. The carrier as described in embodiment 273, wherein the CMV initiator further includes a CMV upstream enhancer.

275. The vector as described in embodiment 274, wherein the CMV initiator and the CMV upstream enhancer comprise nucleotide sequences having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1674.

276. The vector as described in embodiment 273, wherein the CBh promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1675.

277. The vector as described in any of embodiments 261 to 276, wherein the vector further comprises a nucleotide sequence of a coding peptide linker.

278. The carrier as described in embodiment 277, wherein the peptide linker comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with an amino acid sequence as shown in SEQ ID NO: 34 or 1928.

279. The carrier as described in any of embodiments 261 to 278, wherein the carrier further comprises a polyadenylated (polyA) tail.

280. The carrier as described in embodiment 279, wherein the polyA tail comprises an SV40 polyA tail.

281. The vector as described in embodiment 280, wherein the SV40 polyA tail comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1678.

282. The vector as described in any of embodiments 261 to 281, wherein the gRNA comprises at least one CRISPR RNA (crRNA) backbone (bb) and at least one spacer.

283. The vector as described in embodiment 282, wherein the gRNA comprises two crRNA bbs and a spacer, in the form of crRNA bb-spacer-crRNA bb from 5' to 3'.

284. The vector as described in embodiment 282, wherein the gRNA comprises three crRNA bb, a first spacer and a second spacer, in the form of crRNA bb-first spacer-crRNA bb-second spacer-crRNA bb from 5' to 3'.

285. The vector as described in embodiment 282, wherein the gRNA comprises a crRNA bb and a spacer, in the form of crRNA bb-spacer from 5' to 3'.

286. The vector as described in any of embodiments 282 to 284, wherein at least one crRNA bb comprises two or more crRNA bbs, and wherein the two or more crRNA bbs are distinct.

287. The vector as described in any of embodiments 282 to 284, wherein at least one crRNA bb comprises two or more crRNA bbs, and wherein the two or more crRNA bbs are identical.

288. The vector as described in any of embodiments 282 to 284, wherein at least one crRNA bb comprises two or more crRNA bbs, and wherein the two or more crRNA bbs are a mixture of some identical crRNA bbs and some different crRNA bbs.

289. The carrier as described in any of embodiments 282 to 288, wherein at least one septum comprises two or more septum, and wherein the two or more septum target different target sequences.

290. The carrier as described in any of embodiments 282 to 288, wherein at least one septum comprises two or more septums, and wherein the two or more septums target the same target sequence.

291. A cell comprising an RGN polypeptide as described in any one of embodiments 144 to 208, an RNP complex as described in any one of embodiments 209 to 223, a gRNA as described in any one of embodiments 224 to 255, a nucleic acid molecule as described in any one of embodiments 256 to 260, or a vector as described in any one of embodiments 261 to 290.

292. The cell as described in embodiment 291, wherein the cell is a prokaryotic cell.

293. The cell as described in embodiment 291, wherein the cell is a eukaryotic cell.

294. The cell as described in embodiment 293, wherein the eukaryotic cell is a mammalian cell.

295. The cell as described in embodiment 294, wherein the mammalian cell is a human cell.

296. The cell as described in embodiment 295, wherein the human cell is an immune cell.

297. The cell as described in embodiment 296, wherein the immune cell is a stem cell.

298. The cell as described in embodiment 297, wherein the stem cell is an induced pluripotent stem cell.

299. The cell as described in embodiment 291, wherein the eukaryotic cell is an insect or bird cell.

300. The cell as described in embodiment 291, wherein the eukaryotic cell is a fungal cell.

301. The cell as described in embodiment 291, wherein the eukaryotic cell is a plant cell.

302. A plant or a portion of a plant, comprising cells as described in embodiment 301.

303. An RNA-guided nuclease (RGN) system for binding to one or more target sequences, said system comprising: a) one or more guide RNAs (gRNAs), or one or more polynucleotides comprising one or more nucleotide sequences encoding one or more gRNAs, wherein the one or more gRNAs include CRISPR RNA (crRNA) comprising a crRNA backbone and spacers; and b) an RGN polypeptide or a polynucleotide comprising a nucleotide sequence encoding an RGN polypeptide, the RGN polypeptide comprising, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 108 1. The amino acid sequence represented by any one of 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity.

304. The RGN system as described in embodiment 303, wherein the RGN polypeptide comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 108 The amino acid sequence represented by any one of 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687 has an amino acid sequence with at least 95% sequence identity.

305. The RGN system as described in embodiment 303 or 304, wherein the RGN polypeptide comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687.

306. The RGN system as described in any of embodiments 303 to 305, wherein each of one or more gRNAs is capable of hybridizing with a target strand of the target sequence and forming a complex with the RGN polypeptide to induce the RGN polypeptide to bind to the target sequence.

307. The RGN system as described in any of embodiments 303 to 306, wherein the spacer is hybridized with the sequence of the eukaryotic target.

308. The RGN system as described in embodiment 307, wherein the eukaryotic target sequence includes the mammalian target sequence.

309. An RGN system as described in any of embodiments 303 to 308, wherein at least one of the one or more nucleotide sequences encoding one or more gRNAs and the nucleotide sequences encoding an RGN polypeptide is operatively linked to a promoter heterologous to the nucleotide sequences.

310. The RGN system as described in any of embodiments 303 to 309, wherein the one or more nucleotide sequences encoding one or more gRNAs include a nucleotide sequence encoding one or more gRNAs.

311. The RGN system of embodiment 310, wherein the one nucleotide sequence encoding one or more gRNAs is operatively linked to a single promoter.

312. The RGN system as described in any of embodiments 303 to 309, wherein the one or more nucleotide sequences encoding one or more gRNAs comprise two or more nucleotide sequences, each nucleotide sequence encoding a gRNA.

313. The RGN system as described in embodiment 312, wherein each nucleotide sequence encoding the gRNA is operatively linked to a promoter.

314. The RGN system as described in embodiments 311 or 313, wherein the promoter comprises the RNA polymerase III promoter.

315. The RGN system of embodiment 314, wherein the RNA polymerase III promoter comprises the human U6 promoter or the human 7SK promoter.

316. The RGN system of embodiment 315, wherein the human U6 promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1672.

317. The RGN system of embodiment 315, wherein the human 7SK promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1673.

318. The RGN system as described in any of embodiments 303 to 317, wherein the polynucleotide encoding the nucleotide sequence of the RGN polypeptide comprises an mRNA having a heterologous 5' untranslated region (UTR) and/or a heterologous 3' UTR.

319. An RGN system as described in any of embodiments 303 to 318, wherein one or more gRNAs do not include tracrRNA.

320. An RGN system as described in any of embodiments 303 to 319, wherein one or more gRNAs have a total length of 35 to 250 nucleotides (nt), or 35 to 170 nt, or 35 to 125 nt, or 40 to 100 nt, or 40 to 70 nt, or 40 to 60 nt.

321. An RGN system as described in any of embodiments 303 to 319, wherein one or more gRNAs have a maximum length of 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt, 82 nt, 82 nt, 83 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt, 83 nt, 8 ... nt, 83 nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 160 nt, 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt, 180 nt, 190 nt, 200 nt, 205 nt, 210 nt, 215 nt, 220 nt, 225 nt, 230 nt, 235 nt, 240 nt, 245 nt or 250 nt total length.

322. An RGN system as described in any of embodiments 303 to 319, wherein one or more gRNAs comprise a crRNA backbone having a total length of up to 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt.

323. The RGN system as described in any one of embodiments 303 to 322, wherein the one or more gRNAs are selected from the group consisting of: a) gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 1; b) gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 2; c) gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 7 or differing from SEQ ID NO: 8 by 1 to 5 nucleotides. 7) A nucleotide sequence having 1 to 5 nucleotide differences, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; d) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence having 1 to 5 nucleotide differences, as shown in SEQ ID NO: 8 or having 1 to 5 nucleotide differences, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA comprising CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence having 1 to 5 nucleotide differences, as shown in SEQ ID NO: 1617 or having 1 to 5 nucleotide differences, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (f) gRNA comprising CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence having 1 to 5 nucleotide differences, as shown in SEQ ID NO: 1618 or having 1 to 5 nucleotide differences, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; ID NO: 1618 has a nucleotide sequence that is 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (g) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence as shown in SEQ ID NO: 1619 or that is 1 to 5 nucleotides different from SEQ ID NO: 1619, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence as shown in SEQ ID NO: 1620 or that is 1 to 5 nucleotides different from SEQ ID NO: 1620, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, CRISPR The RNA includes a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1631 or differing from SEQ ID NO: 1631 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1; (j) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1957 or differing from SEQ ID NO: 1957 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1930; (k) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1958 or differing from SEQ ID NO: 1958 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1930; The amino acid sequence of SEQ ID NO: 1931 has an amino acid sequence with at least 90% sequence identity; (l) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1959 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1932; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1960 or differing from SEQ ID NO: 1960 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1933; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1961 or differing from SEQ ID NO: 1932 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1933; 1961 has a nucleotide sequence that is 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1962 or having 1 to 5 nucleotides different from SEQ ID NO: 1962, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935; (p) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1963 or having 1 to 5 nucleotides different from SEQ ID NO: 1963, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936; (q) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1963 or having 1 to 5 nucleotides different from SEQ ID NO: 1963; The RNA includes a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1964 or differing from SEQ ID NO: 1964 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1965 or differing from SEQ ID NO: 1965 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938; (s) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1966 or differing from SEQ ID NO: 1966 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938. The amino acid sequence of SEQ ID NO: 1939 has at least 90% sequence identity; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1967 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1968 or differing from SEQ ID NO: 1968 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1969 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941; 1969 has a nucleotide sequence that is 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942; (w) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1970 or having 1 to 5 nucleotides different from SEQ ID NO: 1970, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1971 or having 1 to 5 nucleotides different from SEQ ID NO: 1971, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944; (y) gRNA, including CRISPR RNA, CRISPR... The RNA includes a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1972 or differing from SEQ ID NO: 1972 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1945; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1973 or differing from SEQ ID NO: 1973 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1974 or differing from SEQ ID NO: 1974 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; The amino acid sequence of SEQ ID NO: 1947 has at least 90% sequence identity; (bb) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1975 or differing from SEQ ID NO: 1975 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1976 or differing from SEQ ID NO: 1976 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1977 or differing from SEQ ID NO: 1949 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; 1977 contains nucleotide sequences that are 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1978 or having 1 to 5 nucleotides different from SEQ ID NO: 1978, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951; (ff) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1979 or having 1 to 5 nucleotides different from SEQ ID NO: 1979, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1979 or having 1 to 5 nucleotides different from SEQ ID NO: 1979; The RNA includes a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1980 or differing from SEQ ID NO: 1980 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1981 or differing from SEQ ID NO: 1981 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954; (iii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1982 or differing from SEQ ID NO: 1982 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954. The amino acid sequence of 1955 has an amino acid sequence with at least 90% sequence identity; and (jj)gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1983 or differing from SEQ ID NO: 1983 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1956.

324. The RGN system of embodiment 323, wherein the one or more gRNAs are selected from the group consisting of: a) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; d) gRNA having a crRNA backbone, The crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 8 by 5 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1617 by 5 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1618 by 5 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (g) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 8 by 5 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; 1619 has a nucleotide sequence that is 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1620, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1631, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1; (j) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 5 nucleotides different from SEQ ID NO: 1957, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1957. The amino acid sequence of SEQ ID NO: 1930 has at least 90% sequence identity; (k) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1958 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1960 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932; The amino acid sequence of SEQ ID NO: 1933 has at least 90% sequence identity; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935. The amino acid sequence of SEQ ID NO: 1936 has at least 90% sequence identity; (q) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938; (s) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938. The amino acid sequence of SEQ ID NO: 1939 has at least 90% sequence identity; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941. The amino acid sequence of SEQ ID NO: 1942 has at least 90% sequence identity; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944; The amino acid sequence of SEQ ID NO: 1945 has at least 90% sequence identity; (z) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947; (bb) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947; The amino acid sequence of SEQ ID NO: 1948 has at least 90% sequence identity; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1951 has at least 90% sequence identity; (ff) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953; (hh) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953; The amino acid sequence of 1954 has at least 90% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1956.

325. The RGN system of embodiment 323, wherein the one or more gRNAs are selected from the group consisting of: a) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; d) gRNA having a crRNA backbone, The crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 8 by 4 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1617 by 4 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1618 by 4 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (g) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 8 by 4 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; 1619 has a nucleotide sequence that is 4 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 4 nucleotides different from the SEQ ID NO: 1620, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 4 nucleotides different from the SEQ ID NO: 1631, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1; (j) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 4 nucleotides different from the SEQ ID NO: 1957, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1957. The amino acid sequence of SEQ ID NO: 1930 has at least 90% sequence identity; (k) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1958 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1960 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932; The amino acid sequence of SEQ ID NO: 1933 has at least 90% sequence identity; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935. The amino acid sequence of SEQ ID NO: 1936 has at least 90% sequence identity; (q) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938; (s) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938. The amino acid sequence of SEQ ID NO: 1939 has at least 90% sequence identity; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941. The amino acid sequence of SEQ ID NO: 1942 has at least 90% sequence identity; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944; The amino acid sequence of SEQ ID NO: 1945 has at least 90% sequence identity; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947; (bb) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947. The amino acid sequence of SEQ ID NO: 1948 has at least 90% sequence identity; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1951 has at least 90% sequence identity; (ff) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953; (hh) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953; The amino acid sequence of 1954 has at least 90% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 4 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1956.

326. The RGN system of embodiment 323, wherein the one or more gRNAs are selected from the group consisting of: a) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; d) gRNA having a crRNA backbone, The crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 8 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1617 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1618 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (g) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 8 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; 1619 has a nucleotide sequence that is 3 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 3 nucleotides different from SEQ ID NO: 1620, wherein the RGN polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 3 nucleotides different from SEQ ID NO: 1631, wherein the RGN polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 1; (j) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 3 nucleotides different from SEQ ID NO: 1957, wherein the RGN polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 1957. The amino acid sequence of SEQ ID NO: 1930 has at least 95% sequence identity; (k) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1958 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1960 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932; The amino acid sequence of SEQ ID NO: 1933 has at least 95% sequence identity; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935. The amino acid sequence of SEQ ID NO: 1936 has at least 95% sequence identity; (q) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1964 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1965 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938; (s) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1966 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938. The amino acid sequence of SEQ ID NO: 1939 has at least 95% sequence identity; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941. The amino acid sequence of SEQ ID NO: 1942 has at least 95% sequence identity; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944; The amino acid sequence of SEQ ID NO: 1945 has at least 95% sequence identity; (z) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947; (bb) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947; The amino acid sequence of SEQ ID NO: 1948 has at least 95% sequence identity; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1951 has at least 95% sequence identity; (ff) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1979 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1980 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953; (hh) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1981 by 3 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953; The amino acid sequence of 1954 has at least 95% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 3 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1956.

327. The RGN system of embodiment 323, wherein the one or more gRNAs are selected from the group consisting of: a) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; d) gRNA having a crRNA backbone, The crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 8 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1617 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1618 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (g) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 8 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; 1619 has a nucleotide sequence that differs from SEQ ID NO: 4 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that differs from SEQ ID NO: 4 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that differs from SEQ ID NO: 1631 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1; (j) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that differs from SEQ ID NO: 1957 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; The amino acid sequence of SEQ ID NO: 1930 has at least 95% sequence identity; (k) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1958 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1960 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932; The amino acid sequence of SEQ ID NO: 1933 has at least 95% sequence identity; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935. The amino acid sequence of SEQ ID NO: 1936 has at least 95% sequence identity; (q) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938; (s) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1966 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938. The amino acid sequence of SEQ ID NO: 1939 has at least 95% sequence identity; (t) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1967 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1968 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941; (v) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1969 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941. The amino acid sequence of SEQ ID NO: 1942 has at least 95% sequence identity; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1970 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1971 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1972 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1944 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1945 has at least 95% sequence identity; (z) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1973 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1974 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947; (bb) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1975 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947. The amino acid sequence of SEQ ID NO: 1948 has at least 95% sequence identity; (cc) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1976 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1977 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950; (ee) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1978 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; The amino acid sequence of SEQ ID NO: 1951 has at least 95% sequence identity; (ff) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1979 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1980 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953; (hh) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1981 by 2 nucleotides, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953; The amino acid sequence of 1954 has at least 95% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 2 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1956.

328. The RGN system of embodiment 323, wherein the one or more gRNAs are selected from the group consisting of: a) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 5 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 6 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) gRNA having a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 7 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; d) gRNA having a crRNA backbone, The crRNA backbone includes a nucleotide sequence that differs from SEQ ID NO: 8 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (e) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1617 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1618 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (g) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 8 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; 1619 has a nucleotide sequence that is 1 nucleotide different, wherein the RGN polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 4; (h) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 1 nucleotide different from SEQ ID NO: 1620, wherein the RGN polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 4; (i) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 1 nucleotide different from SEQ ID NO: 1631, wherein the RGN polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 1; (j) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone that comprises a nucleotide sequence that is 1 nucleotide different from SEQ ID NO: 1957, wherein the RGN polypeptide comprises an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 1957. The amino acid sequence of SEQ ID NO: 1930 has at least 95% sequence identity; (k) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1958 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931; (l) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1959 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932; (m) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1960 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1931 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1932 ... The amino acid sequence of SEQ ID NO: 1933 has at least 95% sequence identity; (n) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1961 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1962 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935; (p) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1963 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1934 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1935 ... The amino acid sequence of SEQ ID NO: 1936 has at least 95% sequence identity; (q) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1964 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1965 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938; (s) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1966 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938. The amino acid sequence of SEQ ID NO: 1939 has at least 95% sequence identity; (t) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1967 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940; (u) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1968 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941; (v) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1969 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1939 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1941 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1941 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1969 ... The amino acid sequence of SEQ ID NO: 1942 has at least 95% sequence identity; (w) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1970 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1971 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944; (y) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1972 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1942 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1943 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 194 ...3 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1944 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1943 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1943 by one nucleotide The amino acid sequence of SEQ ID NO: 1945 has at least 95% sequence identity; (z) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1973 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1974 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947; (bb) gRNA, including CRISPR RNA, wherein the crRNA backbone of the CRISPR RNA includes a nucleotide sequence that differs from SEQ ID NO: 1975 by one nucleotide, wherein the RGN polypeptide includes an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947. The amino acid sequence of SEQ ID NO: 1948 has at least 95% sequence identity; (cc) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1976 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1977 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950; (ee) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1978 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1948 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence differing from SEQ ID NO: 1949 ... The amino acid sequence of SEQ ID NO: 1951 has at least 95% sequence identity; (ff) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953; (hh) gRNA, including CRISPR RNA, wherein the CRISPR RNA has a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953; The amino acid sequence of 1954 has at least 95% sequence identity; (ii) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955; and (jj) gRNA, including CRISPR RNA, the CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by one nucleotide, wherein the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1956.

329. The RGN system of embodiment 323, wherein the one or more gRNAs are selected from the group consisting of: a) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 5, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1; b) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 6, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2; c) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 7, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3; d) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 8, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 9. (e) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1617, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4; (f) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1618, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4; (g) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1619, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4; (h) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1620, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4; (i) gRNA having a crRNA backbone, the crRNA backbone comprising the amino acid sequence shown in SEQ ID NO: 4; The nucleotide sequence shown in SEQ ID NO: 1631, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1; (j) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1957, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1930; (k) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1958, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1931; (l) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1959, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1932; (m) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1960, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1932. The following are listed: (n) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1933; (o) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1962; wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1935; (p) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1963; wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1936; (q) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1964; wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1935. The following are listed: (r) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1937; (s) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1965, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1938; (s) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1966, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1939; (t) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1967, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1940; (u) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1968, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1940. The following are the amino acid sequences shown in SEQ ID NO: 1941; (v) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1969, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1942; (w) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1970, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1943; (x) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1971, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1944; (y) gRNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1972, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1944. The following are listed: (z) gRNA with a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1973, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1946; (aa) gRNA with a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1974, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1947; (bb) gRNA with a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1975, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1948; (cc) gRNA with a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1976, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1948. The following are listed: 1949 amino acid sequence; (dd) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1977, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1950; (ee) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1978, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1951; (ff) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1979, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1952; (gg) gRNA having a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1980, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1949; The following are described: (i) an amino acid sequence as shown in SEQ ID NO: 1953; (ii) a gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1981, wherein the RGN polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1954; (ii) a gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1982, wherein the RGN polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1955; and (jj) a gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1983, wherein the RGN polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1956.

330. The RGN system as described in any one of embodiments 303 to 329, wherein the spacer comprises a nucleotide sequence as shown in any one of SEQ ID NO: 294 to 396, 1789 to 1796, 1798 to 1880, 1890 to 1893 and 2082 to 2125.

331. The RGN system as described in any one of embodiments 303 to 330, wherein the one or more gRNAs comprise the nucleotide sequences shown in any one of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658.

332. The RGN system as described in any of embodiments 303 to 331, wherein at least one of the one or more gRNAs includes at least one chemical modification.

333. The RGN system as described in embodiment 332, wherein at least one chemical modification comprises: bridging nucleic acid (BNA) modification; 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluorine (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphosilicate (MSP) modification; 2'-O-methyl 3'-phosphosilicate (MP) modification; and thiophosphate (PS) modification.

334. The RGN system as described in embodiment 333, wherein the BNA includes 2′,4′ BNA modifications.

335. The RGN system of embodiment 334, wherein the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA ^NC [N-Me] modification, 2′-O,4′-C-ethylene bridging nucleic acid (2′,4′-ENA) modification and S-restricted ethyl (cEt) modification.

336. The RGN system as described in embodiment 334, wherein the 2′,4′ BNA is an LNA modification.

337. The RGN system as described in embodiment 334, wherein the 2′,4′ BNA is a cEt modification.

338. The RGN system as described in embodiments 332 or 333, wherein at least one chemical modification includes BNA modification, 2'-O-Me modification or PS modification.

339. The RGN system as described in any of embodiments 303 to 338, wherein the RGN polypeptide is not found to be complexed with the one or more gRNAs in nature.

340. The RGN system as described in any of embodiments 303 to 339, wherein the target sequence is a eukaryotic target sequence.

341. The RGN system as described in any of embodiments 303 to 340, wherein the target sequence is disposed adjacent to and positioned at 3' of the original spacer adjacent motif (PAM).

342. The RGN system of embodiment 341, wherein a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 identifies a PAM having a common nucleotide sequence shown in AYG, wherein the PAM is adjacent to the target sequence and is located at its 5'; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 1933, or 1944 identifies a PAM having a common nucleotide sequence shown in ATTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949 identifies a PAM having a common nucleotide sequence shown in VTTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4. RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is in its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is in its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is in its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1937 or 1950 identify PAMs having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1951 identify PAMs having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1953 identify PAMs having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'.

343. The RGN system as described in any of embodiments 303 to 342, wherein the targeted sequence is intracellular.

344. The RGN system as described in embodiment 343, wherein the cell is a eukaryotic cell.

345. The RGN system as described in embodiment 344, wherein the eukaryotic cell is a mammalian cell.

346. The RGN system as described in any of embodiments 303 to 345, wherein the system guides the cleavage of the target nucleic acid molecule.

347. The RGN system as described in embodiment 346, wherein shearing produces a double-strand fracture.

348. The RGN system of embodiment 347, wherein the double-strand break includes a 5' protrusion of 5 to 12 nt, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

349. The RGN system of embodiment 347, wherein the double-strand break includes a 1 nt 3' protrusion, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO: 1.

350. The RGN system as described in any one of embodiments 303 to 345, wherein the RGN polypeptide is nuclease-inactivating.

351. The RGN system of embodiment 350, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 408, 597, 678, 827, 896, 979, 2686, and 2687.

352. The RGN system as described in any of embodiments 303 to 351, wherein the RGN polypeptide is operatively fused with a base-editing polypeptide.

353. The RGN system as described in embodiment 352, wherein the base-editing polypeptide includes a deaminase.

354. The RGN system as described in any of embodiments 303 to 351, wherein the RGN polypeptide is operatively fused with a polymerase-editing polypeptide.

355. The RGN system of embodiment 354, wherein the polymerase editing polypeptide comprises DNA polymerase.

356. The RGN system as described in embodiment 354, wherein the polymerase editing polypeptide comprises reverse transcriptase.

357. The RGN system as described in any of embodiments 303 to 351, wherein the RGN peptide is operatively fused with an effector domain.

358. The RGN system as described in embodiment 357, wherein the effector subdomain is operatively fused to the N-terminus or C-terminus of the RGN peptide.

359. The RGN system as described in embodiment 357, wherein the effect subdomain is operatively fused to an internal address of the RGN polypeptide.

360. The RGN system as described in any of embodiments 357 to 359, wherein the effect subdomain includes a cleavage domain, a deaminase domain, or a performance regulation subdomain.

361. The RGN system as described in embodiment 360, wherein the expression regulation subdomain includes a transcriptional activation domain, a transcriptional repression domain, or an epigenetic modification domain.

362. The RGN system as described in any of embodiments 303 to 361, wherein the RGN polypeptide includes one or more nuclear localization signals (NLS).

363. The RGN system of embodiment 362, wherein one or more NLSs comprise an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any of SEQ ID NO: 33, 35, 407, and 192.

364. The RGN system as described in any of embodiments 303 to 363, wherein the RGN peptide is codon-optimized for its expression in eukaryotic cells.

365. The RGN system as described in any of embodiments 303 to 364, wherein the system further comprises one or more donor polynucleotides.

366. A cell comprising an RGN system as described in any one of embodiments 303 to 365.

367. The cell as described in embodiment 366, wherein the cell is a prokaryotic cell.

368. The cell as described in embodiment 366, wherein the cell is a eukaryotic cell.

369. The cell as described in embodiment 368, wherein the eukaryotic cell is a mammalian cell.

370. The cell as described in embodiment 369, wherein the mammalian cell is a human cell.

371. The cell as described in embodiment 370, wherein the human cell is an immune cell.

372. The cell as described in embodiment 371, wherein the immune cell is a stem cell.

373. The cell as described in embodiment 372, wherein the stem cell is an induced potential-rich stem cell.

374. The cell as described in embodiment 368, wherein the eukaryotic cell is an insect or bird cell.

375. The cell as described in embodiment 368, wherein the eukaryotic cell is a fungal cell.

376. The cell as described in embodiment 368, wherein the eukaryotic cell is a plant cell.

377. A plant or a portion of a plant, comprising cells as described in embodiment 376.

378. A pharmaceutical composition comprising a nucleic acid molecule as described in any one of embodiments 1 to 78 and 256 to 260; a vector as described in any one of embodiments 79 to 122 and 231 to 290; an RGN polypeptide as described in any one of embodiments 144 to 208; an RNP complex as described in any one of embodiments 209 to 223; a gRNA as described in any one of embodiments 224 to 255; a cell as described in any one of embodiments 125 to 130, 293 to 298 and 368 to 373; or an RGN system as described in any one of embodiments 303 to 365; and a pharmaceutically acceptable vector.

379. A method for binding to a target sequence in a target nucleic acid molecule, comprising delivering an RGN system according to any one of embodiments 303 to 365 to the target sequence or a cell including the target sequence.

380. The method of embodiment 379, wherein the RGN polypeptide or the one or more gRNAs further comprises a detectable tracer, thereby allowing detection of the target sequence.

381. The method of embodiment 379, wherein the RGN polypeptide or the one or more gRNAs further includes an expression regulator, thereby regulating the expression of a target gene including the target sequence.

382. A method for cleaving and/or modifying a target nucleic acid molecule comprising a target sequence, the method comprising delivering a system according to any one of embodiments 303 to 365 to the target sequence or a cell comprising the target sequence, wherein cleaving or modification of the target nucleic acid molecule occurs.

383. The method of embodiment 382, wherein the modified target nucleic acid molecule comprises inserting heterologous DNA into the target nucleic acid molecule.

384. The method of embodiment 382, wherein the modified target nucleic acid molecule comprises a deletion of at least one nucleotide from the target nucleic acid molecule.

385. The method of embodiment 382, wherein the modified target nucleic acid molecule includes a mutation of at least one nucleotide in the target nucleic acid molecule.

386. A method for binding one or more target sequences in a target nucleic acid molecule to an RNA-guided nuclease (RGN) polypeptide, the method comprising: a) assembling a ribonucleoprotein (RNP) complex under conditions suitable for forming an RNP complex by combining: i) a guiding RNA (gRNA); and ii) an RGN polypeptide, including as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 108 1. The amino acid sequence represented by any one of 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687 has at least A) an amino acid sequence with 90% sequence identity; and b) contacting the target nucleic acid molecule or a cell including the target nucleic acid molecule with the assembled RNP complex; thereby binding the one or more target sequences to the RGN polypeptide.

387. A method for binding one or more target sequences in a target nucleic acid molecule to an RNA-guided nuclease (RGN) polypeptide, the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with: i) one or more guiding RNAs (gRNAs); and ii) an RGN polypeptide or a polynucleotide encoding the RGN polypeptide, the RGN polypeptide comprising, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 108 4. The amino acid sequence represented by any one of 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity, thereby enabling the target sequence to bind to RGN.

388. The method as described in embodiment 386 or 387, wherein the RGN polypeptide comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, The amino acid sequence represented by any one of 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 95% sequence identity.

389. The method of any one of embodiments 386-388, wherein the RGN polypeptide comprises as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687.

390. The method of any one of embodiments 386 to 389, wherein the RGN polypeptide is nuclease-inactivated.

391. The method of embodiment 390, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 408, 597, 678, 827, 896, 979, 2686, and 2687.

392. The method of any one of embodiments 386 to 389, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule, thereby allowing cleavage and/or modification of the target nucleic acid molecule.

393. The method of embodiment 392, wherein the cleavage produces a double-strand break in the target nucleic acid molecule, wherein the double-strand break includes a 5' protrusion of 5 to 12 nt, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

394. The method of embodiment 392, wherein the cleavage produces a double-strand break in the target nucleic acid molecule, wherein the double-strand break includes a 1 nt 3' protrusion, and wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

395. The method of any of embodiments 387 to 394, wherein the polynucleotide encoding the RGN polypeptide comprises an mRNA having a heterologous 5' untranslated region (UTR) and/or a heterologous 3' UTR.

396. The method as described in any of embodiments 379 to 394, wherein the method is performed in vitro, in vivo or ex vivo.

397. The method as described in any of embodiments 379 to 396, wherein the cell is a eukaryotic cell.

398. The method as described in embodiment 397, wherein the eukaryotic cell is a mammalian cell.

399. The method of any of embodiments 386 to 394, wherein the RGN polypeptide or the gRNA further comprises a detectable tracer, thereby allowing detection of the target sequence.

400. The method of any of embodiments 386 to 394, wherein the RGN polypeptide or the one or more gRNAs further comprises an expression regulator, thereby allowing regulation of the expression of a target gene including the target sequence.

401. The method of any of embodiments 386 to 394, wherein the RGN polypeptide further comprises a base-editing polypeptide, thereby allowing modification of the target nucleic acid molecule.

402. The method of embodiment 401, wherein the base-editing polypeptide comprises a deaminerase.

403. The method of any of embodiments 386 to 394, wherein the RGN polypeptide further comprises a polymerase editing polypeptide, thereby allowing modification of the target nucleic acid molecule.

404. The method of embodiment 403, wherein the polymerase editing polypeptide comprises DNA polymerase.

405. The method of embodiment 403, wherein the polymerase editing polypeptide comprises reverse transcriptase.

406. The method of any of embodiments 386 to 394, wherein the RGN polypeptide is operatively fused with an effector domain, thereby allowing cleavage and/or modification of the target nucleic acid molecule.

407. The method of embodiment 406, wherein the effect subdomain is operatively fused to the N-terminus or C-terminus of the RGN peptide.

408. The method as described in embodiment 406, wherein the effect subdomain is operatively fused to an internal address of the RGN polypeptide.

409. The method as described in any of embodiments 406 to 408, wherein the effect subdomain includes a cleavage domain, a deaminase domain, or a performance regulation subdomain.

410. The method of embodiment 409, wherein the expression regulation subdomain includes a transcriptional activation domain, a transcriptional repression domain, or an epigenetic modification domain.

411. A method for cleaving and/or modifying a target nucleic acid molecule comprising one or more target sequences, the method comprising contacting the target nucleic acid molecule with: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises, as in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 96 3, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 111 6. The amino acid sequence shown in any one of 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity; and b) a guiding RNA (gRNA) capable of targeting the target sequence with the RGN polypeptide of (a); wherein splicing and/or modification of the target nucleic acid molecule occurs.

412. The method of embodiment 411, wherein the RGN polypeptide comprises, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, The amino acid sequence represented by any one of 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 95% sequence identity.

413. The method as described in embodiments 411 or 412, wherein the RGN polypeptide comprises, as in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687.

414. The method of any one of embodiments 411 to 413, wherein the RGN polypeptide is nuclease-inactivated.

415. The method of embodiment 414, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 408, 597, 678, 827, 896, 979, 2686, and 2687.

416. The method of any one of embodiments 411 to 413, wherein the cleavage of the RGN polypeptide results in double strand breakage.

417. The method of embodiment 416, wherein the double strand breaks include a 5' protrusion of 5 to 12 nt, wherein the RGN polypeptide has at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4.

418. The method of embodiment 416, wherein the double strand break includes a 1 nt 3' protrusion, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO: 1.

419. The method of any one of embodiments 411 to 418, wherein the RGN polypeptide is operatively fused with the base editing polypeptide.

420. The method of embodiment 419, wherein the base-editing polypeptide includes a deaminase.

421. The method of any of embodiments 411 to 418, wherein the RGN polypeptide is operatively fused with the polymerase editing polypeptide.

422. The method of embodiment 421, wherein the polymerase editing polypeptide comprises DNA polymerase.

423. The method of embodiment 421, wherein the polymerase editing polypeptide comprises reverse transcriptase.

424. The method of any of embodiments 411 to 418, wherein the RGN peptide is operatively fused with an effector domain.

425. The method of embodiment 424, wherein the effect subdomain is operatively fused to the N-terminus or C-terminus of the RGN peptide.

426. The method of embodiment 424, wherein the effect subdomain is operatively fused to an internal address of the RGN polypeptide.

427. The method of any of embodiments 424 to 426, wherein the effect subdomain comprises a cleavage domain, a deaminase domain, or a performance regulation subdomain.

428. The method of embodiment 427, wherein the expression regulation subdomain includes a transcriptional activation domain, a transcriptional repression domain, or an epigenetic modification domain.

429. The method of any one of embodiments 411 to 418, wherein the modified target nucleic acid molecule comprises inserting heterologous DNA into the target nucleic acid molecule.

430. The method of any one of embodiments 411 to 418, wherein the modified target nucleic acid molecule comprises the deletion of at least one nucleotide from the target nucleic acid molecule.

431. The method of any one of embodiments 411 to 418, wherein the modified target nucleic acid molecule includes a mutation of at least one nucleotide in the target nucleic acid molecule.

432. The method of any of embodiments 411 to 431, wherein the target sequence is disposed adjacent to and disposed at its 3' of the original spacer adjacent motif (PAM).

433. The method of embodiment 432, wherein a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 identifies a PAM having a common nucleotide sequence shown in AYG, wherein the PAM is adjacent to the target sequence and is in its 5'; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 1933 or 1944 identifies a PAM having a common nucleotide sequence shown in ATTN, wherein the PAM is adjacent to the target sequence and is in its 5'; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947 or 1949 identifies a PAM having a common nucleotide sequence shown in VTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4. RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is in its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is in its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is in its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1937 or 1950 identify PAMs having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1951 identify PAMs having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1953 identify PAMs having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'.

434. The method as described in any of embodiments 411 to 433, wherein the target sequence is a eukaryotic target sequence.

435. The method of embodiment 434, wherein the eukaryotic target sequence is a mammalian target sequence.

436. The method of any one of embodiments 411 to 435, wherein: a) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the one or more gRNAs have a CRISPR RNA (crRNA) backbone having a nucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides; b) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the one or more gRNAs have a crRNA backbone having a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides; c) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the one or more gRNAs have a crRNA backbone having a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides; The amino acid sequence shown in SEQ ID NO: 3 has an amino acid sequence with at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence as shown in SEQ ID NO: 7 or different from SEQ ID NO: 7 by 1 to 5 nucleotides; d) The RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence as shown in SEQ ID NO: 8 or different from SEQ ID NO: 8 by 1 to 5 nucleotides; (e) The RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1617 or different from SEQ ID NO: 1617 by 1 to 5 nucleotides; (f) The RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1617 or different from SEQ ID NO: 1617 by 1 to 5 nucleotides; The amino acid sequence of 4 has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1618 or differing from SEQ ID NO: 1618 by 1 to 5 nucleotides; (g) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1619 or differing from SEQ ID NO: 1619 by 1 to 5 nucleotides; (h) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1619 by 1 to 5 nucleotides. 1620 has a nucleotide sequence that differs by 1 to 5 nucleotides; (i) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1631 or having 1 to 5 nucleotide differences from SEQ ID NO: 1631; (j) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1930, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1957 or having 1 to 5 nucleotide differences from SEQ ID NO: 1957; (k) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1920. (l) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1959 or having 1 to 5 nucleotide differences from SEQ ID NO: 1958; (m) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1960 or having 1 to 5 nucleotide differences from SEQ ID NO: 1960; (n) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1958 or having 1 to 5 nucleotide differences from SEQ ID NO: 1958; The amino acid sequence of SEQ ID NO: 1934 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1961 or differing from SEQ ID NO: 1961 by 1 to 5 nucleotides; (o) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1962 or differing from SEQ ID NO: 1962 by 1 to 5 nucleotides; (p) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1963 or differing from SEQ ID NO: 1964 by 1 to 5 nucleotides; 1963 has a nucleotide sequence that differs from SEQ ID NO: 1964 by 1 to 5 nucleotides; (q) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1964 or having 1 to 5 nucleotide differences from SEQ ID NO: 1964; (r) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1965 or having 1 to 5 nucleotide differences from SEQ ID NO: 1965; (s) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1965 or having 1 to 5 nucleotide differences from SEQ ID NO: 1965; The amino acid sequence of SEQ ID NO: 1939 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1966 or differing from SEQ ID NO: 1966 by 1 to 5 nucleotides; (t) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1967 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides; (u) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1968 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides; (v) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1969 or having 1 to 5 nucleotide differences from SEQ ID NO: 1969; (w) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1970 or having 1 to 5 nucleotide differences from SEQ ID NO: 1970; (x) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1970 or having 1 to 5 nucleotide differences from SEQ ID NO: 1968; The amino acid sequence of SEQ ID NO: 1944 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1971 or differing from SEQ ID NO: 1971 by 1 to 5 nucleotides; (y) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1945, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1972 or differing from SEQ ID NO: 1972 by 1 to 5 nucleotides; (z) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1973 or differing from SEQ ID NO: 1974 by 1 to 5 nucleotides. 1973 has a nucleotide sequence that differs from SEQ ID NO: 1974 by 1 to 5 nucleotides; (aa) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1974 or having 1 to 5 nucleotide differences from SEQ ID NO: 1974; (bb) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1975 or having 1 to 5 nucleotide differences from SEQ ID NO: 1975; (cc) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1975 or having 1 to 5 nucleotide differences from SEQ ID NO: 1975; The amino acid sequence of SEQ ID NO: 1949 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1976 or differing from SEQ ID NO: 1976 by 1 to 5 nucleotides; (dd) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1977 or differing from SEQ ID NO: 1977 by 1 to 5 nucleotides; (ee) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1978 or differing from SEQ ID NO: 1978 has a nucleotide sequence that differs from SEQ ID NO: 1978 by 1 to 5 nucleotides; (ff) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1979 or having 1 to 5 nucleotide differences from SEQ ID NO: 1979; (gg) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1980 or having 1 to 5 nucleotide differences from SEQ ID NO: 1980; (hh) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1978 or having 1 to 5 nucleotide differences from SEQ ID NO: 1979; The amino acid sequence of SEQ ID NO: 1954 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1981 or differing from SEQ ID NO: 1981 by 1 to 5 nucleotides; (ii) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1982 or differing from SEQ ID NO: 1982 by 1 to 5 nucleotides; or (jj) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1956, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1983 or differing from SEQ ID NO: 1984 by 1 to 5 nucleotides; In 1983, there were nucleotide sequences that differed by 1 to 5 nucleotides.

437. The method of embodiment 436, wherein: a) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein one or more gRNAs have a CRISPR RNA (crRNA) backbone having a nucleotide sequence that differs from SEQ ID NO: 5 by 5 nucleotides; b) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 6 by 5 nucleotides; c) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 7 by 5 nucleotides; d) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 7 by 5 nucleotides; The amino acid sequence shown in SEQ ID NO: 4 has an amino acid sequence with at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 8 by 5 nucleotides; (e) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1617 by 5 nucleotides; (f) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by 5 nucleotides; (g) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 4 has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1619 by 5 nucleotides; (h) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1620 by 5 nucleotides; (i) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by 5 nucleotides; (j) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 1930 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1957 by 5 nucleotides; (k) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1958 by 5 nucleotides; (l) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 5 nucleotides; (m) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 1933 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 5 nucleotides; (n) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 5 nucleotides; (o) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 5 nucleotides; (p) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 1936 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 5 nucleotides; (q) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 5 nucleotides; (r) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 5 nucleotides; (s) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 1939 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 5 nucleotides; (t) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 5 nucleotides; (u) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 5 nucleotides; (v) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 1942 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 5 nucleotides; (w) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 5 nucleotides; (x) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 5 nucleotides; (y) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 1945 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1945, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides; (z) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 5 nucleotides; (aa) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 5 nucleotides; (bb) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 1948 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by 5 nucleotides; (cc) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by 5 nucleotides; (dd) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 5 nucleotides; (ee) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 5 nucleotides; The amino acid sequence of SEQ ID NO: 1951 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 5 nucleotides; (ff) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by 5 nucleotides; (gg) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 5 nucleotides; (hh) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 5 nucleotides; (ii) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by 5 nucleotides; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 5 nucleotides; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1956, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 5 nucleotides.

438. The method of embodiment 436, wherein: a) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein one or more gRNAs have a CRISPR RNA (crRNA) backbone having a nucleotide sequence different from SEQ ID NO: 5 by 4 nucleotides; b) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 6 by 4 nucleotides; c) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 7 by 4 nucleotides; d) the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 7 by 4 nucleotides; The amino acid sequence shown in SEQ ID NO: 4 has an amino acid sequence with at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 8 by 4 nucleotides; (e) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1617 by 4 nucleotides; (f) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by 4 nucleotides; (g) the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 4 has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1619 by 4 nucleotides; (h) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1620 by 4 nucleotides; (i) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by 4 nucleotides; (j) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1631 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 1930 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1957 by 4 nucleotides; (k) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1958 by 4 nucleotides; (l) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 4 nucleotides; (m) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 1933 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 4 nucleotides; (n) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 4 nucleotides; (o) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 4 nucleotides; (p) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 1936 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 4 nucleotides; (q) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 4 nucleotides; (r) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 4 nucleotides; (s) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 1939 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 4 nucleotides; (t) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 4 nucleotides; (u) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 4 nucleotides; (v) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 1942 has at least 90% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 4 nucleotides; (w) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 4 nucleotides; (x) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 4 nucleotides; (y) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 1945 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1945, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 4 nucleotides; (z) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 4 nucleotides; (aa) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 4 nucleotides; (bb) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 1948 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1975 by 4 nucleotides; (cc) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1976 by 4 nucleotides; (dd) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 4 nucleotides; (ee) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1977 by 4 nucleotides; The amino acid sequence of SEQ ID NO: 1951 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1978 by 4 nucleotides; (ff) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1979 by 4 nucleotides; (gg) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 4 nucleotides; (hh) the RGN polypeptide comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1980 by 4 nucleotides; (ii) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by 4 nucleotides; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 4 nucleotides; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1956, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 4 nucleotides.

439. The method of embodiment 436, wherein: a) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein one or more gRNAs have a CRISPR RNA (crRNA) backbone having a nucleotide sequence different from SEQ ID NO: 5 by 3 nucleotides; b) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 6 by 3 nucleotides; c) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 7 by 3 nucleotides; d) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 7 by 3 nucleotides; The amino acid sequence shown in SEQ ID NO: 4 has at least 95% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 8 by 3 nucleotides; (e) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1617 by 3 nucleotides; (f) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by 3 nucleotides; (g) the RGN polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 4 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 4 has at least 95% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by 3 nucleotides; (h) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1620 by 3 nucleotides; (i) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1631 by 3 nucleotides; (j) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1631 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 1930 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1957 by 3 nucleotides; (k) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1958 by 3 nucleotides; (l) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 3 nucleotides; (m) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 1933 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 3 nucleotides; (n) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 3 nucleotides; (o) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 3 nucleotides; (p) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 1936 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 3 nucleotides; (q) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 3 nucleotides; (r) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 3 nucleotides; (s) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 1939 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 3 nucleotides; (t) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 3 nucleotides; (u) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 3 nucleotides; (v) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 1942 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 3 nucleotides; (w) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 3 nucleotides; (x) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 3 nucleotides; (y) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 1945 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1945, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 3 nucleotides; (z) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 3 nucleotides; (aa) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 3 nucleotides; (bb) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 1948 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1975 by 3 nucleotides; (cc) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1976 by 3 nucleotides; (dd) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1977 by 3 nucleotides; (ee) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1948, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1977 by 3 nucleotides; The amino acid sequence of SEQ ID NO: 1951 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1978 by 3 nucleotides; (ff) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1979 by 3 nucleotides; (gg) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1980 by 3 nucleotides; (hh) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1980 by 3 nucleotides; (ii) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by 3 nucleotides; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 3 nucleotides; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1956, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 3 nucleotides.

440. The method of embodiment 436, wherein: a) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein one or more gRNAs have a CRISPR RNA (crRNA) backbone having a nucleotide sequence different from SEQ ID NO: 5 by 2 nucleotides; b) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 6 by 2 nucleotides; c) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 7 by 2 nucleotides; d) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 7 by 2 nucleotides; The amino acid sequence shown in SEQ ID NO: 4 has an amino acid sequence with at least 95% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 8 by 2 nucleotides; (e) the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1617 by 2 nucleotides; (f) the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by 2 nucleotides; (g) the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 4 has at least 95% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by 2 nucleotides; (h) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1620 by 2 nucleotides; (i) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1631 by 2 nucleotides; (j) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1631 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1930 has at least 95% sequence identity with the amino acid sequence, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1957 by 2 nucleotides; (k) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1958 by 2 nucleotides; (l) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 2 nucleotides; (m) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1959 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1933 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 2 nucleotides; (n) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1961 by 2 nucleotides; (o) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 2 nucleotides; (p) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1962 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1936 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1963 by 2 nucleotides; (q) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides; (r) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides; (s) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1939 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1966 by 2 nucleotides; (t) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1967 by 2 nucleotides; (u) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 2 nucleotides; (v) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1968 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1942 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1969 by 2 nucleotides; (w) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1970 by 2 nucleotides; (x) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 2 nucleotides; (y) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1971 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1945 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1945, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1972 by 2 nucleotides; (z) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1973 by 2 nucleotides; (aa) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 2 nucleotides; (bb) the RGN polypeptide comprises an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1974 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1948 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1975 by 2 nucleotides; (cc) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1976 by 2 nucleotides; (dd) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1977 by 2 nucleotides; (ee) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1977 by 2 nucleotides; The amino acid sequence of SEQ ID NO: 1951 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1978 by 2 nucleotides; (ff) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1979 by 2 nucleotides; (gg) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1980 by 2 nucleotides; (hh) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1980 by 2 nucleotides; (ii) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1981 by 2 nucleotides; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1982 by 2 nucleotides; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1956, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence that differs from SEQ ID NO: 1983 by 2 nucleotides.

441. The method of embodiment 436, wherein: a) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the one or more gRNAs have a CRISPR RNA (crRNA) backbone having a nucleotide sequence different from SEQ ID NO: 5 by one nucleotide; b) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 6 by one nucleotide; c) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 7 by one nucleotide; d) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the one or more gRNAs have a crRNA backbone having a nucleotide sequence different from SEQ ID NO: 7 by one nucleotide; The amino acid sequence shown in SEQ ID NO: 4 has an amino acid sequence with at least 95% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 8 by one nucleotide; (e) the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1617 by one nucleotide; (f) the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by one nucleotide; (g) the RGN polypeptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone having a nucleotide sequence that differs from SEQ ID NO: 1618 by one nucleotide; The amino acid sequence of SEQ ID NO: 4 has at least 95% sequence identity, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1619 by one nucleotide; (h) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1620 by one nucleotide; (i) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1631 by one nucleotide; (j) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1631 by one nucleotide; The amino acid sequence of SEQ ID NO: 1930 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1957 by one nucleotide; (k) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1958 by one nucleotide; (l) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1959 by one nucleotide; (m) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1959 by one nucleotide; The amino acid sequence of SEQ ID NO: 1933 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1961 by one nucleotide; (n) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1961 by one nucleotide; (o) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1962 by one nucleotide; (p) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1962 by one nucleotide; The amino acid sequence of SEQ ID NO: 1936 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1963 by one nucleotide; (q) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1964 by one nucleotide; (r) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1965 by one nucleotide; (s) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1965 by one nucleotide; The amino acid sequence of SEQ ID NO: 1939 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1966 by one nucleotide; (t) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1967 by one nucleotide; (u) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1968 by one nucleotide; (v) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1968 by one nucleotide; The amino acid sequence of SEQ ID NO: 1942 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1942, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1969 by one nucleotide; (w) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1970 by one nucleotide; (x) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1971 by one nucleotide; (y) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1971 by one nucleotide; The amino acid sequence of SEQ ID NO: 1945 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1945, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1972 by one nucleotide; (z) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1946, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1973 by one nucleotide; (aa) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1974 by one nucleotide; (bb) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1974 by one nucleotide; The amino acid sequence of SEQ ID NO: 1948 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1975 by one nucleotide; (cc) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1976 by one nucleotide; (dd) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1977 by one nucleotide; (ee) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1977 by one nucleotide; The amino acid sequence of SEQ ID NO: 1951 has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1978 by one nucleotide; (ff) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1979 by one nucleotide; (gg) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1980 by one nucleotide; (hh) the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1980 by one nucleotide; (ii) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1954, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1981 by one nucleotide; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1955, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1982 by one nucleotide; or (jj) The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1956, wherein one or more gRNAs have a crRNA backbone comprising a nucleotide sequence differing from SEQ ID NO: 1983 by one nucleotide.

442. The method of embodiment 436, wherein: a) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, and the one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 5; b) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2, and the one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 6; c) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3, and the one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 7; d) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4, and the one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 8; (e) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1; (f) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1617; (g) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1618; (g) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1619; (h) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1620; (i) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1620; (j) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1930, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1957; (k) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1931, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1958; (l) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1932, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1959; (m) The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1930. The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1933, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1960; (n) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1934, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1961; (o) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1935, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1962; (p) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1936, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1963; (q) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1936; The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1937, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1964; (r) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1938, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1965; (s) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1939, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1966; (t) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1940, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1967; (u) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1938; The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1941, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1968; (v) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1942, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1969; (w) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1943, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1970; (x) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1944, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1971; (y) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1944. The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1945, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1972; (z) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1946, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1973; (aa) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1947, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1974; (bb) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1948, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1975; (cc) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1948. The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1949, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1976; (dd) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1950, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1977; (ee) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1951, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1978; (ff) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1952, wherein one or more gRNAs have a crRNA backbone, the crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1979; (gg) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1949; The RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1953, wherein one or more gRNAs have a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1980; (ii) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1954, wherein one or more gRNAs have a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1981; (ii) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1955, wherein one or more gRNAs have a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1982; or (jj) the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1956, wherein one or more gRNAs have a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1983.

443. The method of any of embodiments 411 to 442, wherein the target sequence is inside a cell.

444. The method of embodiment 443 further comprises: culturing cells under conditions in which the RGN polypeptide is expressed and the target nucleic acid molecule is cleaved and/or modified to generate a modified target nucleic acid molecule; and selecting cells comprising the modified target nucleic acid molecule.

445. A cell generated according to the method of embodiment 444, wherein the target nucleic acid molecule has been cleaved and/or modified at the target sequence.

446. A cell as in embodiment 445, wherein the cell is a eukaryotic cell.

447. Cells as in embodiment 446, wherein the eukaryotic cells are mammalian cells.

448. A plant or a part of a plant, comprising cells as described in embodiments 445 or 446.

449. A pharmaceutical composition comprising cells and pharmaceutically acceptable delivery agents as described in any one of embodiments 445 to 447.

450. A lipid nanoparticle (LNP) comprising: (i) an RNA-guided nuclease (RGN) polypeptide, including those described in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 9 63, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 11 The amino acid sequence represented by any one of 16, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity, or a polynucleotide encoding the RGN polypeptide; and (ii) one or more guide RNAs (gRNAs), or a polynucleotide encoding one or more gRNAs.

451. The LNP as described in embodiment 450, wherein the polynucleotide encoding the RGN polypeptide comprises mRNA, the mRNA comprising a heterologous 5' untranslated region (UTR) and/or a heterologous 3' UTR.

452. The LNP as described in embodiment 450, wherein the polynucleotide encoding the RGN polypeptide is a plastid.

453. The LNP as described in any of embodiments 450 to 452, wherein the polynucleotide encoding one or more gRNAs is a plastid.

454. The LNP as described in any of embodiments 450 to 453, wherein one or more gRNAs include two gRNAs, three gRNAs, four gRNAs, five gRNAs, six gRNAs, or more gRNAs.

455. An RNA-guided nuclease (RGN) polypeptide comprising a protoseptate adjacent motif (PAM) interaction domain recognizing a PAM site, the PAM site comprising a nucleotide sequence as shown in any one of AYG, ATTN, VTTN, TTN, STTN, TTH, RTTN, RTYN, and ATG, wherein the RGN polypeptide comprises an interaction domain as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, The amino acid sequence represented by any one of 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity.

456. The RGN polypeptide as described in embodiment 455, wherein the PAM interaction domain is located in the N-terminal region of the RGN polypeptide.

The following examples are provided for illustrative purposes and not for limiting purposes. Table 33, attached to this disclosure, provides descriptions and sequences of sequence identifiers, such as those disclosed herein. Example 1 : Identification of RNA- guided nucleases

Four distinct CRISPR-linked RNA guiding nucleases (RGNs) have been identified and are described in Table 1 below. Table 1 provides the name of each RGN, its amino acid sequence, the source from which it was obtained, and the untreated crRNA (see the identification method in Example 2). For all RGNs described in Table 1, the untreated crRNA can also be used as sgRNA. Table 1 : Overview of SEQ IDs and CRISPR- linked systems RGN ID RGN subtype SEQ ID NO. Source Untreated crRNA sequence ( SEQ ID NO. ) LPG10238 VH 1 Casimicrobiaceae PALSA-1005 sp003153335 5 LPG10239 VJ 2 Virus 6 LPG10240 VJ 3 Virus 7 LPG10241 VJ 4 Virus 8 LPG13090 VI 1930 germ 1957 LPG13091 VI 1931 germ 1958 LPG13092 VI 1932 UBA11063 | UBA11063 sp003962895 1959 LPG13093 VJ 1933 germ 1960 LPG13094 VJ 1934 germ 1961 LPG13095 VJ 1935 germ 1962 LPG13096 VJ 1936 germ 1963 LPG13097 VJ 1937 germ 1964 LPG13098 VJ 1938 germ 1965 LPG13099 VJ 1939 Dokdonella |Dokdonella sp016721015 1966 LPG13100 VJ 1940 germ 1967 LPG13101 VJ 1941 germ 1968 LPG13102 VJ 1942 germ 1969 LPG13103 VJ 1943 germ 1970 LPG13104 VJ 1944 germ 1971 LPG13105 VJ 1945 germ 1972 LPG13106 VJ 1946 germ 1973 LPG13107 VJ 1947 germ 1974 LPG13108 VJ 1948 JAAYLU01 | JAAYLU01 sp002068355 1975 LPG13109 VJ 1949 germ 1976 LPG13110 VJ 1950 germ 1977 LPG13111 VJ 1951 germ 1978 LPG13112 VH 1952 Bacteroidia 1979 LPG13113 VH 1953 germ 1980 LPG13114 VH 1954 germ 1981 LPG13115 VI 1955 germ 1982 LPG13116 VJ 1956 germ 1983 Example 2 : Guided RNA identification and sgRNA construction

Guide RNA is determined by identifying crRNA written at CRISPR loci in the genome. Manual curation of RNA is performed using secondary structure prediction using RNAfold (an RNA folding software). sgRNA cassettes are prepared by DNA synthesis and are generally designed as follows (5'->3'): crRNA, operatively linked to a 25-30 bp spacer sequence at its 3' end.

For in vitro assays, the sgRNAs were synthesized by the service provider (Integrated DNA Technologies). The crRNA sequences of each RGN polypeptide were identified and are listed in Table 1. See below for the sgRNAs used in PAM libraries 1 and 2. Example 3 : Determining the PAM requirements for each RGN .

The PAM requirement for each RGN was determined using an in vitro transductive PAM determination method adapted from that of Karvelis et al. (2015) Genome Biol 253. In short, two plasmoid libraries (L1 and L2) were generated in the pUC18 backbone (ampR), each containing a distinct 30 bp protospacer (target) sequence flanked by 8 random nucleotides (i.e., PAM regions). The target sequences and flanking PAM regions for each RGN's library 1 and library 2 are listed in Table 2.

In short, protein synthesis is initiated in vitro from T7 promoter-driven bacterial phenoplasts using the PURExpress In Vitro Protein Synthesis kit (New England Biolabs). In the presence of appropriate sgRNA, plastids containing RGN-recognizable PAMs are cleaved. The cleaved DNA product is then end-repaired and A-tailed using T4 DNA polymerase and the Klenow fragment (3'-->5' exo-) (New England Biolabs). The transpeptides were ligated with terminally repaired and A-tailed products using T4 DNA polymerase (New England Biolabs). After cleaning, the products were amplified by PCR using primers complementary to the ligated transpeptides and plasmid backbone sequences, resulting in the inclusion of enriched PAM sequences in the amplicon. The service providers (MoGene, St. Louis, Missouri; Elevate Bio, Waltham, Massachusetts) performed deep sequencing (150 bp single terminal reads) on the NextSeq platform (Illumina). Typically, 500,000 reads were obtained per amplicon. For each sample, the PAM region was extracted, counted, and normalized to the total reads. PAMs that induce plasmonic shearing are identified by enrichment compared to the control group (i.e., the initiation frequencies of PAMs in the library). To present the PAM requirements for a new RGN, the deficiency rate (frequency in the sample / frequency in the control group) of all sequences in the region involved is converted into a position-frequency matrix. A sufficient PAM is defined as those PAMs that are significantly enriched compared to the frequencies in the control library. PAM sequences are identified and reported when a consistent pattern exists among the most enriched PAMs. The common PAMs for each RGN are provided in Table 2. PAM orientation is also indicated in Table 2. Table 2 : PAM or PAM Sample Determination RGN ID sgRNA L1 ( SEQ ID NO. ) sgRNA L2 ( SEQ ID NO. ) PAM PAM orientation LPG10238 12, 16 20, 24 AYG 5'-PAM-target-3' LPG10239 11, 15 19, 23 ATTN 5'-PAM-target-3' LPG10240 10, 14 18, 22 VTTN 5'-PAM-target-3' LPG10241 9, 13 17, 21 TTN 5'-PAM-target-3' LPG13090 1984 2011 TTN 5'-PAM-target-3' LPG13091 1985 2012 STTN 5'-PAM-target-3' LPG13092 1986 2013 TTN 5'-PAM-target-3' LPG13093 1987 2014 ATTN 5'-PAM-target-3' LPG13094 1988 2015 TTN 5'-PAM-target-3' LPG13095 1989 2016 TTH 5'-PAM-target-3' LPG13096 1990 2017 TTN 5'-PAM-target-3' LPG13097 1991 2018 RTTN 5'-PAM-target-3' LPG13098 1992 2019 VTTN 5'-PAM-target-3' LPG13099 1993 2020 TTN 5'-PAM-target-3' LPG13100 1994 2021 TTN 5'-PAM-target-3' LPG13101 1995 2022 TTN 5'-PAM-target-3' LPG13102 1996 2023 TTN 5'-PAM-target-3' LPG13103 1997 2024 VTTN 5'-PAM-target-3' LPG13104 1998 2025 ATTN 5'-PAM-target-3' LPG13105 1999 2026 VTTN 5'-PAM-target-3' LPG13106 2000 2027 TTN 5'-PAM-target-3' LPG13107 2001 2028 VTTN 5'-PAM-target-3' LPG13108 2002 2029 TTN 5'-PAM-target-3' LPG13109 2003 2030 VTTN 5'-PAM-target-3' LPG13110 2004 2031 RTTN 5'-PAM-target-3' LPG13111 2005 2032 RTYN 5'-PAM-target-3' LPG13112 2006 2033 TTN 5'-PAM-target-3' LPG13113 2007 2034 ATG 5'-PAM-target-3' LPG13114 2008 2035 TTN 5'-PAM-target-3' LPG13115 2009 2036 TTN 5'-PAM-target-3' LPG13116 2010 2037 TTN 5'-PAM-target-3' N is A, C, T/U, or G; Y is C or T/U; V is A, G, or C; S is C or G; H is A, C, or T/U; R is A or G. Example 4 : Evidence of gene editing activity in mammalian cells

RGN expression cassettes were generated and introduced into a vector for mammalian expression. The LPG10238 to LPG10241 RGN peptides were codon-optimized for human expression (SEQ ID NO: 29 to 32) and were operatively fused at the 5' end to the SV40 nuclear localization sequence (NLS; SEQ ID NO: 33) and the Gly-Ser conjugate (SEQ ID NO: 34), and at the 3' end to the conjugate (SEQ ID NO: 34) and the nucleoprotein NLS sequence (SEQ ID NO: 35). The LPG13090 to LPG13116 RGN peptides are codon-optimized for human expression (SEQ ID NO: 2659 to 2685) and are operatively fused at the 5' end to the SV40 nuclear localization sequence (NLS; SEQ ID NO: 33) and the Gly-Ser connective (SEQ ID NO: 1928), and at the 3' end to the cMYC NLS sequence (SEQ ID NO: 1927). Each expression cassette is under the control of a cytomegalovirus (CMV) promoter (SEQ ID NO: 36). CMV transcription enhancers (SEQ ID NO: 37), known in the art, can also be included in a construct including a CMV promoter. Each guide RNA expression construct encoding a single gRNA was generated under the control of the human RNA polymerase III U6 promoter (SEQ ID NO: 38) and introduced into a plasmid vector. The sequence of the target sequence for each guide RNA is listed in Table 3.

Several of the structures described above were introduced into mammalian cells. One day prior to transfection, ^{1 x 10⁴} HEK293T cells (Sigma) were plated in 96-well dishes in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). The following day, when cells reached 50-60% confluency, 160 ng of RGN expression ploids and 40 ng of a single gRNA expression ploidy system were co-transfected using a Lipofectamine 3000 kit (Thermo Scientific) according to the manufacturer's instructions. After 72 hours of growth, total genome DNA was harvested using the QuickExtract DNA extraction protocol (Biosearch Technologies/Lucigen) according to the manufacturer's instructions.

Then, the total genomic DNA was analyzed to determine the editing rate of each RGN for each genomic target. First, oligonucleotides were generated for PCR amplification and subsequent analysis of the amplified genomic target sites. The oligonucleotide sequences used are listed in Table 4.

All PCR reactions were performed using 12.5 μL of 2X Platinum Superfi II PCR Master Mix (Thermo Scientific) in 25 μL of reaction mixture (containing 0.5 μM per primer). First, large genomic regions covering each target gene were amplified using the following program: 98°C, 1 min; 35 cycles of [98°C, 10 s; 60°C, 15 s; 72°C, 15 min]; 72°C, 5 min; 12°C, indefinite. These primers contained Nextera reads 1 and 2 for the Illumina transducer and barcode addition for Illumina sequencing.

Two different genes in the human genome are targeted for RNA-guided cleavage. These loci, along with the SEQ ID NOs of the mentioned sgRNAs, are listed in Table 3 below.surface 3 Used to test gene editing activity in mammalian cells. Guidance RNA The target and sgRNA sequence RGN ID Gene Name Guide body name sgRNA ( SEQ ID NO. ) Target sequence ( SEQ ID NO. ) Septal sequence ( SEQ ID NO. ) LPG10238 B2M SGN008512 39 183 294 LPG10238 B2M SGN008513 40 184 295 LPG10238 B2M SGN008514 41 185 296 LPG10238 B2M SGN008515 42 186 297 LPG10238 B2M SGN008516 43 187 298 LPG10238 B2M SGN008517 44 188 299 LPG10238 B2M SGN008518 45 189 300 LPG10238 B2M SGN008519 46 190 301 LPG10238 B2M SGN008520 47 191 302 LPG10238 B2M SGN008521 48 192 303 LPG10239 B2M SGN008522 49 193 304 LPG10239 B2M SGN008523 50 194 305 LPG10239 B2M SGN008524 51 195 306 LPG10239 B2M SGN008525 52 196 307 LPG10239 B2M SGN008526 53 197 308 LPG10239 B2M SGN008527 54 198 309 LPG10239 B2M SGN008528 55 199 310 LPG10239 B2M SGN008529 56 200 311 LPG10239 B2M SGN008530 57 201 312 LPG10239 B2M SGN008531 58 202 313 LPG10241 B2M SGN008532 59 193 304 LPG10241 B2M SGN008533 60 194 305 LPG10241 B2M SGN008534 61 195 306 LPG10241 B2M SGN008535 62 196 307 LPG10241 B2M SGN008536 63 203 314 LPG10241 B2M SGN008537 64 204 315 LPG10241 B2M SGN008538 65 205 316 LPG10241 B2M SGN008539 66 206 317 LPG10241 B2M SGN008540 67 207 318 LPG10241 B2M SGN008541 68 208 319 LPG10240 B2M SGN008542 69 193 304 LPG10240 B2M SGN008543 70 194 305 LPG10240 B2M SGN008544 71 195 306 LPG10240 B2M SGN008545 72 196 307 LPG10240 B2M SGN008546 73 203 314 LPG10240 B2M SGN008547 74 204 315 LPG10240 B2M SGN008548 75 205 316 LPG10240 B2M SGN008549 76 206 317 LPG10240 B2M SGN008550 77 207 318 LPG10240 B2M SGN008551 78 208 319 LPG10241 B2M SGN008552 79 209 320 LPG10241 B2M SGN008553 80 210 321 LPG10241 B2M SGN008554 81 211 322 LPG10241 B2M SGN008555 82 212 323 LPG10241 B2M SGN008556 83 213 324 LPG10241 B2M SGN008557 84 214 325 LPG10241 B2M SGN008558 85 215 326 LPG10241 B2M SGN008559 86 216 327 LPG10241 B2M SGN008560 87 217 328 LPG10241 B2M SGN008561 88 218 329 LPG10240 B2M SGN008562 89 209 320 LPG10240 B2M SGN008563 90 210 321 LPG10240 B2M SGN008564 91 211 322 LPG10240 B2M SGN008565 92 212 323 LPG10240 B2M SGN008566 93 213 324 LPG10240 B2M SGN008567 94 214 325 LPG10240 B2M SGN008568 95 215 326 LPG10240 B2M SGN008569 96 216 327 LPG10240 B2M SGN008570 97 217 328 LPG10240 B2M SGN008571 98 218 329 LPG10239 B2M SGN008572 99 209 320 LPG10239 B2M SGN008573 100 210 321 LPG10239 B2M SGN008574 101 211 322 LPG10239 B2M SGN008575 102 213 324 LPG10239 B2M SGN008576 103 219 330 LPG10239 B2M SGN008577 104 220 331 LPG10239 B2M SGN008578 105 221 332 LPG10239 B2M SGN008579 106 222 333 LPG10239 B2M SGN008580 107 223 334 LPG10239 B2M SGN008581 108 224 335 LPG10238 B2M SGN008592 109 226 337 LPG10238 B2M SGN008593 110 227 338 LPG10238 B2M SGN008594 111 228 339 LPG10238 B2M SGN008595 112 229 340 LPG10238 B2M SGN008596 113 230 341 LPG10238 B2M SGN008597 114 231 342 LPG10238 B2M SGN008598 115 232 343 LPG10238 B2M SGN008599 116 233 344 LPG10238 B2M SGN008600 117 234 345 LPG10238 B2M SGN008601 118 235 346 LPG10238 B2M SGN008602 119 236 347 LPG10238 B2M SGN008603 120 237 348 LPG10238 B2M SGN008604 121 238 349 LPG10238 B2M SGN008605 122 239 350 LPG10238 B2M SGN008609 123 225 336 LPG10238 B2M SGN008610 124 240 351 LPG10238 B2M SGN008611 125 241 352 LPG10238 B2M SGN008612 126 242 353 LPG10238 TRAC SGN008613 127 243 354 LPG10238 TRAC SGN008614 128 244 355 LPG10238 TRAC SGN008615 129 245 356 LPG10238 TRAC SGN008616 130 246 357 LPG10238 TRAC SGN008617 131 247 358 LPG10238 TRAC SGN008618 132 248 359 LPG10238 TRAC SGN008619 133 249 360 LPG10238 TRAC SGN008620 134 250 361 LPG10238 TRAC SGN008621 135 251 362 LPG10241 B2M SGN008636 136 252 363 LPG10241 B2M SGN008637 137 253 364 LPG10241 B2M SGN008638 138 254 365 LPG10241 B2M SGN008639 139 255 366 LPG10241 B2M SGN008640 140 256 367 LPG10241 B2M SGN008641 141 257 368 LPG10241 B2M SGN008642 142 258 369 LPG10241 B2M SGN008643 143 259 370 LPG10241 TRAC SGN008645 144 260 371 LPG10241 TRAC SGN008646 145 261 372 LPG10241 TRAC SGN008647 146 262 373 LPG10241 TRAC SGN008648 147 263 374 LPG10241 TRAC SGN008649 148 264 375 LPG10241 TRAC SGN008650 149 265 376 LPG10241 TRAC SGN008651 150 266 377 LPG10241 TRAC SGN008652 151 267 378 LPG10241 TRAC SGN008653 152 268 379 LPG10241 TRAC SGN008654 153 269 380 LPG10240 B2M SGN008655 154 270 381 LPG10240 B2M SGN008656 155 257 368 LPG10240 B2M SGN008657 156 219 330 LPG10240 B2M SGN008658 157 224 335 LPG10240 B2M SGN008659 158 271 382 LPG10240 B2M SGN008660 159 259 370 LPG10240 B2M SGN008661 160 272 383 LPG10240 TRAC SGN008662 161 265 376 LPG10240 TRAC SGN008663 162 273 384 LPG10240 TRAC SGN008664 163 274 385 LPG10240 TRAC SGN008665 164 275 386 LPG10240 TRAC SGN008666 165 276 387 LPG10240 TRAC SGN008668 166 269 380 LPG10240 TRAC SGN008669 167 267 378 LPG10240 TRAC SGN008670 168 268 379 LPG10240 TRAC SGN008671 169 277 388 LPG10239 B2M SGN008672 170 278 389 LPG10239 B2M SGN008673 171 270 381 LPG10239 B2M SGN008674 172 257 368 LPG10239 TRAC SGN008677 173 279 390 LPG10239 TRAC SGN008678 174 265 376 LPG10239 TRAC SGN008679 175 280 391 LPG10239 TRAC SGN008680 176 281 392 LPG10239 TRAC SGN008681 177 282 393 LPG10239 TRAC SGN008682 178 274 385 LPG10239 TRAC SGN008683 179 283 394 LPG10239 TRAC SGN008684 180 284 395 LPG10239 TRAC SGN008685 181 285 396 LPG10239 TRAC SGN008686 182 273 384 LPG13090 B2M SGN013365 2126 193 304 LPG13090 B2M SGN013366 2127 194 305 LPG13090 B2M SGN013367 2128 195 306 LPG13090 B2M SGN013368 2129 203 314 LPG13090 B2M SGN013369 2130 2038 2082 LPG13090 B2M SGN013370 2131 2039 2083 LPG13090 B2M SGN013371 2132 205 316 LPG13090 B2M SGN013372 2133 206 317 LPG13090 B2M SGN013373 2134 2040 2084 LPG13090 B2M SGN013374 2135 2041 2085 LPG13090 B2M SGN013375 2136 207 318 LPG13090 B2M SGN013376 2137 2042 2086 LPG13090 B2M SGN013377 2138 2043 2087 LPG13090 B2M SGN013378 2139 2044 2088 LPG13090 B2M SGN013379 2140 198 309 LPG13090 B2M SGN013380 2141 2045 2089 LPG13090 B2M SGN013381 2142 2046 2090 LPG13090 B2M SGN013382 2143 199 310 LPG13090 TRAC SGN013383 2144 1775 1880 LPG13090 TRAC SGN013384 2145 2047 2091 LPG13092 B2M SGN013385 2146 193 304 LPG13092 B2M SGN013386 2147 194 305 LPG13092 B2M SGN013387 2148 195 306 LPG13092 B2M SGN013388 2149 203 314 LPG13092 B2M SGN013389 2150 2038 2082 LPG13092 B2M SGN013390 2151 2039 2083 LPG13092 B2M SGN013391 2152 205 316 LPG13092 B2M SGN013392 2153 206 317 LPG13092 B2M SGN013393 2154 2040 2084 LPG13092 B2M SGN013394 2155 2041 2085 LPG13092 B2M SGN013395 2156 207 318 LPG13092 B2M SGN013396 2157 2042 2086 LPG13092 B2M SGN013397 2158 2043 2087 LPG13092 B2M SGN013398 2159 2044 2088 LPG13092 B2M SGN013399 2160 198 309 LPG13092 B2M SGN013400 2161 2045 2089 LPG13092 B2M SGN013401 2162 2046 2090 LPG13092 B2M SGN013402 2163 199 310 LPG13092 TRAC SGN013403 2164 1775 1880 LPG13092 TRAC SGN013404 2165 2047 2091 LPG13093, LPG13106 B2M SGN013405 2166 193 304 LPG13093, LPG13106 B2M SGN013406 2167 194 305 LPG13093, LPG13106 B2M SGN013407 2168 195 306 LPG13093, LPG13106 B2M SGN013408 2169 2039 2083 LPG13093, LPG13106 B2M SGN013409 2170 2041 2085 LPG13093, LPG13106 B2M SGN013410 2171 198 309 LPG13093, LPG13106 B2M SGN013411 2172 199 310 LPG13093 B2M SGN013412 2173 196 307 LPG13093 B2M SGN013413 2174 197 308 LPG13093 B2M SGN013414 2175 200 311 LPG13093 B2M SGN013415 2176 201 312 LPG13093 B2M SGN013416 2177 202 313 LPG13093 B2M SGN013417 2178 2048 2092 LPG13093 TRAC SGN013418 2179 2049 2093 LPG13093 TRAC SGN013419 2180 1771 1876 LPG13093 TRAC SGN013420 2181 2050 2094 LPG13093 TRAC SGN013421 2182 2051 2095 LPG13093 TRAC SGN013422 2183 1766 1871 LPG13093 TRAC SGN013423 2184 2052 2096 LPG13093 TRAC SGN013424 2185 2053 2097 LPG13094 B2M SGN013425 2186 193 304 LPG13094 B2M SGN013426 2187 194 305 LPG13094 B2M SGN013427 2188 195 306 LPG13094 B2M SGN013428 2189 203 314 LPG13094 B2M SGN013429 2190 2038 2082 LPG13094 B2M SGN013430 2191 2039 2083 LPG13094 B2M SGN013431 2192 205 316 LPG13094 B2M SGN013432 2193 206 317 LPG13094 B2M SGN013433 2194 2040 2084 LPG13094 B2M SGN013434 2195 2041 2085 LPG13094 B2M SGN013435 2196 207 318 LPG13094 B2M SGN013436 2197 2042 2086 LPG13094 B2M SGN013437 2198 2043 2087 LPG13094 B2M SGN013438 2199 2044 2088 LPG13094 B2M SGN013439 2200 198 309 LPG13094 B2M SGN013440 2201 2045 2089 LPG13094 B2M SGN013441 2202 2046 2090 LPG13094 B2M SGN013442 2203 199 310 LPG13094 TRAC SGN013443 2204 1775 1880 LPG13094 TRAC SGN013444 2205 2047 2091 LPG13095 B2M SGN013445 2206 193 304 LPG13095 B2M SGN013446 2207 194 305 LPG13095 B2M SGN013447 2208 195 306 LPG13095 B2M SGN013448 2209 203 314 LPG13095 B2M SGN013449 2210 2038 2082 LPG13095 B2M SGN013450 2211 2039 2083 LPG13095 B2M SGN013451 2212 205 316 LPG13095 B2M SGN013452 2213 206 317 LPG13095 B2M SGN013453 2214 2040 2084 LPG13095 B2M SGN013454 2215 2041 2085 LPG13095 B2M SGN013455 2216 207 318 LPG13095 B2M SGN013456 2217 2042 2086 LPG13095 B2M SGN013457 2218 2043 2087 LPG13095 B2M SGN013458 2219 2044 2088 LPG13095 B2M SGN013459 2220 198 309 LPG13095 B2M SGN013460 2221 2045 2089 LPG13095 B2M SGN013461 2222 2046 2090 LPG13095 B2M SGN013462 2223 199 310 LPG13095 TRAC SGN013463 2224 1775 1880 LPG13095 TRAC SGN013464 2225 2047 2091 LPG13096 B2M SGN013465 2226 193 304 LPG13096 B2M SGN013466 2227 194 305 LPG13096 B2M SGN013467 2228 195 306 LPG13096 B2M SGN013468 2229 203 314 LPG13096 B2M SGN013469 2230 2038 2082 LPG13096 B2M SGN013470 2231 2039 2083 LPG13096 B2M SGN013471 2232 205 316 LPG13096 B2M SGN013472 2233 206 317 LPG13096 B2M SGN013473 2234 2040 2084 LPG13096 B2M SGN013474 2235 2041 2085 LPG13096 B2M SGN013475 2236 207 318 LPG13096 B2M SGN013476 2237 2042 2086 LPG13096 B2M SGN013477 2238 2043 2087 LPG13096 B2M SGN013478 2239 2044 2088 LPG13096 B2M SGN013479 2240 198 309 LPG13096 B2M SGN013480 2241 2045 2089 LPG13096 B2M SGN013481 2242 2046 2090 LPG13096 B2M SGN013482 2243 199 310 LPG13096 TRAC SGN013483 2244 1775 1880 LPG13096 TRAC SGN013484 2245 2047 2091 LPG13097 B2M SGN013485 2246 193 304 LPG13097 B2M SGN013486 2247 194 305 LPG13097 B2M SGN013487 2248 195 306 LPG13097 B2M SGN013488 2249 203 314 LPG13097 B2M SGN013489 2250 198 309 LPG13097 B2M SGN013490 2251 199 310 LPG13097 B2M SGN013491 2252 2054 2098 LPG13097 B2M SGN013492 2253 208 319 LPG13097 B2M SGN013493 2254 196 307 LPG13097 B2M SGN013494 2255 197 308 LPG13097 B2M SGN013495 2256 200 311 LPG13097 B2M SGN013496 2257 201 312 LPG13097 B2M SGN013497 2258 202 313 LPG13097 B2M SGN013498 2259 2048 2092 LPG13097 TRAC SGN013499 2260 1776 1881 LPG13097 TRAC SGN013500 2261 2052 2096 LPG13097 TRAC SGN013501 2262 2055 2099 LPG13097 TRAC SGN013502 2263 2056 2100 LPG13097 TRAC SGN013503 2264 2057 2101 LPG13097 TRAC SGN013504 2265 2058 2102 LPG13098 B2M SGN013505 2266 193 304 LPG13098 B2M SGN013506 2267 194 305 LPG13098 B2M SGN013507 2268 195 306 LPG13098 B2M SGN013508 2269 203 314 LPG13098 B2M SGN013509 2270 2038 2082 LPG13098 B2M SGN013510 2271 2039 2083 LPG13098 B2M SGN013511 2272 205 316 LPG13098 B2M SGN013512 2273 206 317 LPG13098 B2M SGN013513 2274 2040 2084 LPG13098 B2M SGN013514 2275 2041 2085 LPG13098 B2M SGN013515 2276 207 318 LPG13098 B2M SGN013516 2277 2042 2086 LPG13098 B2M SGN013517 2278 2043 2087 LPG13098 B2M SGN013518 2279 198 309 LPG13098 B2M SGN013519 2280 2045 2089 LPG13098 B2M SGN013520 2281 199 310 LPG13098 TRAC SGN013521 2282 1775 1880 LPG13098 TRAC SGN013522 2283 2047 2091 LPG13098 TRAC SGN013523 2284 2059 2103 LPG13098 TRAC SGN013524 2285 2060 2104 LPG13099 B2M SGN013525 2286 193 304 LPG13099 B2M SGN013526 2287 194 305 LPG13099 B2M SGN013527 2288 195 306 LPG13099 B2M SGN013528 2289 203 314 LPG13099 B2M SGN013529 2290 2038 2082 LPG13099 B2M SGN013530 2291 2039 2083 LPG13099 B2M SGN013531 2292 205 316 LPG13099 B2M SGN013532 2293 206 317 LPG13099 B2M SGN013533 2294 2040 2084 LPG13099 B2M SGN013534 2295 2041 2085 LPG13099 B2M SGN013535 2296 207 318 LPG13099 B2M SGN013536 2297 2042 2086 LPG13099 B2M SGN013537 2298 2043 2087 LPG13099 B2M SGN013538 2299 2044 2088 LPG13099 B2M SGN013539 2300 198 309 LPG13099 B2M SGN013540 2301 2045 2089 LPG13099 B2M SGN013541 2302 2046 2090 LPG13099 B2M SGN013542 2303 199 310 LPG13099 TRAC SGN013543 2304 1775 1880 LPG13099 TRAC SGN013544 2305 2047 2091 LPG13100 B2M SGN013545 2306 193 304 LPG13100 B2M SGN013546 2307 194 305 LPG13100 B2M SGN013547 2308 195 306 LPG13100 B2M SGN013548 2309 203 314 LPG13100 B2M SGN013549 2310 2038 2082 LPG13100 B2M SGN013550 2311 2039 2083 LPG13100 B2M SGN013551 2312 205 316 LPG13100 B2M SGN013552 2313 206 317 LPG13100 B2M SGN013553 2314 2040 2084 LPG13100 B2M SGN013554 2315 2041 2085 LPG13100 B2M SGN013555 2316 207 318 LPG13100 B2M SGN013556 2317 2042 2086 LPG13100 B2M SGN013557 2318 2043 2087 LPG13100 B2M SGN013558 2319 2044 2088 LPG13100 B2M SGN013559 2320 198 309 LPG13100 B2M SGN013560 2321 2045 2089 LPG13100 B2M SGN013561 2322 2046 2090 LPG13100 B2M SGN013562 2323 199 310 LPG13100 TRAC SGN013563 2324 1775 1880 LPG13100 TRAC SGN013564 2325 2047 2091 LPG13101 B2M SGN013565 2326 193 304 LPG13101 B2M SGN013566 2327 194 305 LPG13101 B2M SGN013567 2328 195 306 LPG13101 B2M SGN013568 2329 203 314 LPG13101 B2M SGN013569 2330 2038 2082 LPG13101 B2M SGN013570 2331 2039 2083 LPG13101 B2M SGN013571 2332 205 316 LPG13101 B2M SGN013572 2333 206 317 LPG13101 B2M SGN013573 2334 2040 2084 LPG13101 B2M SGN013574 2335 2041 2085 LPG13101 B2M SGN013575 2336 207 318 LPG13101 B2M SGN013576 2337 2042 2086 LPG13101 B2M SGN013577 2338 2043 2087 LPG13101 B2M SGN013578 2339 2044 2088 LPG13101 B2M SGN013579 2340 198 309 LPG13101 B2M SGN013580 2341 2045 2089 LPG13101 B2M SGN013581 2342 2046 2090 LPG13101 B2M SGN013582 2343 199 310 LPG13101 TRAC SGN013583 2344 1775 1880 LPG13101 TRAC SGN013584 2345 2047 2091 LPG13102 B2M SGN013585 2346 193 304 LPG13102 B2M SGN013586 2347 194 305 LPG13102 B2M SGN013587 2348 195 306 LPG13102 B2M SGN013588 2349 203 314 LPG13102 B2M SGN013589 2350 2038 2082 LPG13102 B2M SGN013590 2351 2039 2083 LPG13102 B2M SGN013591 2352 205 316 LPG13102 B2M SGN013592 2353 206 317 LPG13102 B2M SGN013593 2354 2040 2084 LPG13102 B2M SGN013594 2355 2041 2085 LPG13102 B2M SGN013595 2356 207 318 LPG13102 B2M SGN013596 2357 2042 2086 LPG13102 B2M SGN013597 2358 2043 2087 LPG13102 B2M SGN013598 2359 2044 2088 LPG13102 B2M SGN013599 2360 198 309 LPG13102 B2M SGN013600 2361 2045 2089 LPG13102 B2M SGN013601 2362 2046 2090 LPG13102 B2M SGN013602 2363 199 310 LPG13102 TRAC SGN013603 2364 1775 1880 LPG13102 TRAC SGN013604 2365 2047 2091 LPG13103 B2M SGN013605 2366 193 304 LPG13103 B2M SGN013606 2367 194 305 LPG13103 B2M SGN013607 2368 195 306 LPG13103 B2M SGN013608 2369 203 314 LPG13103 B2M SGN013609 2370 2038 2082 LPG13103 B2M SGN013610 2371 2039 2083 LPG13103 B2M SGN013611 2372 205 316 LPG13103 B2M SGN013612 2373 206 317 LPG13103 B2M SGN013613 2374 2040 2084 LPG13103 B2M SGN013614 2375 2041 2085 LPG13103 B2M SGN013615 2376 207 318 LPG13103 B2M SGN013616 2377 2042 2086 LPG13103 B2M SGN013617 2378 2043 2087 LPG13103 B2M SGN013618 2379 198 309 LPG13103 B2M SGN013619 2380 2045 2089 LPG13103 B2M SGN013620 2381 199 310 LPG13103 TRAC SGN013621 2382 1775 1880 LPG13103 TRAC SGN013622 2383 2047 2091 LPG13103 TRAC SGN013623 2384 2059 2103 LPG13103 TRAC SGN013624 2385 2060 2104 LPG13104 B2M SGN013625 2386 193 304 LPG13104 B2M SGN013626 2387 194 305 LPG13104 B2M SGN013627 2388 195 306 LPG13104 B2M SGN013628 2389 2039 2083 LPG13104 B2M SGN013629 2390 2041 2085 LPG13104 B2M SGN013630 2391 198 309 LPG13104 B2M SGN013631 2392 199 310 LPG13104 B2M SGN013632 2393 196 307 LPG13104 B2M SGN013633 2394 197 308 LPG13104 B2M SGN013634 2395 200 311 LPG13104 B2M SGN013635 2396 201 312 LPG13104 B2M SGN013636 2397 202 313 LPG13104 B2M SGN013637 2398 2048 2092 LPG13104 TRAC SGN013638 2399 2049 2093 LPG13104 TRAC SGN013639 2400 1771 1876 LPG13104 TRAC SGN013640 2401 2050 2094 LPG13104 TRAC SGN013641 2402 2051 2095 LPG13104 TRAC SGN013642 2403 1776 1881 LPG13104 TRAC SGN013643 2404 2052 2096 LPG13104 TRAC SGN013644 2405 2053 2097 LPG13105 B2M SGN013645 2406 193 304 LPG13105 B2M SGN013646 2407 194 305 LPG13105 B2M SGN013647 2408 195 306 LPG13105 B2M SGN013648 2409 203 314 LPG13105 B2M SGN013649 2410 2038 2082 LPG13105 B2M SGN013650 2411 2039 2083 LPG13105 B2M SGN013651 2412 205 316 LPG13105 B2M SGN013652 2413 206 317 LPG13105 B2M SGN013653 2414 2040 2084 LPG13105 B2M SGN013654 2415 2041 2085 LPG13105 B2M SGN013655 2416 207 318 LPG13105 B2M SGN013656 2417 2042 2086 LPG13105 B2M SGN013657 2418 2043 2087 LPG13105 B2M SGN013658 2419 198 309 LPG13105 B2M SGN013659 2420 2045 2089 LPG13105 B2M SGN013660 2421 199 310 LPG13105 TRAC SGN013661 2422 1775 1880 LPG13105 TRAC SGN013662 2423 2047 2091 LPG13105 TRAC SGN013663 2424 2059 2103 LPG13105 TRAC SGN013664 2425 2060 2104 LPG13106 B2M SGN013668 2426 203 314 LPG13106 B2M SGN013669 2427 2038 2082 LPG13106 B2M SGN013671 2428 205 316 LPG13106 B2M SGN013672 2429 206 317 LPG13106 B2M SGN013673 2430 2040 2084 LPG13106 B2M SGN013675 2431 207 318 LPG13106 B2M SGN013676 2432 2042 2086 LPG13106 B2M SGN013677 2433 2043 2087 LPG13106 B2M SGN013678 2434 2044 2088 LPG13106 B2M SGN013680 2435 2045 2089 LPG13106 B2M SGN013681 2436 2046 2090 LPG13106 TRAC SGN013683 2437 1775 1880 LPG13106 TRAC SGN013684 2438 2047 2091 LPG13107 B2M SGN013685 2439 193 304 LPG13107 B2M SGN013686 2440 194 305 LPG13107 B2M SGN013687 2441 195 306 LPG13107 B2M SGN013688 2442 203 314 LPG13107 B2M SGN013689 2443 2038 2082 LPG13107 B2M SGN013690 2444 2039 2083 LPG13107 B2M SGN013691 2445 205 316 LPG13107 B2M SGN013692 2446 206 317 LPG13107 B2M SGN013693 2447 2040 2084 LPG13107 B2M SGN013694 2448 2041 2085 LPG13107 B2M SGN013695 2449 207 318 LPG13107 B2M SGN013696 2450 2042 2086 LPG13107 B2M SGN013697 2451 2043 2087 LPG13107 B2M SGN013698 2452 198 309 LPG13107 B2M SGN013699 2453 2045 2089 LPG13107 B2M SGN013700 2454 199 310 LPG13107 TRAC SGN013701 2455 1775 1880 LPG13107 TRAC SGN013702 2456 2047 2091 LPG13107 TRAC SGN013703 2457 2059 2103 LPG13107 TRAC SGN013704 2458 2060 2104 LPG13108 B2M SGN013705 2459 193 304 LPG13108 B2M SGN013706 2460 194 305 LPG13108 B2M SGN013707 2461 195 306 LPG13108 B2M SGN013708 2462 203 314 LPG13108 B2M SGN013709 2463 2038 2082 LPG13108 B2M SGN013710 2464 2039 2083 LPG13108 B2M SGN013711 2465 205 316 LPG13108 B2M SGN013712 2466 206 317 LPG13108 B2M SGN013713 2467 2040 2084 LPG13108 B2M SGN013714 2468 2041 2085 LPG13108 B2M SGN013715 2469 207 318 LPG13108 B2M SGN013716 2470 2042 2086 LPG13108 B2M SGN013717 2471 2043 2087 LPG13108 B2M SGN013718 2472 2044 2088 LPG13108 B2M SGN013719 2473 198 309 LPG13108 B2M SGN013720 2474 2045 2089 LPG13108 B2M SGN013721 2475 2046 2090 LPG13108 B2M SGN013722 2476 199 310 LPG13108 TRAC SGN013723 2477 1775 1880 LPG13108 TRAC SGN013724 2478 2047 2091 LPG13109 B2M SGN013725 2479 193 304 LPG13109 B2M SGN013726 2480 194 305 LPG13109 B2M SGN013727 2481 195 306 LPG13109 B2M SGN013728 2482 203 314 LPG13109 B2M SGN013729 2483 2038 2082 LPG13109 B2M SGN013730 2484 2039 2083 LPG13109 B2M SGN013731 2485 205 316 LPG13109 B2M SGN013732 2486 206 317 LPG13109 B2M SGN013733 2487 2040 2084 LPG13109 B2M SGN013734 2488 2041 2085 LPG13109 B2M SGN013735 2489 207 318 LPG13109 B2M SGN013736 2490 2042 2086 LPG13109 B2M SGN013737 2491 2043 2087 LPG13109 B2M SGN013738 2492 198 309 LPG13109 B2M SGN013739 2493 2045 2089 LPG13109 B2M SGN013740 2494 199 310 LPG13109 TRAC SGN013741 2495 1775 1880 LPG13109 TRAC SGN013742 2496 2047 2091 LPG13109 TRAC SGN013743 2497 2059 2103 LPG13109 TRAC SGN013744 2498 2060 2104 LPG13110 B2M SGN013745 2499 193 304 LPG13110 B2M SGN013746 2500 194 305 LPG13110 B2M SGN013747 2501 195 306 LPG13110 B2M SGN013748 2502 203 314 LPG13110 B2M SGN013749 2503 198 309 LPG13110 B2M SGN013750 2504 199 310 LPG13110 B2M SGN013751 2505 2054 2098 LPG13110 B2M SGN013752 2506 208 319 LPG13110 B2M SGN013753 2507 196 307 LPG13110 B2M SGN013754 2508 197 308 LPG13110 B2M SGN013755 2509 200 311 LPG13110 B2M SGN013756 2510 201 312 LPG13110 B2M SGN013757 2511 202 313 LPG13110 B2M SGN013758 2512 2048 2092 LPG13110 TRAC SGN013759 2513 1776 1881 LPG13110 TRAC SGN013760 2514 2052 2096 LPG13110 TRAC SGN013761 2515 2055 2099 LPG13110 TRAC SGN013762 2516 2056 2100 LPG13110 TRAC SGN013763 2517 2057 2101 LPG13110 TRAC SGN013764 2518 2058 2102 LPG13111 B2M SGN013765 2519 193 304 LPG13111 B2M SGN013766 2520 194 305 LPG13111 B2M SGN013767 2521 195 306 LPG13111 B2M SGN013768 2522 203 314 LPG13111 B2M SGN013769 2523 2039 2083 LPG13111 B2M SGN013770 2524 2041 2085 LPG13111 B2M SGN013771 2525 2042 2086 LPG13111 B2M SGN013772 2526 198 309 LPG13111 B2M SGN013773 2527 199 310 LPG13111 B2M SGN013774 2528 2061 2105 LPG13111 B2M SGN013775 2529 2062 2106 LPG13111 B2M SGN013776 2530 2063 2107 LPG13111 B2M SGN013777 2531 2064 2108 LPG13111 B2M SGN013778 2532 2065 2109 LPG13111 B2M SGN013779 2533 2066 2110 LPG13111 B2M SGN013780 2534 2067 2111 LPG13111 TRAC SGN013781 2535 2068 2112 LPG13111 TRAC SGN013782 2536 2069 2113 LPG13111 TRAC SGN013783 2537 2070 2114 LPG13111 TRAC SGN013784 2538 2071 2115 LPG13112 B2M SGN013785 2539 193 304 LPG13112 B2M SGN013786 2540 194 305 LPG13112 B2M SGN013787 2541 195 306 LPG13112 B2M SGN013788 2542 203 314 LPG13112 B2M SGN013789 2543 2038 2082 LPG13112 B2M SGN013790 2544 2039 2083 LPG13112 B2M SGN013791 2545 205 316 LPG13112 B2M SGN013792 2546 206 317 LPG13112 B2M SGN013793 2547 2040 2084 LPG13112 B2M SGN013794 2548 2041 2085 LPG13112 B2M SGN013795 2549 207 318 LPG13112 B2M SGN013796 2550 2042 2086 LPG13112 B2M SGN013797 2551 2043 2087 LPG13112 B2M SGN013798 2552 2044 2088 LPG13112 B2M SGN013799 2553 198 309 LPG13112 B2M SGN013800 2554 2045 2089 LPG13112 B2M SGN013801 2555 2046 2090 LPG13112 B2M SGN013802 2556 199 310 LPG13112 TRAC SGN013803 2557 1775 1880 LPG13112 TRAC SGN013804 2558 2047 2091 LPG13114 B2M SGN013805 2559 193 304 LPG13114 B2M SGN013806 2560 194 305 LPG13114 B2M SGN013807 2561 195 306 LPG13114 B2M SGN013808 2562 203 314 LPG13114 B2M SGN013809 2563 2038 2082 LPG13114 B2M SGN013810 2564 2039 2083 LPG13114 B2M SGN013811 2565 205 316 LPG13114 B2M SGN013812 2566 206 317 LPG13114 B2M SGN013813 2567 2040 2084 LPG13114 B2M SGN013814 2568 2041 2085 LPG13114 B2M SGN013815 2569 207 318 LPG13114 B2M SGN013816 2570 2042 2086 LPG13114 B2M SGN013817 2571 2043 2087 LPG13114 B2M SGN013818 2572 2044 2088 LPG13114 B2M SGN013819 2573 198 309 LPG13114 B2M SGN013820 2574 2045 2089 LPG13114 B2M SGN013821 2575 2046 2090 LPG13114 B2M SGN013822 2576 199 310 LPG13114 TRAC SGN013823 2577 1775 1880 LPG13114 TRAC SGN013824 2578 2047 2091 LPG13115 B2M SGN013825 2579 193 304 LPG13115 B2M SGN013826 2580 194 305 LPG13115 B2M SGN013827 2581 195 306 LPG13115 B2M SGN013828 2582 203 314 LPG13115 B2M SGN013829 2583 2038 2082 LPG13115 B2M SGN013830 2584 2039 2083 LPG13115 B2M SGN013831 2585 205 316 LPG13115 B2M SGN013832 2586 206 317 LPG13115 B2M SGN013833 2587 2040 2084 LPG13115 B2M SGN013834 2588 2041 2085 LPG13115 B2M SGN013835 2589 207 318 LPG13115 B2M SGN013836 2590 2042 2086 LPG13115 B2M SGN013837 2591 2043 2087 LPG13115 B2M SGN013838 2592 2044 2088 LPG13115 B2M SGN013839 2593 198 309 LPG13115 B2M SGN013840 2594 2045 2089 LPG13115 B2M SGN013841 2595 2046 2090 LPG13115 B2M SGN013842 2596 199 310 LPG13115 TRAC SGN013843 2597 1775 1880 LPG13115 TRAC SGN013844 2598 2047 2091 LPG13116 B2M SGN013845 2599 193 304 LPG13116 B2M SGN013846 2600 194 305 LPG13116 B2M SGN013847 2601 195 306 LPG13116 B2M SGN013848 2602 203 314 LPG13116 B2M SGN013849 2603 2038 2082 LPG13116 B2M SGN013850 2604 2039 2083 LPG13116 B2M SGN013851 2605 205 316 LPG13116 B2M SGN013852 2606 206 317 LPG13116 B2M SGN013853 2607 2040 2084 LPG13116 B2M SGN013854 2608 2041 2085 LPG13116 B2M SGN013855 2609 207 318 LPG13116 B2M SGN013856 2610 2042 2086 LPG13116 B2M SGN013857 2611 2043 2087 LPG13116 B2M SGN013858 2612 2044 2088 LPG13116 B2M SGN013859 2613 198 309 LPG13116 B2M SGN013860 2614 2045 2089 LPG13116 B2M SGN013861 2615 2046 2090 LPG13116 B2M SGN013862 2616 199 310 LPG13116 TRAC SGN013863 2617 1775 1880 LPG13116 TRAC SGN013864 2618 2047 2091 LPG13091 B2M SGN013865 2619 203 314 LPG13091 B2M SGN013866 2620 2038 2082 LPG13091 B2M SGN013867 2621 205 316 LPG13091 B2M SGN013868 2622 206 317 LPG13091 B2M SGN013869 2623 207 318 LPG13091 B2M SGN013870 2624 2045 2089 LPG13091 B2M SGN013871 2625 1757 1862 LPG13091 B2M SGN013872 2626 2054 2098 LPG13091 B2M SGN013873 2627 2072 2116 LPG13091 B2M SGN013874 2628 204 315 LPG13091 B2M SGN013875 2629 208 319 LPG13091 B2M SGN013876 2630 2073 2117 LPG13091 B2M SGN013877 2631 2074 2118 LPG13091 B2M SGN013878 2632 2075 2119 LPG13091 B2M SGN013879 2633 2076 2120 LPG13091 B2M SGN013880 2634 2077 2121 LPG13091 B2M SGN013881 2635 2078 2122 LPG13091 B2M SGN013882 2636 2079 2123 LPG13091 TRAC SGN013883 2637 2080 2124 LPG13091 TRAC SGN013884 2638 1691 1796 LPG13113 B2M SGN013885 2639 2081 2125 LPG13113 B2M SGN013886 2640 1738 1843 LPG13113 B2M SGN013887 2641 183 294 LPG13113 B2M SGN013888 2642 184 295 LPG13113 B2M SGN013889 2643 185 296 LPG13113 B2M SGN013890 2644 186 297 LPG13113 B2M SGN013891 2645 187 298 LPG13113 B2M SGN013892 2646 188 299 LPG13113 B2M SGN013893 2647 189 300 LPG13113 B2M SGN013894 2648 190 301 LPG13113 B2M SGN013895 2649 191 302 LPG13113 B2M SGN013896 2650 1736 1841 LPG13113 TRAC SGN013897 2651 1724 1829 LPG13113 TRAC SGN013898 2652 1723 1828 LPG13113 TRAC SGN013899 2653 1721 1826 LPG13113 TRAC SGN013900 2654 1720 1825 LPG13113 TRAC SGN013901 2655 1717 1822 LPG13113 TRAC SGN013902 2656 1718 1823 LPG13113 TRAC SGN013903 2657 1719 1824 LPG13113 TRAC SGN013904 2658 1722 1827 surface 4 Oligonucleotides used to detect gene editing activity in mammalian cells Introduction group Forward induction (SEQ ID NO) Reverse primer (SEQ ID NO) Target of primer amplification ( SEQ ID NOs. ) 1 286 287 225, 236, 237, 238, 239, 1757, 2081 2 288 289 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 240, 241, 242, 252, 253, 254, 255, 256, 257, 258, 259, 270, 271, 272, 278, 1736, 1738, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2048, 2054, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079 3 290 291 243, 244, 245, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 273, 274, 275, 276, 277, 279, 280, 281, 282, 283, 284, 285, 1691, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1766, 1771, 1775, 1776, 2047, 2049, 2050, 2051, 2052, 2053, 2055, 2056, 2057, 2058, 2059, 2060, 2068, 2069, 2070, 2071, 2080 4 292 293 246, 247, 248, 249, 250, 251

Deep sequencing was performed on PCR products containing Nextera reads 1 and 2. Library preparation and deep sequencing were performed by the service provider (MoGene) on the Illumina NextSeq platform. Typically, each amplicon generated 200,000 250 bp paired-end reads (2 x 100,000 reads). Reads were analyzed using CRISPResso (Pinello et al. 2016 Nature Biotech, 34:695-697) to calculate edit rates. Output alignments were hand-curated to identify insertion and deletion (INDEL) sites and to identify microhomological sites at recombination sites. Edit rates are shown in Table 5. All experiments were performed in human cells. "Target sequence" is the target sequence within a gene target. For each target sequence, depending on the RGN used, the guide RNA includes a spacer and an appropriate crRNA backbone. Examples of selected mammalian genome editing are shown in Table 5. Table 5 : Editing rates of RGNs in mammalian cells . RGN ID Guide body name Percentage of missing insertions LPG10238 SGN008512 0.33 LPG10238 SGN008513 1.16 LPG10238 SGN008514 0.07 LPG10238 SGN008515 0.33 LPG10238 SGN008516 12.59 LPG10238 SGN008517 6.28 LPG10238 SGN008518 24.63 LPG10238 SGN008519 8.98 LPG10238 SGN008520 3.79 LPG10238 SGN008521 2.84 LPG10239 SGN008522 0.10 LPG10239 SGN008523 0.00 LPG10239 SGN008524 0.40 LPG10239 SGN008525 0.00 LPG10239 SGN008526 0.05 LPG10239 SGN008527 0.05 LPG10239 SGN008528 0.00 LPG10239 SGN008529 0.00 LPG10239 SGN008530 0.00 LPG10239 SGN008531 0.20 LPG10241 SGN008532 0.00 LPG10241 SGN008533 0.00 LPG10241 SGN008534 3.55 LPG10241 SGN008535 0.05 LPG10241 SGN008536 0.00 LPG10241 SGN008537 0.05 LPG10241 SGN008538 0.00 LPG10241 SGN008539 1.85 LPG10241 SGN008540 0.00 LPG10241 SGN008541 1.60 LPG10240 SGN008542 0.05 LPG10240 SGN008543 0.00 LPG10240 SGN008544 0.00 LPG10240 SGN008545 0.00 LPG10240 SGN008546 0.00 LPG10240 SGN008547 0.00 LPG10240 SGN008548 0.00 LPG10240 SGN008549 0.00 LPG10240 SGN008550 0.00 LPG10240 SGN008551 1.25 LPG10241 SGN008552 0.00 LPG10241 SGN008553 0.10 LPG10241 SGN008554 3.35 LPG10241 SGN008555 0.00 LPG10241 SGN008556 0.10 LPG10241 SGN008557 0.15 LPG10241 SGN008558 0.00 LPG10241 SGN008559 2.30 LPG10241 SGN008560 0.00 LPG10241 SGN008561 2.30 LPG10240 SGN008562 0.00 LPG10240 SGN008563 0.00 LPG10240 SGN008564 0.07 LPG10240 SGN008565 0.00 LPG10240 SGN008566 0.00 LPG10240 SGN008567 0.00 LPG10240 SGN008568 0.00 LPG10240 SGN008569 0.05 LPG10240 SGN008570 0.00 LPG10240 SGN008571 2.45 LPG10239 SGN008572 0.15 LPG10239 SGN008573 0.00 LPG10239 SGN008574 0.27 LPG10239 SGN008575 0.00 LPG10239 SGN008576 0.00 LPG10239 SGN008577 0.00 LPG10239 SGN008578 0.00 LPG10239 SGN008579 0.00 LPG10239 SGN008580 0.00 LPG10239 SGN008581 0.00 LPG10238 SGN008592 0.42 LPG10238 SGN008593 0.73 LPG10238 SGN008594 0.26 LPG10238 SGN008595 0.09 LPG10238 SGN008596 10.22 LPG10238 SGN008597 5.34 LPG10238 SGN008598 24.97 LPG10238 SGN008599 9.15 LPG10238 SGN008600 4.76 LPG10238 SGN008601 4.94 LPG10238 SGN008602 3.90 LPG10238 SGN008603 21.15 LPG10238 SGN008604 7.95 LPG10238 SGN008605 30.65 LPG10238 SGN008609 4.90 LPG10238 SGN008610 9.40 LPG10238 SGN008611 0.35 LPG10238 SGN008612 4.85 LPG10238 SGN008613 1.30 LPG10238 SGN008614 2.10 LPG10238 SGN008615 0.00 LPG10238 SGN008616 0.35 LPG10238 SGN008617 0.00 LPG10238 SGN008618 10.85 LPG10238 SGN008619 0.45 LPG10238 SGN008620 0.00 LPG10238 SGN008621 0.60 LPG10241 SGN008636 0.00 LPG10241 SGN008637 0.08 LPG10241 SGN008638 0.62 LPG10241 SGN008639 0.21 LPG10241 SGN008640 0.00 LPG10241 SGN008641 0.00 LPG10241 SGN008642 0.00 LPG10241 SGN008643 0.00 LPG10241 SGN008645 0.00 LPG10241 SGN008646 0.00 LPG10241 SGN008647 0.00 LPG10241 SGN008648 0.05 LPG10241 SGN008649 0.60 LPG10241 SGN008650 0.95 LPG10241 SGN008651 0.05 LPG10241 SGN008652 0.07 LPG10241 SGN008653 0.62 LPG10241 SGN008654 8.66 LPG10240 SGN008655 0.00 LPG10240 SGN008656 0.00 LPG10240 SGN008657 0.00 LPG10240 SGN008658 0.00 LPG10240 SGN008659 0.00 LPG10240 SGN008660 0.88 LPG10240 SGN008661 0.00 LPG10240 SGN008662 0.00 LPG10240 SGN008663 0.00 LPG10240 SGN008664 0.07 LPG10240 SGN008665 0.00 LPG10240 SGN008666 2.97 LPG10240 SGN008668 0.00 LPG10240 SGN008669 0.00 LPG10240 SGN008670 0.20 LPG10240 SGN008671 0.00 LPG10239 SGN008672 0.10 LPG10239 SGN008673 0.00 LPG10239 SGN008674 0.00 LPG10239 SGN008677 1.33 LPG10239 SGN008678 0.00 LPG10239 SGN008679 0.04 LPG10239 SGN008680 0.52 LPG10239 SGN008681 0.00 LPG10239 SGN008682 0.25 LPG10239 SGN008683 0.05 LPG10239 SGN008684 0.00 LPG10239 SGN008685 0.00 LPG10239 SGN008686 0.29 LPG13090 SGN013365 0.2025 LPG13090 SGN013366 0 LPG13090 SGN013367 0 LPG13090 SGN013368 0.0425 LPG13090 SGN013369 0.0175 LPG13090 SGN013370 0 LPG13090 SGN013371 4.0125 LPG13090 SGN013372 0.705 LPG13090 SGN013373 0.0775 LPG13090 SGN013374 0.69 LPG13090 SGN013375 5.3025 LPG13090 SGN013376 10.1975 LPG13090 SGN013377 0 LPG13090 SGN013378 2.8975 LPG13090 SGN013379 14.3075 LPG13090 SGN013380 0.1275 LPG13090 SGN013381 0.01 LPG13090 SGN013382 0.41 LPG13090 SGN013383 0.11 LPG13090 SGN013384 0 LPG13091 SGN013865 0 LPG13091 SGN013866 0 LPG13091 SGN013867 0.0225 LPG13091 SGN013868 0 LPG13091 SGN013869 0 LPG13091 SGN013870 0 LPG13091 SGN013871 0.0825 LPG13091 SGN013872 0 LPG13091 SGN013873 0 LPG13091 SGN013874 0 LPG13091 SGN013875 0.8725 LPG13091 SGN013876 0 LPG13091 SGN013877 0 LPG13091 SGN013878 0 LPG13091 SGN013879 0 LPG13091 SGN013880 0 LPG13091 SGN013881 0 LPG13091 SGN013882 0 LPG13091 SGN013883 0 LPG13091 SGN013884 0 LPG13092 SGN013385 0.05 LPG13092 SGN013386 0 LPG13092 SGN013387 0.02 LPG13092 SGN013388 0.12 LPG13092 SGN013389 0.22 LPG13092 SGN013390 0 LPG13092 SGN013391 4.135 LPG13092 SGN013392 2.165 LPG13092 SGN013393 0.6425 LPG13092 SGN013394 0.1525 LPG13092 SGN013395 0.5175 LPG13092 SGN013396 0.56 LPG13092 SGN013397 0.14 LPG13092 SGN013398 0.2775 LPG13092 SGN013399 0.605 LPG13092 SGN013400 0.255 LPG13092 SGN013401 0 LPG13092 SGN013402 0.015 LPG13092 SGN013403 0.465 LPG13092 SGN013404 0.115 LPG13093 SGN013405 0 LPG13093 SGN013406 0 LPG13093 SGN013407 0 LPG13093 SGN013408 0 LPG13093 SGN013409 0 LPG13093 SGN013410 0 LPG13093 SGN013411 0 LPG13093 SGN013412 0 LPG13093 SGN013413 0 LPG13093 SGN013414 0 LPG13093 SGN013415 0 LPG13093 SGN013416 0 LPG13093 SGN013417 0.0125 LPG13093 SGN013418 0 LPG13093 SGN013419 0 LPG13093 SGN013420 0 LPG13093 SGN013421 0 LPG13093 SGN013422 0 LPG13093 SGN013423 0.0075 LPG13093 SGN013424 0 LPG13094 SGN013425 0 LPG13094 SGN013426 0 LPG13094 SGN013427 0.08 LPG13094 SGN013428 0 LPG13094 SGN013429 0 LPG13094 SGN013430 0 LPG13094 SGN013431 0 LPG13094 SGN013432 0 LPG13094 SGN013433 0.0325 LPG13094 SGN013434 0 LPG13094 SGN013435 0 LPG13094 SGN013436 2.11 LPG13094 SGN013437 0 LPG13094 SGN013438 0 LPG13094 SGN013439 0 LPG13094 SGN013440 0 LPG13094 SGN013441 0 LPG13094 SGN013442 0 LPG13094 SGN013443 0 LPG13094 SGN013444 0 LPG13095 SGN013445 0.7425 LPG13095 SGN013446 0 LPG13095 SGN013447 1.2875 LPG13095 SGN013448 0.5 LPG13095 SGN013449 0 LPG13095 SGN013450 0.1325 LPG13095 SGN013451 1.0575 LPG13095 SGN013452 0.05 LPG13095 SGN013453 0.0975 LPG13095 SGN013454 0.1175 LPG13095 SGN013455 0.1125 LPG13095 SGN013456 3.6025 LPG13095 SGN013457 0 LPG13095 SGN013458 0.2 LPG13095 SGN013459 2.4725 LPG13095 SGN013460 0.01 LPG13095 SGN013461 0 LPG13095 SGN013462 0.47 LPG13095 SGN013463 0.0075 LPG13095 SGN013464 0 LPG13096 SGN013465 0 LPG13096 SGN013466 0 LPG13096 SGN013467 0.01 LPG13096 SGN013468 0.02 LPG13096 SGN013469 0 LPG13096 SGN013470 0 LPG13096 SGN013471 0.4125 LPG13096 SGN013472 0.155 LPG13096 SGN013473 0.045 LPG13096 SGN013474 0.01 LPG13096 SGN013475 0.015 LPG13096 SGN013476 0.015 LPG13096 SGN013477 0 LPG13096 SGN013478 0.0425 LPG13096 SGN013479 0.02 LPG13096 SGN013480 0.03 LPG13096 SGN013481 0 LPG13096 SGN013482 0 LPG13096 SGN013483 0.01 LPG13096 SGN013484 0 LPG13097 SGN013485 0.1575 LPG13097 SGN013486 0 LPG13097 SGN013487 6.3525 LPG13097 SGN013488 0.1275 LPG13097 SGN013489 1.05 LPG13097 SGN013490 0 LPG13097 SGN013491 2.4625 LPG13097 SGN013492 3.725 LPG13097 SGN013493 0.0575 LPG13097 SGN013494 0.0075 LPG13097 SGN013495 0 LPG13097 SGN013496 0 LPG13097 SGN013497 0.1375 LPG13097 SGN013498 0 LPG13097 SGN013499 0.12 LPG13097 SGN013500 1.515 LPG13097 SGN013501 0 LPG13097 SGN013502 6.2775 LPG13097 SGN013503 6.3875 LPG13097 SGN013504 1.8775 LPG13098 SGN013505 0 LPG13098 SGN013506 0 LPG13098 SGN013507 0 LPG13098 SGN013508 0 LPG13098 SGN013509 0 LPG13098 SGN013510 0 LPG13098 SGN013511 0 LPG13098 SGN013512 0 LPG13098 SGN013513 0 LPG13098 SGN013514 0 LPG13098 SGN013515 0 LPG13098 SGN013516 0.27 LPG13098 SGN013517 0 LPG13098 SGN013518 0 LPG13098 SGN013519 0 LPG13098 SGN013520 0 LPG13098 SGN013521 0 LPG13098 SGN013522 0 LPG13098 SGN013523 0 LPG13098 SGN013524 0 LPG13099 SGN013525 0 LPG13099 SGN013526 0 LPG13099 SGN013527 0 LPG13099 SGN013528 0 LPG13099 SGN013529 0 LPG13099 SGN013530 0 LPG13099 SGN013531 0 LPG13099 SGN013532 0 LPG13099 SGN013533 0 LPG13099 SGN013534 0 LPG13099 SGN013535 0 LPG13099 SGN013536 0.01 LPG13099 SGN013537 0 LPG13099 SGN013538 0 LPG13099 SGN013539 0 LPG13099 SGN013540 0 LPG13099 SGN013541 0 LPG13099 SGN013542 0 LPG13099 SGN013543 0 LPG13099 SGN013544 0 LPG13100 SGN013545 0 LPG13100 SGN013546 0.0125 LPG13100 SGN013547 0.235 LPG13100 SGN013548 0 LPG13100 SGN013549 0 LPG13100 SGN013550 0 LPG13100 SGN013551 0 LPG13100 SGN013552 0 LPG13100 SGN013553 0 LPG13100 SGN013554 0 LPG13100 SGN013555 0 LPG13100 SGN013556 0.0425 LPG13100 SGN013557 0 LPG13100 SGN013558 0.0675 LPG13100 SGN013559 0.02 LPG13100 SGN013560 0 LPG13100 SGN013561 0 LPG13100 SGN013562 0 LPG13100 SGN013563 0 LPG13100 SGN013564 0 LPG13101 SGN013565 3.235 LPG13101 SGN013566 0 LPG13101 SGN013567 0.0175 LPG13101 SGN013568 0 LPG13101 SGN013569 0 LPG13101 SGN013570 0 LPG13101 SGN013571 0 LPG13101 SGN013572 0.0825 LPG13101 SGN013573 0.2725 LPG13101 SGN013574 0.0475 LPG13101 SGN013575 0 LPG13101 SGN013576 2.6325 LPG13101 SGN013577 0.01 LPG13101 SGN013578 4.6075 LPG13101 SGN013579 4.425 LPG13101 SGN013580 0 LPG13101 SGN013581 1.2875 LPG13101 SGN013582 0.595 LPG13101 SGN013583 0.11 LPG13101 SGN013584 0 LPG13102 SGN013585 0 LPG13102 SGN013586 0 LPG13102 SGN013587 0 LPG13102 SGN013588 0.0075 LPG13102 SGN013589 0 LPG13102 SGN013590 0 LPG13102 SGN013591 0 LPG13102 SGN013592 0 LPG13102 SGN013593 0 LPG13102 SGN013594 0 LPG13102 SGN013595 0 LPG13102 SGN013596 0 LPG13102 SGN013597 0 LPG13102 SGN013598 0 LPG13102 SGN013599 0 LPG13102 SGN013600 0 LPG13102 SGN013601 0 LPG13102 SGN013602 0 LPG13102 SGN013603 0 LPG13102 SGN013604 0 LPG13103 SGN013605 0 LPG13103 SGN013606 0 LPG13103 SGN013607 1.635 LPG13103 SGN013608 0 LPG13103 SGN013609 0 LPG13103 SGN013610 0 LPG13103 SGN013611 0 LPG13103 SGN013612 0.095 LPG13103 SGN013613 0 LPG13103 SGN013614 0 LPG13103 SGN013615 0 LPG13103 SGN013616 2.9725 LPG13103 SGN013617 0 LPG13103 SGN013618 0 LPG13103 SGN013619 0 LPG13103 SGN013620 0.0175 LPG13103 SGN013621 0 LPG13103 SGN013622 0 LPG13103 SGN013623 0 LPG13103 SGN013624 0 LPG13104 SGN013625 0 LPG13104 SGN013626 0 LPG13104 SGN013627 0.0075 LPG13104 SGN013628 0 LPG13104 SGN013629 0 LPG13104 SGN013630 0 LPG13104 SGN013631 0 LPG13104 SGN013632 0 LPG13104 SGN013633 0 LPG13104 SGN013634 0 LPG13104 SGN013635 0 LPG13104 SGN013636 0 LPG13104 SGN013637 0.15 LPG13104 SGN013638 0 LPG13104 SGN013639 0 LPG13104 SGN013640 0 LPG13104 SGN013641 0 LPG13104 SGN013642 0 LPG13104 SGN013643 0 LPG13104 SGN013644 0 LPG13105 SGN013645 0 LPG13105 SGN013646 0 LPG13105 SGN013647 0 LPG13105 SGN013648 0 LPG13105 SGN013649 0 LPG13105 SGN013650 0 LPG13105 SGN013651 0 LPG13105 SGN013652 0 LPG13105 SGN013653 0 LPG13105 SGN013654 0 LPG13105 SGN013655 0 LPG13105 SGN013656 0 LPG13105 SGN013657 0 LPG13105 SGN013658 0 LPG13105 SGN013659 0 LPG13105 SGN013660 0 LPG13105 SGN013661 0 LPG13105 SGN013662 0 LPG13105 SGN013663 0 LPG13105 SGN013664 0 LPG13106 SGN013405 0 LPG13106 SGN013406 0 LPG13106 SGN013407 0 LPG13106 SGN013668 0 LPG13106 SGN013669 0 LPG13106 SGN013408 0 LPG13106 SGN013671 0 LPG13106 SGN013672 0 LPG13106 SGN013673 0 LPG13106 SGN013409 0 LPG13106 SGN013675 0 LPG13106 SGN013676 0 LPG13106 SGN013677 0 LPG13106 SGN013678 0 LPG13106 SGN013410 0 LPG13106 SGN013680 0 LPG13106 SGN013681 0 LPG13106 SGN013411 0 LPG13106 SGN013683 0 LPG13106 SGN013684 0 LPG13107 SGN013685 0 LPG13107 SGN013686 0.04 LPG13107 SGN013687 0 LPG13107 SGN013688 0 LPG13107 SGN013689 0 LPG13107 SGN013690 0 LPG13107 SGN013691 0 LPG13107 SGN013692 0 LPG13107 SGN013693 0 LPG13107 SGN013694 0 LPG13107 SGN013695 0 LPG13107 SGN013696 0 LPG13107 SGN013697 0 LPG13107 SGN013698 0 LPG13107 SGN013699 0 LPG13107 SGN013700 0 LPG13107 SGN013701 0 LPG13107 SGN013702 0 LPG13107 SGN013703 0 LPG13107 SGN013704 0 LPG13108 SGN013705 0 LPG13108 SGN013706 0 LPG13108 SGN013707 0 LPG13108 SGN013708 0 LPG13108 SGN013709 0 LPG13108 SGN013710 0 LPG13108 SGN013711 0 LPG13108 SGN013712 0 LPG13108 SGN013713 0 LPG13108 SGN013714 0.01 LPG13108 SGN013715 0 LPG13108 SGN013716 0.4975 LPG13108 SGN013717 0 LPG13108 SGN013718 0 LPG13108 SGN013719 0 LPG13108 SGN013720 0 LPG13108 SGN013721 0 LPG13108 SGN013722 0 LPG13108 SGN013723 0 LPG13108 SGN013724 0 LPG13109 SGN013725 0 LPG13109 SGN013726 0 LPG13109 SGN013727 0.02 LPG13109 SGN013728 0 LPG13109 SGN013729 0 LPG13109 SGN013730 0 LPG13109 SGN013731 0 LPG13109 SGN013732 0 LPG13109 SGN013733 0.0075 LPG13109 SGN013734 0 LPG13109 SGN013735 0 LPG13109 SGN013736 0.0075 LPG13109 SGN013737 0 LPG13109 SGN013738 0 LPG13109 SGN013739 0 LPG13109 SGN013740 0 LPG13109 SGN013741 0 LPG13109 SGN013742 0 LPG13109 SGN013743 0 LPG13109 SGN013744 0 LPG13110 SGN013745 0.06 LPG13110 SGN013746 0 LPG13110 SGN013747 0.6075 LPG13110 SGN013748 0.0375 LPG13110 SGN013749 0.785 LPG13110 SGN013750 0 LPG13110 SGN013751 0.0225 LPG13110 SGN013752 0.6875 LPG13110 SGN013753 0.03 LPG13110 SGN013754 0 LPG13110 SGN013755 0 LPG13110 SGN013756 0 LPG13110 SGN013757 0 LPG13110 SGN013758 0.0825 LPG13110 SGN013759 0 LPG13110 SGN013760 0 LPG13110 SGN013761 0 LPG13110 SGN013762 0 LPG13110 SGN013763 0.0275 LPG13110 SGN013764 0 LPG13111 SGN013765 0 LPG13111 SGN013766 0 LPG13111 SGN013767 0 LPG13111 SGN013768 0 LPG13111 SGN013769 0 LPG13111 SGN013770 0 LPG13111 SGN013771 0.315 LPG13111 SGN013772 0 LPG13111 SGN013773 0.0075 LPG13111 SGN013774 0 LPG13111 SGN013775 0 LPG13111 SGN013776 0 LPG13111 SGN013777 0 LPG13111 SGN013778 0 LPG13111 SGN013779 0 LPG13111 SGN013780 0 LPG13111 SGN013781 0 LPG13111 SGN013782 0 LPG13111 SGN013783 0 LPG13111 SGN013784 0 LPG13112 SGN013785 0 LPG13112 SGN013786 0.02 LPG13112 SGN013787 0 LPG13112 SGN013788 0 LPG13112 SGN013789 0 LPG13112 SGN013790 0 LPG13112 SGN013791 0 LPG13112 SGN013792 0 LPG13112 SGN013793 0 LPG13112 SGN013794 0.015 LPG13112 SGN013795 0 LPG13112 SGN013796 0 LPG13112 SGN013797 0 LPG13112 SGN013798 0 LPG13112 SGN013799 0 LPG13112 SGN013800 0 LPG13112 SGN013801 0 LPG13112 SGN013802 0 LPG13112 SGN013803 0 LPG13112 SGN013804 0 LPG13113 SGN013885 3.125 LPG13113 SGN013886 1.625 LPG13113 SGN013887 21.0725 LPG13113 SGN013888 14.3375 LPG13113 SGN013889 33.325 LPG13113 SGN013890 3.285 LPG13113 SGN013891 30.7925 LPG13113 SGN013892 3.2225 LPG13113 SGN013893 17.2075 LPG13113 SGN013894 0.8 LPG13113 SGN013895 19.175 LPG13113 SGN013896 36.025 LPG13113 SGN013897 36.0525 LPG13113 SGN013898 29.965 LPG13113 SGN013899 1.7325 LPG13113 SGN013900 7.0075 LPG13113 SGN013901 0 LPG13113 SGN013902 1.5825 LPG13113 SGN013903 7.2525 LPG13113 SGN013904 0.13 LPG13114 SGN013805 0 LPG13114 SGN013806 0 LPG13114 SGN013807 0 LPG13114 SGN013808 0 LPG13114 SGN013809 0 LPG13114 SGN013810 0 LPG13114 SGN013811 0 LPG13114 SGN013812 0 LPG13114 SGN013813 0 LPG13114 SGN013814 0 LPG13114 SGN013815 0 LPG13114 SGN013816 0 LPG13114 SGN013817 0 LPG13114 SGN013818 0 LPG13114 SGN013819 0 LPG13114 SGN013820 0 LPG13114 SGN013821 0 LPG13114 SGN013822 0 LPG13114 SGN013823 0 LPG13114 SGN013824 0 LPG13115 SGN013825 3.785 LPG13115 SGN013826 5.305 LPG13115 SGN013827 17.1025 LPG13115 SGN013828 15.03 LPG13115 SGN013829 8.58 LPG13115 SGN013830 3.4875 LPG13115 SGN013831 35.675 LPG13115 SGN013832 29.9575 LPG13115 SGN013833 9.8325 LPG13115 SGN013834 3.3275 LPG13115 SGN013835 0.515 LPG13115 SGN013836 27.4225 LPG13115 SGN013837 0 LPG13115 SGN013838 10.4575 LPG13115 SGN013839 24.8475 LPG13115 SGN013840 1.3825 LPG13115 SGN013841 1.4575 LPG13115 SGN013842 25.1775 LPG13115 SGN013843 3.8325 LPG13115 SGN013844 0.36 LPG13116 SGN013845 1.0825 LPG13116 SGN013846 0 LPG13116 SGN013847 3.81 LPG13116 SGN013848 0 LPG13116 SGN013849 0.0325 LPG13116 SGN013850 0 LPG13116 SGN013851 0.375 LPG13116 SGN013852 0.01 LPG13116 SGN013853 0.635 LPG13116 SGN013854 0 LPG13116 SGN013855 0.2475 LPG13116 SGN013856 0.84 LPG13116 SGN013857 0.2675 LPG13116 SGN013858 2.5575 LPG13116 SGN013859 1.09 LPG13116 SGN013860 0 LPG13116 SGN013861 0.01 LPG13116 SGN013862 0.015 LPG13116 SGN013863 0.0175 LPG13116 SGN013864 0

Table 6 summarizes the V-type systems tested in the examples. LPG10238 and LPG13113 are the best-performing VH-type nucleases and the only known highly active VH-type nuclease. LPG13115 is the best-performing VI-type nuclease. LPG10241 is the best-performing VJ-type nuclease. Table 6 : Gene Editing Overview of the Tested V -type Systems RGN ID Subtype PAM Protein size ( aa ) Main chain length (guide RNA length with or without spacers) The largest observed percentage of missing edit insertions LPG10241 Vj TTN 770 ≤ 36 8.7 LPG10240 Vj VTTN 786 ≤ 36 3.0 LPG10239 Vj ATTN 770 ≤ 36 1.3 LPG10238 Vh AYG 889 ≤ 36 30.7 LPG13090 VI TTN 1086 35 14.3075 LPG13091 VI STTN 1093 36 0.8725 LPG13092 VI TTN 1032 36 4.135 LPG13093 VJ ATTN 790 37 0.0125 LPG13094 VJ TTN 796 36 2.11 LPG13095 VJ TTH 782 36 3.6025 LPG13096 VJ TTN 870 36 0.4125 LPG13097 VJ RTTN 718 36 6.3875 LPG13098 VJ VTTN 761 36 0.27 LPG13099 VJ TTN 781 36 0.01 LPG13100 VJ TTN 725 36 0.235 LPG13101 VJ TTN 769 36 4.6075 LPG13102 VJ TTN 752 36 0.0075 LPG13103 VJ VTTN 737 36 2.9725 LPG13104 VJ ATTN 749 36 0.15 LPG13105 VJ VTTN 781 36 0 LPG13106 VJ TTN 790 37 0 LPG13107 VJ VTTN 793 37 0.04 LPG13108 VJ TTN 775 36 0.4975 LPG13109 VJ VTTN 747 36 0.02 LPG13110 VJ RTTN 708 36 0.785 LPG13111 VJ RTYN 775 36 0.315 LPG13112 VH TTN 872 36 0.02 LPG13113 VH ATG 882 36 36.0525 LPG13114 VH TTN 924 39 0 LPG13115 VI TTN 1045 36 35.675 LPG13116 VJ TTN 717 36 3.81 N is A, C, T/U, or G; Y is C or T/U; V is A, G, or C; S is C or G; H is A, C, or T/U; R is A or G. Example 5 : Domain mapping of type V RGN peptides .

Based on sequence alignment with publicly available sequences, the domains of LPG10241 were located. Due to the availability of both the sequence and crystal structure of VJ-type Cas enzymes, the domains of LPG10241 were identified as: N-terminal PAM interaction (PI) domain, RecI and RecII domains, oligomer binding domain (OBD), tripartite RuvC domain, and zinc finger domain (ZF). The domains are located as shown in Figure 4A. In the case of the unknown structure and lack of publicly available sequences for VH-type Cas enzymes, the domains of LPG10238 were located based on sequence similarity with other V-type enzymes, particularly those in the A, F, and I subtype families. The domains identified in LPG10238 include: bipartite REC domains (RecI and RecII) and tripartite RuvC domains. These domains are located as shown in Figure 4B. Based on these locations, the three acidic active site residues of each enzyme were identified, as described in Table 7, which include two aspartate residues and one glutamate residue. Table 7 : Active site residues of LPG10238 and LPG10241 LPG10238 LPG10241 Active site residue 1 D474 D396 Active site residue 2 E672 E622 Active site residue 3 D759 D711 Example 6 : Identification of arginine-substituted LPG10238 and LPG10241 mutants with enhanced activity

The amino acid substitution mutation system was generated by GenScript Biotech Corporation (Piscataway, NJ), in which each residue in the C-terminal half of the amino acid sequence of each of LPG10238 and LPG10241 mutates to arginine. C-terminal half mutation of each protein aims to avoid mutation of the DNA colloid recognition region (PI and REC domains). Due to the lack of VH-type enzyme structure, the entire C-terminal half of the LPG10238 protein mutates, starting with RuvC-1. In contrast, for LPG10241, only the presumed RuvC domain is targeted for arginine mutation. For both enzymes, targeted mutation involves mutating the active site residue (described in Example 5) to arginine. The mutation system for each enzyme is summarized in Table 8. The sequences of the LPG10238 mutant are shown in SEQ ID NOs: 408 to 802 and 1929. The sequences of the LPG10241 mutant are shown in SEQ ID NOs: 803 to 1033. Table 8 : LPG10238 and LPG10241 arginine mutants. Parent RGN ID Number of mutants Mutant IDs SEQ ID NO of LPG10238 or LPG10241 mutant LPG10238 396 LPG10427 to LPG10821 and LPG12222 SEQ ID NO: 408 to 802 and SEQ ID NO: 1929 LPG10241 231 LPG11059 to LPG11289 SEQ ID NO: 803 to SEQ ID NO: 1033

Mutant systems with mammalian gene editing activity were selected using plasmid-lipoplastin transfection and insertion/deletion NGS quantification methods described in Example 4. To account for inter-experimental variability, editing efficiency was normalized to wild-type LPG10238 or LPG10241 per plate (Figures 5A and 5C, respectively). For each of the initial mutant groups, editing of two primers and two genomic loci was evaluated (Figures 5A and 5C). Approximately 40 to 90 of the most active mutants were selected from these mutants for further testing in validation rounds, with the LPG10238 mutant having two additional primers and the LPG10241 mutant having one primer (Figures 5B and 5D). Table 9 : Selection-guided RNAs of LPG10238 and LPG10241 arginine mutants Parent RGN ID Preliminary screening Verification Screening LPG10238 396 mutants SGN008516 (SEQ ID NO: 43) 87 mutants SGN010508 (SEQ ID NO: 1405) SGN008518 (SEQ ID NO: 45) SGN010571 (SEQ ID NO: 1406) LPG10241 231 mutants SGN008534 (SEQ ID NO: 61) 43 mutants SGN008539 (SEQ ID NO: 66) 24 mutants SGN013008 (SEQ ID NO: 1414) SGN009798 (SEQ ID NO: 43) SGN013009 (SEQ ID NO: 1415)

The screening included arginine mutants with active site residues identified in Example 5. These mutants did not show activity in the initial screening, confirming their importance in the activity of both LPG10238 (Fig. 6A) and LPG10241 (Fig. 6B). Similarly, for LPG10241, cysteine residues coordinated with zinc in the ZF domain were identified, conforming to the canonical CXXC motif (Figs. 7A and 7B). Example 7 : Combined mutants exhibit improved editing.

Based on the screening data in Example 6, 16 of the most active LPG10238 single-arginine mutants were identified, exhibiting enhanced editing relative to wild-type LPG10238; and 15 of the most active LPG10241 single-arginine mutants were identified, also exhibiting enhanced editing relative to wild-type LPG10241 (Figures 8A and 8B, Table 10). Six mutants from each of these two best-performing mutant groups were selected for combinatorial mutant evaluation (Figures 8A and 8B, Table 10). All 63 mutant combinations exhibiting enhanced activity were screened. These mutant combinations included: 6 single-, 15 two-, 20 three-, 15 four-, 6 five-, and 1 six-mutant variants. For LPG10238, 63 mutants are LPG12953 to LPG13009 (SEQ ID NO: 1034 to 1090). In addition, for LPG10238, 15 additional combinatorial mutants were generated by combining some of the most active unselected mutants with selected mutants (LPG13010 to LPG13024, SEQ ID NO: 1091 to 1105). For LPG10241, all 63 combinations of the six selected mutants (LPG13212 to LPG13268; SEQ ID NO: 1106 to 1162) with enhanced activity were screened, along with 11 additional combinations of unselected and selected mutants covering the ZF domain and C-terminal tail (LPG13269 to LPG13279; SEQ ID NO: 1163 to 1173). Table 10 : Best-performing single arginine mutants of LPG10238 and LPG10241. Parent RGN ID Mutant RGN ID mutation SEQ ID NO of mutant RGN LPG10238 LPG10469* N517R 450 LPG10482* M530R 463 LPG10490* E538R 471 LPG10500 L548R 481 LPG10503 D551R 484 LPG10504* I552R 485 LPG10505 E553R 486 LPG10519 H568R 500 LPG10521 G570R 502 LPG10524 V574R 505 LPG10527 N577R 508 LPG10558 V611R 539 LPG10621* E677R 602 LPG10726 D789R 707 LPG10727 K790R 708 LPG10736* K801R 717 LPG10241 LPG11065* L377R 809 LPG11066 A378R 810 LPG11122 M438R 866 LPG11154* S625R 898 LPG11165* L638R 909 LPG11168 N641R 912 LPG11172 Q645R 916 LPG11216* A691R 960 LPG11217 N692R 961 LPG11219 S695R 963 LPG11222* D698R 966 LPG11226 L702R 970 LPG11231 M707R 975 LPG11268* V747R 1012 LPG11275 D755R 1019 *Selecting mutants for combination mutant assessment

These combined mutant systems were screened using another set of five to six leads (Figs. 9A and 9B, Table 11). On average, the 18 most active LPG10238 mutants exhibited greater than or equal to twice the editing capacity of the parental LPG10238 (Fig. 10A). On average, the 18 most active LPG10238 mutants had an editing efficiency of 18.8% for these leads, compared to an average editing efficiency of 9.3% for the parental LPG10238 (Table 12). Similarly, the 17 most active LPG10241 combined mutants exhibited editing capacity more than 1.3 times greater than that of the parental LPG10241 (Fig. 10B). On average, for these leads, the 17 most active LPG10241 mutants exhibited an average editing efficiency of 16.4%, compared to 5.7% for wild-type LPG10241 (Table 13). Table 11 : Screening lead sets of combined mutants LPG10238 Filtering Guide LPG10241 Filtering Guide SGN009748 (SEQ ID NO: 1407) SGN013150 (SEQ ID NO: 1416) SGN009756 (SEQ ID NO: 1408) SGN013151 (SEQ ID NO: 1417) SGN010546 (SEQ ID NO: 1409) SGN013152 (SEQ ID NO: 1418) SGN010579 (SEQ ID NO: 1410) SGN013154 (SEQ ID NO: 1419) SGN010605 (SEQ ID NO: 1411) SGN013155 (SEQ ID NO: 1420) SGN013156 (SEQ ID NO: 1421) Table 12 : Average edit percentage of the 18 best-performing LPG10238 combination mutants RGN ID RGN polypeptide SEQ ID NO mutation Average editing percentage LPG10238 1 None (parent copy) 9.3 LPG13001 1082 M530R, I552R, E677R, K801R 21.9 LPG12991 1072 N517R, M530R, I552R, E677R 21.2 LPG12999 1080 M530R, E538R, I552R, K801R 20.9 LPG13024 1105 M530R, N577R, K790R 19.2 LPG13003 1084 N517R, M530R, E538R, I552R, E677R 21.6 LPG13000 1081 M530R, E538R, E677R, K801R 19.4 LPG13002 1083 E538R, I552R, E677R, K801R 18.9 LPG12988 1069 N517R, M530R, E538R, I552R 20.4 LPG12953 1034 N517R, M530R 20.3 LPG12978 1059 M530R, E538R, I552R 18.5 LPG12989 1070 N517R, M530R, E538R, E677R 18.9 LPG12997 1078 N517R, I552R, E677R, K801R 16.8 LPG12983 1064 M530R, E677R, K801R 16.5 LPG12993 1074 N517R, M530R, E677R, K801R 17.4 LPG12970 1051 N517R, M530R, E677R 17.4 LPG12998 1079 M530R, E538R, I552R, E677R 16.9 LPG12975 1056 N517R, I552R, E677R 16.3 LPG13006 1087 N517R, M530R, I552R, E677R, K801R 16.2 Table 13 : Average edit percentage of the 17 best-performing LPG10241 combination mutants RGN ID RGN polypeptide SEQ ID NO mutation Average editing percentage LPG10241 4 None (parent copy) 5.7 LPG13264 1158 L377R, S625R, L638R, D698R, V747R 19.0 LPG13254 1148 L377R, L638R, A691R, V747R 19.0 LPG13265 1159 L377R, S625R, A691R, D698R, V747R 18.6 LPG13266 1160 L377R, L638R, A691R, D698R, V747R 19.4 LPG13228 1122 L377R, S625R, A691R 17.1 LPG13252 1146 L377R, S625R, D698R, V747R 17.3 LPG13229 1123 L377R, S625R, D698R 17.0 LPG13253 1147 L377R, L638R, A691R, D698R 16.9 LPG13227 1121 L377R, S625R, L638R 16.4 LPG13255 1149 L377R, L638R, D698R, V747R 16.2 LPG13262 1156 L377R, S625R, L638R, A691R, D698R 15.4 LPG13238 1132 S625R, L638R, D698R 15.2 LPG13250 1144 L377R, S625R, A691R, D698R 14.0 LPG13232 1126 L377R, L638R, D698R 14.8 LPG13260 1154 S625R, A691R, D698R, V747R 13.8 LPG13222 1116 L638R, D698R 14.2 LPG13214 1108 L377R, A691R 14.1

For LPG10241, additional combined mutant systems are formed at the C-terminal tail and in the zinc-binding domain. Complete deletion of the C-terminal tail improves activity relative to the parental LPG10241 (Fig. 11A). Mutation of the C-terminal tail to a V-J type concordant sequence (eight arginine or lysine mutations) also improves editing. Finally, combined mutants with eight single arginine mutations from the C-terminal region improve editing by more than 2X relative to the parent (Fig. 11A). Similarly, mutation of the zinc finger domain (i.e., the zinc-binding domain) to a concordant sequence improves editing by more than 2X; while combining four single arginine mutations improves editing to nearly 4X (Fig. 11B).

Eighteen top-performing mutagenic combinations for each enzyme were ranked according to the average activity of the tested primers. The three most active mutagenic systems for each enzyme were evaluated using a set of 43 LPG10238 primers (SEQ ID NO: 1422 to 1464) and 37 LPG10241 primers (SEQ ID NO: 1465 to 1501) for editing (Figures 12A to 12D). For the primer sets, all selected mutagens showed an average editing efficiency improvement of at least 2.7x relative to the parent enzyme. Based on the same metric, the top-performing LPG10238 mutagen showed a 3.5x improvement relative to the parent enzyme, while the top-performing LPG10241 mutagen showed a 3.1x improvement relative to the parent enzyme (see Tables 14A and 14B, respectively).

The three most active LPG10238-derived mutants have four mutations from their parent enzyme; while the three most active LPG10241-derived mutants have three or five mutations. The percentage of sequence identity for each mutant is provided in Tables 14A and 14B. All three LPG10238 mutants share the M530R mutation. The three mutations I552R, E677R, and K801R occur in different combinations of the two mutations in the three mutants. All three LPG10241 mutants contain the mutations found in LPG13229 (L377R, S625R, D698R). Furthermore, LPG13264 and LPG13265 share the V747R mutation. The mutations that distinguish LPG13264 from LPG13265 are L638R (LPG13264) and A691R (LPG13265). Table 14A : The three most active mutants of LPG10238 outperformed the parent enzyme by 2.9 times or more. RGN ID RGN SEQ ID NO mutation Sequence consistency (%) Average edits (%) Fold Improvement LPG10238 1 Parent Unobtainable 3.2 LPG12991 1072 N517R, M530R, I552R, E677R 99.55 11.2 3.5 LPG12999 1080 M530R, E538R, I552R, K801R 99.55 9.7 3.0 LPG13001 1082 M530R, I552R, E677R, K801R 99.55 9.7 3.0 Table 14B : The three most active mutants of LPG10241 outperformed the parent enzyme by 2.7 times or more. RGN ID RGN SEQ ID NO mutation Sequence consistency (%) Average edits (%) Improvement factor LPG10241 4 Parent Unobtainable 3.7 LPG13229 1123 L377R, S625R, D698R 99.61 9.9 2.7 LPG13264 1158 L377R, S625R, L638R, D698R, V747R 99.35 11.6 3.1 LPG13265 1159 L377R, S625R, A691R, D698R, V747R 99.35 10.9 2.9 Example 8 : Guiding Body Engineering

The VJ subtype is known to utilize a relatively short primer backbone (bb) in its CRISPR RNA (crRNA). To evaluate the ability of LPG10241 to utilize a shortened primer backbone, a backbone variant was generated in plassomic form, shortening the backbone from the 5' end of the primer (Fig. 13A). Another set of primer systems was generated using a plassomic coding primer form of crRNA bb-septum-crRNA bb, with a shortened sequence on either crRNA bb (Fig. 13A). These primer backbones are summarized in Table 15. Table 15 : LPG10241 Primer Shortening Design Main chain name Architecture crRNA bb length *crRNA bb length Guide Guide body SEQ ID NO Design 1 crRNA bb-septum 36 nt SGN008539_Design 1 1567 SGN008541_Design 1 1568 SGN009798_Design 1 1569 Design 2 *crRNA bb-septum 34 nt SGN008539_Design 2 1570 SGN008541_Design 2 1571 SGN009798_Design 2 1572 Design 3 *crRNA bb-septum 32 nt SGN008539_Design 3 1573 SGN008541_Design 3 1574 SGN009798_Design 3 1575 Design 4 *crRNA bb-septum 30 nt SGN008539_Design 4 1576 SGN008541_Design 4 1577 SGN009798_Design 4 1578 Design 5 *crRNA bb-septum 28 nt SGN008539_Design 5 1579 SGN008541_Design 5 1580 SGN009798_Design 5 1581 Design 6 crRNA bb-septum-*crRNA bb 36 nt 34 nt SGN008539_Design 6 1582 SGN008541_Design 6 1583 SGN009798_Design 6 1584 Design 7 crRNA bb-septum-*crRNA bb 36 nt 32 nt SGN008539_Design 7 1585 SGN008541_Design 7 1586 SGN009798_Design 7 1587 Design 8 crRNA bb-septum-*crRNA bb 36 nt 30 nt SGN008539_Design 8 1588 SGN008541_Design 8 1589 SGN009798_Design 8 1590 Design 9 crRNA bb-septum-*crRNA bb 36 nt 28 nt SGN008539_Design 9 1591 SGN008541_Design 9 1592 SGN009798_Design 9 1593 Design 10 *crRNA bb-septum-*crRNA bb 34 nt SGN008539_Design 10 1594 SGN008541_Design 10 1595 SGN009798_Design 10 1596 Design 11 *crRNA bb-septum-*crRNA bb 32 nt SGN008539_Design 11 1597 SGN008541_Design 11 1598 SGN009798_Design 11 1599 Design 12 *crRNA bb-septum-*crRNA bb 30 nt SGN008539_Design 12 1600 SGN008541_Design 12 1601 SGN009798_Design 12 1602 Design 13 *crRNA bb-septum-*crRNA bb 28 nt SGN008539_Design 13 1603 SGN008541_Design 13 1604 SGN009798_Design 13 1605 * indicates a shortened crRNA backbone (bb).

The leader system was evaluated using plastid-lipoplastic transfection and editing efficiency of HEK293T cells, as described in Example 4. Compared to the leader host design, shortening the LPG10241 host from 36 nt to 28 nt in length produced enhanced editing (Fig. 13B).

The VH-type primer backbone has not been adequately characterized in the literature. The wild-type crRNA backbone is 36 nt long. A shortened backbone, trimmed by 2 nt from the 5' end, was evaluated and summarized in Table 16. Shortening the LPG10238 crRNA backbone did not maintain or enhance the editing efficiency of the wild-type 36 nt backbone (Figure 14). Backbone extension was not evaluated. Table 16 : LPG10238 primer backbone shortening Architecture crRNA bb length *crRNA bb length Guide Guide body SEQ ID NO *crRNA bb-septum-crRNA bb 36 nt 34 nt SGN013029 1511 SGN013036 1518 *crRNA bb-septum-crRNA bb 36 nt 32 nt SGN013030 1512 SGN013037 1519 *crRNA bb-septum-crRNA bb 36 nt 30 nt SGN013031 1513 SGN013038 1520 *crRNA bb-septum-crRNA bb 36 nt 28 nt SGN013032 1514 SGN013039 1521 *crRNA bb-septum-crRNA bb 36 nt 26 nt SGN013033 1515 SGN013040 1522 *crRNA bb-septum-crRNA bb 36 nt 24 nt SGN013034 1516 SGN013041 1523 *crRNA bb-septum-crRNA bb 36 nt 22 nt SGN013035 1517 SGN013042 1524 *crRNA bb-septum-*crRNA bb 34 nt SGN013043 1525 SGN013050 1532 *crRNA bb-septum-*crRNA bb 32 nt SGN013044 1526 SGN013051 1533 *crRNA bb-septum-*crRNA bb 30 nt SGN013045 1527 SGN013052 1534 *crRNA bb-septum-*crRNA bb 28 nt SGN013046 1528 SGN013053 1535 *crRNA bb-septum-*crRNA bb 26 nt SGN013047 1529 SGN013054 1536 *crRNA bb-septum-*crRNA bb 24 nt SGN013048 1530 SGN013055 1537 *crRNA bb-septum-*crRNA bb 22 nt SGN013049 1531 SGN013056 1538 crRNA bb-septum 36 nt SGN008516_DR-Sectional 1553 SGN008518_DR-Separator 1554 *crRNA bb-septum 34 nt SGN013057 1539 SGN013064 1546 *crRNA bb-septum 32 nt SGN013058 1540 SGN013065 1547 *crRNA bb-septum 30 nt SGN013059 1541 SGN013066 1548 *crRNA bb-septum 28 nt SGN013060 1542 SGN013067 1549 *crRNA bb-septum 26 nt SGN013061 1543 SGN013068 1550 *crRNA bb-septum 24 nt SGN013062 1544 SGN013069 1551 *crRNA bb-septum 22 nt SGN013063 1545 SGN013070 1552 * indicates a shortened crRNA backbone (bb).

Several mutations were introduced into the leader host chain, and editing improvements of the host chain variants were measured (Table 17 and Figure 15). Host chain variant 2 showed more promising editing improvements than the wild type. Table 17 : LPG10238 leader host chain sequence variants Main chain variant compartment 1 compartment 2 Guide body (SEQ ID NO) Average edits (%) Guide body (SEQ ID NO) Average edits (%) Parent SGN008516 (SEQ ID NO: 43) 3.68 SGN008518 (SEQ ID NO: 45) 9.1125 Variant 1 SGN013071 (SEQ ID NO: 1555) 0.05 SGN013077 (SEQ ID NO: 1561) 3.015 Variant 2 SGN013072 (SEQ ID NO: 1556) 6.055 SGN013078 (SEQ ID NO: 1562) 13.865 Variant 3 SGN013073 (SEQ ID NO: 1557) 0 SGN013079 (SEQ ID NO: 1563) 0 Variant 4 SGN013074 (SEQ ID NO: 1558) 0 SGN013080 (SEQ ID NO: 1564) 0 Variant 5 SGN013075 (SEQ ID NO: 1559) 1.22 SGN013081 (SEQ ID NO: 1565) 6.665 Variant 6 SGN013076 (SEQ ID NO: 1560) 0 SGN013082 (SEQ ID NO: 1566) 0 Example 9 : Editing the insertion / substitution of glycine to add LPG10238 and LPG10241

In their catalytic loops, Cas enzymes exhibit dynamism, opening and closing, and rearrangement to adapt to the substrate. Optimal flexibility may be the essence of high performance. To assess the variability of LPG10238 and LPG10241, the amino acid sequences of these enzymes were analyzed for amide bond order/disorder using the web tool DynaMine (Cilia et al. (2013) From protein sequence to dynamics and disorder with DynaMine, Nature Communications 4:2741 doi: 10.1038/ncomms3741; Cilia et al. (2014) The DynaMine webserver: predicting protein dynamics from sequence, Nucleic Acid Research doi: 10.1093/nar/gku270). ^S² score maps of each residue were used to depict the protein's variability along its sequence. An ^S² value of 1 indicates complete order/structure, while a value of 0 indicates complete disorder/randomness.

Most wild-type LPG10238 proteins exhibit limited mutagenesis, while LPG10241 is already a flexible protein. Mutant sequences for these enzymes are generated to enhance their mutagenesis. Based on the ^S2 diagram, residues exhibiting local minima are selected for glycine insertion or substitution. In the absence of R groups, the amide bond with glycine is mutable. To enhance mutagenesis, single, double, or triple glycine motifs are inserted into and/or substituted into the protein coding sequence. Twenty-four local minima were selected in LPG10238 for glycine mutation. Thirteen local minima were selected in LPG10241 for glycine mutation. The mutants are summarized in Table 18. Table 18 : Glycine Motif Insertion / Substitution Mutants RGN ID Count of local minima The number of mutants produced Mutant RGN ID SEQ ID NOs LPG10238 twenty four 148 LPG12074 to LPG12221 1257 to 1404 LPG10241 13 83 LPG11991 to LPG12073 1174 to 1256

Glycine mutants were evaluated in the same manner as arginine mutants in Example 6. Initial screening and validation screening were performed using the same leads. Alternatively, for validation of the LPG10241 mutant, only SGN013008 and SGN013009 were used (Figures 16A and 16B).

The LPG10241 glycine mutant screening yielded two mutants, LPG12040 and LPG12045, with enhanced editing capabilities (Table 19). Table 19 : LPG10241 glycine mutants exhibit enhanced editing capabilities . Mutant RGN ID (SEQ ID NO) mutation Average improvement factor of the parent lines LPG12040 (SEQ ID NO: 1223) D566G+E567G 1.9x LPG12045 (SEQ ID NO: 1228) E584G 3.0x

For LPG10238, glycine mutation screening yielded several mutant families with enhanced editing (Table 20, Figures 16C and 16D). Table 20 : LEG10238 glycine mutants exhibit enhanced editing. Mutant RGN ID (SEQ ID NO) mutation Average improvement factor of the parent lines LPG12121 (SEQ ID NO: 1304) K293_N294insGGG 1.4x LPG12139 (SEQ ID NO: 1322) G359_S360insGG 1.5x LPG12141 (SEQ ID NO: 1324) K361G 1.5x LPG12145 (SEQ ID NO: 1328) G402_N403insG 1.4x LPG12147 (SEQ ID NO: 1330) N401G 1.4x LPG12178 (SEQ ID NO: 1361) T596G 1.3x LPG12217 (SEQ ID NO: 1400) D862G 1.4x Example 10 : Arg-Gly combination mutation (predictive)

The arginine and glycine mutations of LPG10238 and LPG10241 described in Examples 6, 7 and 10 are combined to create LPG10238 and LPG10241 mutant enzymes including arginine substitution and glycine substitution and/or insertion.

Mutant systems with mammalian gene editing activity were screened using plasmid-lipoplastin transfection and insertion/deletion NGS quantification methods described in Example 4. To account for inter-experimental variability, editing efficiency was normalized to the parental LPG10238 or LPG10241 per plate. Initial screening of arginine + glycine mutants was performed using two primers targeting two dissimilar genomic loci. The best-performing mutants were selected for further testing in validation rounds with additional primers. Example 11 : Multiple Editing and Removal

Type V enzymes are attractive because, compared to Cas II enzymes, they possess the general ability to process their own guide RNA while being smaller in both size and size. For delivery methods using vectors such as adeno-associated virus (AAV), the reduction in enzyme and guide RNA size leaves more sequence space for writing additional guide RNA. The multiplex editing and DNA excision capabilities of LPG10238 and LPG10241 were evaluated. The previously described guide plasmid was modified to encode tandem spacers in an array. This form includes the human U6 promoter (SEQ ID NO: 38), followed by a crRNA backbone; spacer 1; another crRNA backbone; spacer 2; and finally terminated with a crRNA backbone (for LPG10238 only) (Table 21).

For LPG10238, septa were selected such that both target the same amplicon and produce an amplicon with a small excision. The LPG10241 construct was designed to excise large regions of the genome by targeting two exons of the same gene; the excision products cause the exons to aggregate and form amplifiable repair product. These guide systems were evaluated using the LPG10241 variant LPG13264. The guide systems are described in Tables 21 and 22. The evaluation was performed as described in Example 4. Table 21 : LPG10238 tandem guide plasmids used for genome excision . Guiding body array RGN ID target amplicon primer set Excised products SGN008516+ SGN008518 (SEQ ID NO: 1606) LPG10238 B2M_ex2 707/700 About 200 bp SGN008518+ SGN008516 (SEQ ID NO: 1607) LPG10238 B2M_ex2 707/700 About 200 bp SGN009694+ SGN0010530 (SEQ ID NO: 1608) LPG10238 LDHA_2 About 140 bp SGN010530+ SGN009694 (SEQ ID NO: 1609) LPG10238 LDHA_2 About 140 bp SGN010530+ SGN0010531 (SEQ ID NO: 1610) LPG10238 LDHA_2 About 85 bp SGN010531+ SGN0010530 (SEQ ID NO: 1611) LPG10238 LDHA_2 About 85 bp

For both LPG10238 and the B2M and LDHA targeting guide plasmid arrays, the excision products were observed through depth sequencing, confirming that LPG10238 not only has multiplex editing capabilities but also the ability to excise small fragments of the genome. Selected alignments are shown in Figures 17A and 17B. Table 22 : LPG10241 tandem guide plasmids used for genome excision . Guiding body array RGN ID Target amplicon Excised products Repairing the amplicon SGN013151+ SGN013156 (SEQ ID NO: 1612) LPG13264 LDHA_6-10 Approximately 6 kb About 400 bp SGN013156+ SGN013151 (SEQ ID NO: 1613) LPG13264 LDHA_6-10 Approximately 6 kb About 400 bp

For LPG10241, which targets a pair of exons of the LDHA gene, the excision product was detected and sequenced, confirming multiple targeting to both upstream and downstream exons. Furthermore, the repair excision caused the two exons to aggregate (Figures 18A and 18B). Example 12 : Induction Treatment

As discussed in this paper, an attractive feature of type V Cas enzymes is the ability of this enzyme family to process its own guide RNA, unlike type II Cas enzymes which require a separate host-provided RNase III. The self-processing capacity of wild-type and non-catalytically active LPG10238 and LPG10241 was assessed by in vitro biochemical assays.

The E. coli codon-optimized architecture in Example 3 was modified to add 2xStrep tracer (SEQ ID NO: 1637) to the 5' of the existing His10 tracer (SEQ ID NO: 1638), thereby generating triple-trace LPG10238 and LPG10241 RGN (SEQ ID NO: 1639 and 1640, respectively). The nuclease-inactivating (non-nuclease-active) architecture is generated by inducing the three active site residues of each enzyme to alanine (non-nuclease-active LPG10238 includes the amino acid sequence shown in SEQ ID NO: 1641; non-nuclease-active LPG10241 includes the amino acid sequence shown in SEQ ID NO: 1642). Each of the four structures was electroporated into an E. coli EXPRESS BL21 (DE3) competitive cell (Biosearch Technologies) and screened at 37 °C on LB agar plates containing 50 μg/mL kanamycin sulfate (Teknova). After overnight incubation, single colonies were selected and grown in 50 mL LB agar supplemented with 50 μg/mL kanamycin sulfate, and then shaken overnight at 37 °C in a 250 mL Ultra Yield Flask (Thomson Scientific) sealed with an AirTop seal. Thirty mL of overnight culture was inoculated into 500 mL of MagicMedia (Thermo Scientific) broth in a 2.5 L Ultra Yield Flask (Thomson Scientific) sealed with an AirTop seal and supplemented with 50 μg/mL kanamycin. The culture was grown at 30 °C with shaking at 300 rpm for 6 hours, followed by growth at 18 °C for 18 hours. Cells were harvested by centrifugation and frozen until use.

Each cell pellet was thawed and resuspended on ice in 25 mL of lysis buffer (Table 23). Cells were lysed by sonication; the lysates were then clarified by centrifugation. The clarified lysates were separated from the precipitated cell debris and incubated with pre-equilibrated Streptactin XT Sepharose Resin (Cytiva). The lysate-resin slurry was applied to an empty PD-10 column (Cytiva), and the flow through was collected. Prior to elution, the column was washed with 5 column volumes (CV) of binding buffer (Table 23). To elute the target protein, a 0.5 CV was applied six times, and each fraction was collected. The fractions were analyzed by SDS-PAGE prior to pooling. Following the manufacturer's instructions, the pooled fraction buffer was exchanged for storage buffer (Table 23) via a PD-10 column (Cytiva). The buffer-exchanged samples were concentrated using an Amicon Ultra – 4 50 kDa MWCO centrifugal concentrator (Amicon). The concentrated protein was aliquoted and stored in a Matrix Tube (Thermo Scientific). Table 23 : RGN Purification Buffer Composition buffer solution Components Combined buffer 100 mM HEPES, pH 8, 500 mM NaCl, sterilized by filtration Lysis buffer 100 mM HEPES, pH 8, 500 mM NaCl, 1 tablet per 50 mL of protease inhibitor, filtered and sterilized. Eluent and buffer solution 100 mM HEPES, pH 8, 500 mM NaCl, 50 mM d-Biotin, filtered and sterilized. Storage buffer 50 mM HEPES, pH 8, 300 mM NaCl, 20% glycerol, filtered and sterilized. Diluted buffer solution 50 mM HEPES, pH 8, 300 mM NaCl, 1 mM DTT, filtered and sterilized.

Enzyme activity was assessed by in vitro shear assay. In short, dsDNA receptacles containing nuclease PAMs at the 5' end of two 25 bp heterologous spacers (targeted L1 (SEQ ID NO: 1659) and L2 (SEQ ID NO: 1660)) were amplified using primers traced by 5'-fluorescence (Table 24). The dsDNA receptacles were identical for both LPG10238 and LPG10241, with appropriate PAMs for each nuclease. Purified enzymes were diluted in a dilution buffer, and RNPs were formed by incubation at room temperature with a 1.5x molar excess of synthetic primers targeting L1 or L2. Reactions containing RNPs, fluorescently traced dsDNA receptors, and rCutsmart buffer (New England Biolabs) were prepared. The reactions were incubated at 37°C, and time points were obtained by removing aliquots and quenching in 50 mM EDTA. The quenched reactions were analyzed by capillary electrophoresis fragment analysis on a 3730xl DNA analyzer (Thermo Fisher Scientific) using the provider (EtonBio). Results were processed using the instrument manufacturer's software (Thermo Fisher Scientific) and then further analyzed using custom Python code. Cleavage activity was determined by the ratio of the signal from the cleaved DNA to the signal from the full-length DNA. An exemplary set of data is illustrated in Figures 19A and 19B (LPG10238) and Figures 20A and 20B (LPG10241). Table 24 : Plasmins of in vitro shear tactile receptors used for amplified fluorescent tracing . dsDNA template RGN ID dsDNA substrate Target Introduction Fluorescent pUC19-GAATATTC-PAM-v2 (SEQ ID NO: 1651) LPG10241 TTC-L1 (SEQ ID NO: 1653), TTC-L2 (SEQ ID NO: 1654) L1, L2 FAM-M13F-40 (SEQ ID NO: 1643), HEX-M13R-48 (SEQ ID NO: 1644) 6-FAM, HEX pUC19-GAACTATG-PAM-v2 (SEQ ID NO: 1652) LPG10238 ATG-L1 (SEQ ID NO: 1656), ATG-L2 (SEQ ID NO: 1657) L1, L2 FAM-M13F-40 (SEQ ID NO: 1643), HEX-M13R-48 (SEQ ID NO: 1644) 6-FAM, HEX

The synthetic tandem primer arrays were designed with 5'-6 FAM tracers and spacers (arrays 47 (SEQ ID NO: 1662), 48 (SEQ ID NO: 1663), 49 (SEQ ID NO: 1664), and 50 (SEQ ID NO: 1665)) targeting L3 (SEQ ID NO: 1661) and L2 (SEQ ID NO: 1660) in two different configurations. Similarly, the dsDNA receptors had PAMs at the 5' of the target L3 and L2. Each target was amplified using a primer pair containing a unique set of fluorescent molecules (Table 25). RNP formation was performed at 37°C in rCutsmart buffer. Shearing reactions and analysis were performed as described previously. The nuclease activity using synthetic tandem primer arrays was confirmed by comparison with similar reactions using synthetic single primers (sg238.1[25], sg238.2[25] and sg238.3[25] (SEQ ID NOs: 1666 to 1668); sg241.1[25], sg241.2[25] and sg241.3[25] (SEQ ID NO: 1669 to 1671)). To monitor primer treatment, the FAM signal of the full-length primer array was tracked throughout the reaction time. Smaller FAM trace fragments appearing with the loss of the full-length fragment indicate leader treatment; however, due to limitations in assays/detection, high levels of noise exist in these small fragments, and the precise size of the fragment cannot be determined. A set of reactions is summarized and shown in Figures 21A and 21B. Table 25 : gBlocks used to generate fluorescently traced dsDNA receptors . dsDNA template RGN ID dsDNA substrate Target Introduction Fluorescent body GAACATTC-PAM-v3.2 gBlock (SEQ ID NO: 1649) LPG10241 TTC-L3 (SEQ ID NO: 1655) L3 FAM-M13F-40 (SEQ ID NO: 1643), PET-MidR (SEQ ID NO: 1645) 6-FAM, PET TTC-L2 (SEQ ID NO: 1654) L2 NED-MidF (SEQ ID NO: 1646), HEX-M13R-48 (SEQ ID NO: 1644) NED, HEX GAACTATG-PAM-v3.2 gBlock (SEQ ID NO: 1650) LPG10238 ATG-L3 (SEQ ID NO: 1658) L3 FAM-M13F-40 (SEQ ID NO: 1643), PET-MidR (SEQ ID NO: 1645) 6-FAM, PET ATG-L2 (SEQ ID NO: 1657) L2 NED-MidF (SEQ ID NO: 1646), HEX-M13R-48 (SEQ ID NO: 1644) NED, HEX Example 13 : AAV delivery of multiple leaders for multiple knockout

The type V nucleases described here are small in size, allowing for efficient, integrated cartridge-type AAV delivery via a ubiquitous strong polymerase II promoter and one or more (if the target is multiple targets or excisions) RNA-guided nucleases. Furthermore, the self-processing of these RNA-guided nucleases allows for the expression of multiple promoters using a single polymerase III promoter, thereby providing the necessary space for the regulation and expression elements required when using AAV vectors (see Table 26 for component sizes within the AAV cartridge).

Several different all-in-one AAV cartridges were tested to identify the most efficient way to deliver these editing systems. For nuclease expression, two different pol II promoters were tested: the CMV promoter + CMV upstream enhancer (SEQ ID NO: 1674) and CBh (SEQ ID NO: 1675). The nuclease coding sequences were optimized for expression in mammalian cells. For expression of (one or more) guide RNAs, two different systems were explored. First, all guide RNAs were expressed in a single transcript using the human U6 promoter (SEQ ID NO: 1672), in which case tandem repeat-septum arrays were designed for each nuclease to target the leader pathways of LDHA and B2M. Secondly, the human U6 pol III promoter was used to express one guide RNA targeting B2M, while the human 7SK pol III promoter was used to express another guide RNA targeting LDHA. Specifically, the target sequences are shown in Table 27a. TRAC and B2M target sequences that can be targeted using the LPG10238, LPG10239, LPG10240, and LPG10241 systems are shown in Table 27b. Additional components are included for regulation and expression. "Singlet" controls were also generated and tested with the multiplexing architecture, where only one guide RNA was used for each given target. The sizes of the nuclease-specific components are shown in Table 28. Table 26 : Sizes of Fixed Components in the AAV Box Composition Size ( nt ) Human U6 pol III activator (SEQ ID NO: 1672) 249 Human 7SK pol III promoter (SEQ ID NO: 1673) 243 CMV pol II initiator + CMV upstream enhancer (SEQ ID NO: 1674) 530 CBh pol II promoter (SEQ ID NO: 1675) 804 N-terminal NLS (cMYC) + GSSGGSSG connector (SEQ ID NO: 1676) 51 C-terminal GSSGGSSG connector + NLS (cMYC) (SEQ ID NO: 1677) 51 SV40 polyA signal (SEQ ID NO: 1678) 183 Table 27a : Target sequences of repeater - septum arrays target sequence B2M target LDHA target LPG10238 GTGCTTATTCAATTCGCTAGAAATGAGTAAAAGCAC (SEQ ID NO: 5) TCGGATGGATGAAACCCAGACACAT (SEQ ID NO: 189) GTGGAAACTGGGTGCACCCGCCTAA (SEQ ID NO: 1685) Table 27b : Target sequences of repeater - spacer arrays repeating sequence TRAC target B2M target LPG10241 CCTGCAAAGCTCAAAGATTGCTCGATTCGTCGAGAC (SEQ ID NO: 8) GCATGTGCAAACGCCTTCAACAACAGCATT (SEQ ID NO: 269) AGTGGGGTAAGTCTTACATTCTTTTGTAAG (SEQ ID NO: 218) LPG10240 GTGGGAGTTCCTCTTGAAGGCTCGGTTCGCCGAGAC (SEQ ID NO: 7) GCAGGCTGTTTCCTTGCTTCAGGAATGGCC (SEQ ID NO: 276) AGTGGGGTAAGTCTTACATTCTTTTGTAAG (SEQ ID NO: 218) LPG10239 GCCGCAACAGCTATTGATTGCTCGATACGTCGAGAC (SEQ ID NO: 6) CTGAAGCAAGGAAACAGCCTGCGAAGGCAC (SEQ ID NO: 281) CTATGTGTCTGGGTTTCATCCATCCGACAT (SEQ ID NO: 211) LPG10238 GTGCTTATTCAATTCGCTAGAAATGAGTAAAAGCAC (SEQ ID NO: 5) CAGGCCTGGGACATGCAAGCCCATAACCGC (SEQ ID NO: 248) TCGGATGGATGAAACCCAGACACATAGCAA (SEQ ID NO: 232) Table 28 : Nuclease-specific composition in AAV cartridges The length (nt) of the encoded sequence of the write-code RGN containing the termination key. Size of the repeater-septum array (nt) Total size (nt) of the AAV genome with two guideways LPG10241 2310 168 3695 LPG10240 2358 168 3743 LPG10239 2310 168 3695 LPG10482 (a mutant of LPG10238) 2667 168 4052

These multiplexed AAV cassettes are reduced to less than the 4.7 kb size limit of an effective genome package (Table 29a). Tables 29a and 29b show the AAV architectures of the LPG10238 system. Table 29b provides a description of the primers used for writing codes in these AAV architectures and their forms.

In short, each construct was generated in HEK293 suspension-adapted cells (Gibco virus-generating cells) under platform conditions via a transient transfection-based procedure. On day 0, production shake flasks were seeded at 1.6 x ^10⁶ VCD (live cells/mL) in the required amount of production medium (Gibco LV-MAX Production Medium). After 24 hours, cells were transfected at approximately 3.0 x ^10⁶ VCD as follows: each transfection mixture was prepared in 5% culture volume (v/v) using DMEM medium as a diluent. pDNA was added at a 1:1:1 molar ratio of 0.7 pg/viable cells and three plastids (pTransgene, pRC, and pHelper). After mixing the pDNA/diluent medium, FectoVIR-AAV (Polyplus) was added to the solution at a ratio of 0.8:1 (mg:mg) to the total pDNA. The solution was manually and vigorously mixed for 15 seconds, the precipitate was checked, and the solution was allowed to incubate for 5 minutes before being added as a push to a flask.

On day 3 post-transfection, the culture was obtained by detergent lysis with 0.5% (v/v) Devilon 13-S9 (MilliporeSigma) + 30 U/ml Denarase nuclease (C-Lecta). The lysis was performed at 37°C in a shaker incubator for 3 hours. After lysis, the lysate was clarified by centrifugation at 5000 xg for 45 min, and the supernatant was filtered through a 0.2 μm PES vacuum flask filter. The clarified lysate containing the carrier was loaded onto a pre-packed column containing AAVX affinity resin (A36651; ThermoFisher) after a two-minute retention time. After washing the resin with a equilibration buffer, the capsid was eluted at a low pH (50 mM glycine/250 mM NaCl/0.01% (v/v) poloxamer 188 (pH 2.0)) with a retention time of 4 minutes. The eluent was immediately neutralized with Tris buffer. The neutralized eluent was then buffer-exchanged and concentrated using a Pierce Concentrator 100KDA MWCO column, 2–6 mL. The support was frozen and stored at -80°C. Table 29a : AAV Structures Structure The length (nt) of the encoded sequence of the write-code RGN containing the termination key. Subject 1 (SEQ ID NO) Subject 2 (SEQ ID NO) Repeater-septum array size (nt) Total size (nt) of the AAV genome with two guideways pLE259 2661 TCGGATGGATGAAACCCAGACACAT (SEQ ID NO: 189) GTGGAAACTGGGTGCACCCGCCTAA (SEQ ID NO: 1685) 158 4474 pLE291 2661 TCGGATGGATGAAACCCAGACACAT (SEQ ID NO: 189) GTGGAAACTGGGTGCACCCGCCTAA (SEQ ID NO: 1685) 158 4203 pLE292 2661 TCGGATGGATGAAACCCAGACACAT (SEQ ID NO: 189) GTGGAAACTGGGTGCACCCGCCTAA (SEQ ID NO: 1685) 97 + 97 4507 pLE296 2661 TCGGATGGATGAAACCCAGACACAT (SEQ ID NO: 189) Unobtainable 97 4142 pLE297 2661 GTGGAAACTGGGTGCACCCGCCTAA (SEQ ID NO: 1685) Unobtainable 97 4142 Table 29b : Description of AAV Structures Structure describe Guide body form pLE259 hU6 starter-SGN008518-SGN010571-Cbh starter-cMycNLS-LPG10482-cMycNLS-SV40 poly A crRNA bb-septum-crRNA bb-septum-crRNA bb pLE291 hU6 starter - SGN008518 - SGN010571 - CMV starter + enhancer - cMycNLS - LPG10482 - cMycNLS - SV40 poly A crRNA bb-septum-crRNA bb-septum-crRNA bb pLE292 hU6 starter - SGN008518 - h7SK starter - SGN010571 - CMV starter + enhancer - cMycNLS - LPG10482 - cMycNLS - SV40 poly A crRNA bb-septum-crRNA bb pLE296 hU6 starter-SGN008518-CMV starter + enhancer-cMycNLS-LPG10482-cMycNLS-SV40 poly A crRNA bb-septum-crRNA bb pLE297 hU6 starter-SGN010571-CMV starter + enhancer-cMycNLS-LPG10482-cMycNLS-SV40 poly A crRNA bb-septum-crRNA bb

Once the vectors are generated, their functionality is tested via transduction with HEK293T cells. In short, HEK293T cells are plated at 25,000 cells per well in 24-well plates. Four to six hours after cell platening, each vector is transduced at 125,000, 250,000, 500,000, and 1,000,000 vector genomes per cell (also known as the multiplicity of infection (MOI)). After 24 hours, the medium (DMEM + 10% FBS + 1% PenStrep) is aspirated and replaced with 1 mL of fresh medium. 120 hours after transduction, genomic DNA is extracted from the cells, as outlined in Example 4. Multiplex editing at two different loci using an integrated AAV box is feasible and effective. Compared to single editing, the optimal multiplex design retains >50% activity at each locus. The CMV+ enhancer pol II promoter outperforms the CBh pol II promoter. Adding pol III (+7SK) to drive the second primer is more advantageous than having two tandem primers driven by a single pol III promoter (Figures 22A and 22B, Tables 30 and 31). Table 30 : Editing at B2M of AAV integrated plasmids transfected into HEK293T cells ( % ) MOI pLE296 pLE259 pLE291 pLE292 1.25e5 8.19 0.22 0.12 1.62 2.5e5 23.27 0.87 1.39 9.15 5e5 39.36 2.59 7.14 23.46 1e6 49.94 4.47 13.6 32.51 Table 31 : Editing ( % ) of LDHA sites on AAV monoclonal plasmids transfected into HEK293T cells MOI pLE297 pLE259 pLE291 pLE292 1.25e5 10.52 0.03 0.13 5.95 2.5e5 29.57 1.39 3.11 18.62 5e5 51.27 4.72 11.2 38.88 1e6 67.47 9.25 21.45 49.17 Example 14 : Characterization of cut points

The cleavage sites of LPG10238, LPG10239, LPG10240, and LPG10241 were characterized by biochemical assays. RNPs, as in Example 3, were generated using the PURExpress system and appropriate sgRNA. The difference from Example 3 is that the recipient plasmid has a defined PAM with L1 and L2 marker sequences on both sides (SEQ ID NO: 1649 and 1650, respectively). Post-cleavage reactants were prepared by NGS in the same manner as in Example 3, but the cleavage plasmids on the proximal and distal sides of the PAM were amplified. Deep sequencing was performed by the service provider (Elevate-Bio, Waltham, MA) on the NextSeq platform (Illumina). Transplant sequences were trimmed, and reads were aligned with the recipient plasmid to determine the cleavage pattern. LPG10239, LPG10240, and LPG10241 exhibit staggered cut sites with significant protrusions of 5 to 12 nt at 5' (Table 32). In contrast, LPG10238 produces cut sites, with the most enriched reads corresponding to a 1 nt protrusion at 3' (Table 32). For all RGNs evaluated, the shearing patterns differ slightly between L1 and L2 targets; however, the overall trend remains the same (Figures 23A and 23B). Table 32 : Characterization of cut sites from LPG10238 to LPG10241 RGN Subtype L1 is subject to mass L2 mass Shear point location (TS/NTS) Types of protrusions Shear point location (TS/NTS) Types of protrusions LPG10241 Vj 22/10 5' – 12 nt 22/14 5' – 8 nt LPG10240 Vj 23/18 5' – 5 nt 21/16 5' – 5 nt LPG10239 Vj 22/12 5' – 10 nt 22/12 5' – 10 nt LPG10238 Vh 22/23 3' – 1 nt 23/24 3' – 1 nt TS = Target Stock; NTS = Non-Target Stock

AS: Active site; C: Carboxyl group terminus; N: Amine group terminus; OBD: Oligobinding domain; PAM: Protoseptate adjacent motif; PI: PAM interaction domain; ZF: Zinc finger domain.

Figures 1A through 1D show the structures of the LPG10238 (Figure 1A), LPG10239 (Figure 1B), LPG10240 (Figure 1C), and LPG10241 (Figure 1D) guiding RNAs (i.e., crRNAs) disclosed herein. Figure 2 shows the insertion/deletion (Indel) efficiency % of the LPG10238, LPG10241, LPG13113, and LPG13115 systems. Gene editing efficiency was determined as described in Example 4. Figures 3A to 3EE show the sequence logos of the adjacent motif (PAM) sites of the protoseptum identified by the following: LPG10238 (having the amino acid sequence as shown in SEQ ID NO: 1) (Figure 3A), LPG10239 (having the amino acid sequence as shown in SEQ ID NO: 2) (Figure 3B), LPG10240 (having the amino acid sequence as shown in SEQ ID NO: 3) (Figure 3C), LPG10241 (having the amino acid sequence as shown in SEQ ID NO: 4) (Figure 3D), LPG13090 (having the amino acid sequence as shown in SEQ ID NO: 1930) (Figure 3E), LPG13091 (having the amino acid sequence as shown in SEQ ID NO: 1931) (Figure 3F), LPG13092 (having the amino acid sequence as shown in SEQ ID NO: 1932) (Figure 3G), LPG13093 (having the amino acid sequence as shown in SEQ ID NO: 1932) (Figure 3G), LPG13093 (having the amino acid sequence as shown in SEQ ID NO: 1932) (Figure 3D), LPG10239 (having the amino acid sequence as shown in SEQ ID NO: 1932) (Figure 3E), LPG10240 (having the amino acid sequence as shown in SEQ ID NO: 1932) (Figure 3G), LPG10241 (having the amino acid sequence as shown in SEQ ID NO: 1932) (Figure 3D ... The amino acid sequences shown in SEQ ID NO: 1933 (Figure 3H), LPG13094 (with the amino acid sequence shown in SEQ ID NO: 1934) (Figure 3I), LPG13095 (with the amino acid sequence shown in SEQ ID NO: 1935) (Figure 3J), LPG13096 (with the amino acid sequence shown in SEQ ID NO: 1936) (Figure 3K), LPG13097 (with the amino acid sequence shown in SEQ ID NO: 1937) (Figure 3L), LPG13098 (with the amino acid sequence shown in SEQ ID NO: 1938) (Figure 3M), LPG13099 (with the amino acid sequence shown in SEQ ID NO: 1939) (Figure 3N), LPG13100 (with the amino acid sequence shown in SEQ ID NO: 1940) (Figure 3O), LPG13101 (with the amino acid sequence shown in SEQ ID NO: 1940) (Figure 3O), LPG13101 (with the amino acid sequence shown in SEQ ID NO: 1934) (Figure 3H), LPG13094 (with the amino acid sequence shown in SEQ ID NO: 1934) (Figure 3I), LPG13095 (with the amino acid sequence shown in SEQ ID NO: 1935) (Figure 3J), LPG13096 (with the amino acid sequence shown in SEQ ID NO: 1936) (Figure 3K), LPG13097 (with the amino acid sequence shown in SEQ ID NO: 1937) (Figure 3L), LPG13098 (with the amino acid sequence shown in SEQ ID NO: 1938) (Figure 3M), LPG13099 (with the amino acid sequence shown in SEQ ID NO: 1939) (Figure 3N), LPG13100 (with The amino acid sequences shown in SEQ ID NO: 1941 (Figure 3P), LPG13102 (with the amino acid sequence shown in SEQ ID NO: 1942) (Figure 3Q), LPG13103 (with the amino acid sequence shown in SEQ ID NO: 1943) (Figure 3R), LPG13104 (with the amino acid sequence shown in SEQ ID NO: 1944) (Figure 3S), LPG13105 (with the amino acid sequence shown in SEQ ID NO: 1945) (Figure 3T), LPG13106 (with the amino acid sequence shown in SEQ ID NO: 1946) (Figure 3U), LPG13107 (with the amino acid sequence shown in SEQ ID NO: 1947) (Figure 3V), LPG13108 (with the amino acid sequence shown in SEQ ID NO: 1948) (Figure 3W), LPG13109 (with the amino acid sequence shown in SEQ ID NO: 1949) (Figure 3W), LPG13109 (with the amino acid sequence shown in SEQ ID NO: 1949) (Figure 3Q), LPG13103 (with the amino acid sequence shown in SEQ ID NO: 1943) (Figure 3R), LPG13104 (with the amino acid sequence shown in SEQ ID NO: 1944) (Figure 3S), LPG13105 (with the amino acid sequence shown in SEQ ID NO: 1945) (Figure 3T), LPG13106 (with the amino acid sequence shown in SEQ ID NO: 1946) (Figure 3U), LPG13107 (with the amino acid sequence shown in SEQ ID NO: 1947) (Figure 3V), LPG13108 (with the amino acid sequence shown in SEQ ID NO: 1948) (Figure 3W), LPG13109 (with The amino acid sequences shown in SEQ ID NO: 1949 (Figure 3X), LPG13110 (with the amino acid sequence shown in SEQ ID NO: 1950) (Figure 3Y), LPG13111 (with the amino acid sequence shown in SEQ ID NO: 1951) (Figure 3Z), LPG13112 (with the amino acid sequence shown in SEQ ID NO: 1952) (Figure 3AA), LPG13113 (with the amino acid sequence shown in SEQ ID NO: 1953) (Figure 3bb), LPG13114 (with the amino acid sequence shown in SEQ ID NO: 1954) (Figure 3CC), LPG13115 (with the amino acid sequence shown in SEQ ID NO: 1955) (Figure 3DD), and LPG13116 (with the amino acid sequence shown in SEQ ID NO: 1956) (Figure 3EE). Each position in the PAM sequence has a nucleotide stack, where the total height of each stack indicates the sequence conservation at that position (measured in units of bits), and the height of each nucleotide within the stack reflects the relative frequency of that nucleotide at that position. Figures 4A and 4B show the mapping domain organization of LPG10241 (Figure 4A) and LPG10238 (Figure 4B) as described in Example 5. N = amino terminus; C = carboxyl terminus; PI = PAM interaction domain; RecI and RecII domains of the recognition lobe; OBD = oligo binding domain; RuvC-I, RuvC-II, RuvC-III and tripartite RuvC domains of the nuclease lobe; ZF = zinc finger domain; AS = active site. The three acidic active site residues in the RuvC-I, RuvC-II, and RuvC-III domains indicate the editing activity of each RGN polypeptide. Figures 5A through 5D show the gene editing activity of LPG10238 mutants (Figures 5A and 5B) and LPG10241 mutants (Figures 5C and 5D) normalized to the parental LPG10238 or LPG10241 gene editing activities, respectively. Preliminary screening (Figure 5A for LPG10238 and Figure 5C for LPG10241) evaluated the editing of the two promoters and two genomic loci. In the initial screening, 40 to 90 of the most active mutants were selected for further testing in the validation rounds. Two additional lead syntheses were used for the LPG10238 mutant, and one for the LPG10241 mutant (Figure 5B shows LPG10238, and Figure 5D shows LPG10241). Figures 6A and 6B show that LPG10238 (Figure 6A) and LPG10241 (Figure 6B) with mutations at the active site residues in the RuvC domain do not exhibit editing activity (the active site residues are indicated in Figures 4A and 4B and described in Example 5). Figures 7A and 7B show that the cysteine residue of the coordinating zinc in the ZF domain of LPG10241 conforms to the canonical CxxC motif (Figure 7A) and is important for editing activity. A mutation of the cysteine at position 687, which is not part of the CxxC motif, still allows for editing activity. Figures 8A and 8B show the average improvement in editing activity of the 16 best-performing LPG10238 mutants (Figure 8A) and the 15 best-performing LPG10241 mutants (Figure 8B) relative to the parental LPG10238 and LPG10241 RGN peptides, respectively. Six mutants indicated by checkmarks for each of LPG10238 and LPG10241 were selected for combinatorial mutant evaluation. Figures 9A and 9B show the editing activity of LPG10238 (Figure 9A) and LPG10241 (Figure 9B) combination mutants normalized to the parental LPG10238 and LPG10241 activities, respectively. The combination mutants comprise all 63 possible combinations (6 single mutants, 15 double mutants, 20 triple mutants, 15 quadruple mutants, 6 quintuple mutants, and 1 hexaple mutant) selected from the 6 mutants in Figures 8A and 8B, respectively, as well as other combination mutants as described in Example 7. These combination mutants were screened using an additional set of five to six leaders. Figures 10A and 10B show the percentage of editing efficiency for the 18 best-performing LPG10238 (Figure 10A) and 17 best-performing LPG10241 (Figure 10B) hybrid mutant combinations. On average, the 18 most active LPG10238 mutants showed greater than or equal to 2-fold editing enhancement compared to the parental LPG10238 (Figure 10A). Similarly, the 17 most active LPG10241 hybrid mutant combinations showed greater than 1.3-fold editing enhancement relative to the parental LPG10241 (Figure 10B). The five guide RNAs and the average percentage of editing efficiency for each guide RNA are shown in Figure 10A. The average percentage of editing efficiency for each of the six guide RNAs is shown in Figure 10B. Figure 10B shows parental LPG10241 controls including parental LPG10241 with the parental guide RNA (indicated by circles on the x-axis) and parental LPG10241 with an engineered guide RNA (indicated by "WT" on the x-axis). The percentages of editing efficiency indicated above the "WT" bars are 3.8%, 1.3%, 2.9%, 3.6%, 4.9%, and 17.7% from left to right. The LPG10241 hybrid mutant system was tested using engineered guide RNA. Figures 11A and 11B show the improvements in editing by the LPG10241 mutant compared to parental LPG10241. The complete deletion of the C-terminal tail relative to parental LPG10241 (Figure 11A) improves activity (Figure 11A, "C-terDel1"). The missing amino acids are 751 to 769 (GQTPDSDEESEVALHTATA (SEQ ID NO: 1927)). C-terminal tail mutation to the V-j type consensus sequence ("Con.Cter1", 8 arginine or lysine mutations) also improves editing. Finally, the combined mutant with 8 single arginine mutations from the C-terminal region improves editing by more than 2-fold relative to the parent ("R.ScanCter"). (Figure 11B) Zinc finger domain (i.e., zinc binding domain) mutation to the consensus sequence improves editing by more than 2-fold ("Cons.Zn1"), while the combination of four single arginine mutations improves editing by nearly 4-fold ("R.ScanZn1"). Figures 12A through 12D show the percentage of editing efficiency of the three most active combined mutants of LPG10238 on a set of 43 primers (Figures 12A and 12B) and the three most active combined mutants of LPG10241 on a set of 37 primers (Figures 12C and 12D). Figures 13A and 13B show the strategy for generating the shortened main-chain variant of the LPG10241 primer (Figure 13A) and the percentage of editing of the shortened LPG10241 primer (NHEJ = non-homologous terminal link) compared to the percentage of editing using LPG10241 primers with three different septum parental lines, as described in Example 8 and Table 15. *Refers to shortened crRNA bb. The arrows in Figure 13A indicate the direction of crRNA bb shortening, i.e., the nucleotides are deleted from the 5' end of the crRNA bb. The "WT" control in Figure 13B has an LPG10241 primer in the form of crRNA bb-septum-cRNA bb (where the cRNA bb is not shortened). Figure 14 shows the percentage of editing of the shortened LPG10238 primer compared to the percentage of editing of the parental LPG10238 primer using two different septums. The shortening design of the LPG10238 primer is described in Example 8 and Table 16. The crRNA backbone of LPG10238 should not be shortened to maintain or enhance the editing efficiency of the parental 36 nt backbone. A schematic diagram of the parental LPG10238 backbone is on the left side of the figure. * crRNA bb = shortened crRNA backbone; S = spacer. Figure 15 shows the average percentage of editing in the backbone of LPG10238 backbone variants with mutations compared to parental LPG10238 backbones using two different spacers. Backbone variant 2 shows a more promising editing improvement than the parental one. Figures 16A through 16D show the editing activities of LPG10241 (Figures 16A and 16B) and LPG10238 (Figures 16C and 16D) glycine mutants normalized to parental LPG10241 and LPG10238 editing activities, respectively. Preliminary screening (Figures 16A and 16C) evaluates the editing of the two backbones. The validation screening test included two additional primers (Figs. 16B and 16D). Figs. 17A and 17B show the alignment of the LDHA marker (Fig. 17A) and B2M marker (Fig. 17B) with the highest-frequency excision amplicon sequences of their respective parental sequences. The primer marker sites are indicated by arrows. Excision was performed using the LPG10238 system described in Example 11. Figs. 18A and 18B show the genome excision activity of mutants LPG10241 and LPG13264. (Fig. 18A) Agarose gel of the repair product amplicon. A and B are from single primer edits that were not expected to be excised, while samples C and D are from excision edits. (-) Lanes without primers. (Figure 18B) Frequency distribution of deletion size in the excision product. Excision was performed using the LPG10241 system described in Example 11. Figures 19A and 19B. (Figure 19A) In vitro cleavage progression of LPG10238 using synthetic tandem guide arrays targeting two double-stranded DNA (dsDNA) receptors (L3, L2). (Figure 19B) In vitro cleavage progression of LPG10238 using a mixture of two synthetic single guides targeting dsDNA receptors (L3, L2), as described in Example 12. NTS = non-targeted strand; TS = target strand. Figures 20A and 20B. (Figure 20A) In vitro cleavage progression of LPG10241 using synthetic tandem primer arrays targeting two dsDNA receptors (L3, L2). (Figure 20B) In vitro cleavage progression of LPG10241 using a mixture of two synthetic single primers targeting dsDNA receptors (L3, L2), as described in Example 12. NTS = non-targeted stock; TS = target stock. Figures 21A and 21B. (Figure 21A) Primer treatment curves for active LPG10238 (circular, square) on two synthetic primer arrays and catalytically inactive LPG10238 (triangular, inverted triangular) on the same arrays. (Fig. 21B) Guideway treatment curves for active LPG10241 (circular, square) on two synthetic guideway arrays and for non-catalytically inactive LPG10241 (triangular, inverted triangular) on the same array. Figs. 22A and 22B show all-in-one AAV plasmids delivered to HEK293T cells. Editing of the LPG10238 system at B2M (Fig. 22A) and LDHA (Fig. 22B) is shown. Figs. 23A and 23B show a summary of the cleavage site features of LPG10241 and LPG10238 as described in Example 14. (Fig. 23A) Cleavage site features of label 1 (L1), and (Fig. 23B) Cleavage site features of label 2 (L2).

TW202536173A_113139690_SEQL.xml

Claims (213)

A nucleic acid molecule comprising a polynucleotide encoding a writing-RNA guiding nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding the writing-RGN polypeptide, the RGN polypeptide comprising, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1 The amino acid sequence represented by any one of 083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has a monoamino acid sequence with at least 90% sequence identity. The nucleic acid molecule as described in claim 1, wherein the RGN polypeptide comprises, as in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein the amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 1. The nucleic acid molecule as claimed in claim 3, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are positively charged amino acid residues. The nucleic acid molecule as described in claim 3 or claim 4, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are arginine (R). The nucleic acid molecule as claimed in claim 5, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 450; (b) an amino acid sequence as shown in SEQ ID NO: 463; (c) an amino acid sequence as shown in SEQ ID NO: 471; (d) an amino acid sequence as shown in SEQ ID NO: 481; (e) an amino acid sequence as shown in SEQ ID NO: 484; (f) an amino acid sequence as shown in SEQ ID NO: 485; (g) an amino acid sequence as shown in SEQ ID NO: 486; (h) an amino acid sequence as shown in SEQ ID NO: 500; (i) an amino acid sequence as shown in SEQ ID NO: 502; (j) an amino acid sequence as shown in SEQ ID NO: 505; (k) an amino acid sequence as shown in SEQ ID NO: 508; (l) an amino acid sequence as shown in SEQ ID NO: 509; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v ...0; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v) an amino acid sequence as shown in The amino acid sequence shown in NO: 539; (m) the amino acid sequence shown in SEQ ID NO: 602; (n) the amino acid sequence shown in SEQ ID NO: 707; (o) the amino acid sequence shown in SEQ ID NO: 708; and (p) the amino acid sequence shown in SEQ ID NO: 717. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein, at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; (c) amino acid positions 530, 552, 677, and 801; (d) amino acid positions 530, 577, and 790; (e) amino acid positions 517, 530, 538, 552, and 677; (f) amino acid positions 530, 538, 677, and 801; (g) amino acid positions 538, 552, 677, and 801. 1; (h) amino acid positions 517, 530, 538 and 552; (i) amino acid positions 517 and 530; (j) amino acid positions 530, 538 and 552; (k) amino acid positions 517, 530, 538 and 677; (l) amino acid positions 517, 552, 677 and 801; (m) amino acid positions 530, 677 and 801; (n) amino acid positions 517, 530, 677 and 801; (o) amino acid positions 517, 530 and 677; (p) amino acid positions 530, 538, 552 and 677; (q) amino acid positions 517, 552 and 677; and (r) amino acid positions 517, 530, 552, 677 and 801. The nucleic acid molecule as claimed in claim 7, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 1072; (b) an amino acid sequence as shown in SEQ ID NO: 1080; (c) an amino acid sequence as shown in SEQ ID NO: 1082; (d) an amino acid sequence as shown in SEQ ID NO: 1105; (e) an amino acid sequence as shown in SEQ ID NO: 1084; (f) an amino acid sequence as shown in SEQ ID NO: 1081; (g) an amino acid sequence as shown in SEQ ID NO: 1083; (h) an amino acid sequence as shown in SEQ ID NO: 1069; (i) an amino acid sequence as shown in SEQ ID NO: 1034; (j) an amino acid sequence as shown in SEQ ID NO: 1059; (k) an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 1070; (l) the amino acid sequence shown in SEQ ID NO: 1078; (m) the amino acid sequence shown in SEQ ID NO: 1064; (n) the amino acid sequence shown in SEQ ID NO: 1074; (o) the amino acid sequence shown in SEQ ID NO: 1051; (p) the amino acid sequence shown in SEQ ID NO: 1079; (q) the amino acid sequence shown in SEQ ID NO: 1056; and (r) the amino acid sequence shown in SEQ ID NO: 1087. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein, at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 517, 530, 552 and 677; (b) amino acid positions 530, 538, 552 and 801; and (c) amino acid positions 530, 552, 677 and 801. The nucleic acid molecule as claimed in claim 9, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1072; (b) an amino acid sequence as shown in SEQ ID NO: 1080; and (c) an amino acid sequence as shown in SEQ ID NO: 1082. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4. The nucleic acid molecule as claimed in claim 11, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are positively charged amino acid residues. The nucleic acid molecule as described in claim 11 or claim 12, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of the amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are an R. The nucleic acid molecule as claimed in claim 13, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 809; (b) an amino acid sequence as shown in SEQ ID NO: 810; (c) an amino acid sequence as shown in SEQ ID NO: 866; (d) an amino acid sequence as shown in SEQ ID NO: 898; (e) an amino acid sequence as shown in SEQ ID NO: 909; (f) an amino acid sequence as shown in SEQ ID NO: 912; (g) an amino acid sequence as shown in SEQ ID NO: 916; (h) an amino acid sequence as shown in SEQ ID NO: 960; (i) an amino acid sequence as shown in SEQ ID NO: 961; (j) an amino acid sequence as shown in SEQ ID NO: 963; (k) an amino acid sequence as shown in SEQ ID NO: 966; (l) an amino acid sequence as shown in SEQ ID NO: 963; (d) an amino acid sequence as shown in SEQ ID NO: 898; (e) an amino acid sequence as shown in SEQ ID NO: 909; (f) an amino acid sequence as shown in SEQ ID NO: 912; (g) an amino acid sequence as shown in SEQ ID NO: 916; (h) an amino acid sequence as shown in SEQ ID NO: 960; (i) an amino acid sequence as shown in SEQ ID NO: 961; (j) an amino acid sequence as shown in SEQ ID NO: 963; (k) an amino acid sequence as shown in SEQ ID NO: 966; (l) an amino acid sequence as shown in SEQ ID NO: 969; (d) an amino acid sequence as shown in SEQ ID NO: 969; (f) an amino acid sequence as shown in SEQ ID NO: 969; (g) an amino acid sequence as shown in The amino acid sequence shown in NO: 970; (m) the amino acid sequence shown in SEQ ID NO: 975; (n) the amino acid sequence shown in SEQ ID NO: 1012; and (o) the amino acid sequence shown in SEQ ID NO: 1019. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein, at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid positions 377, 625, and 691; (g) amino acid Positions 377, 625, 698 and 747; (h) amino acid positions 377, 638, 691 and 698; (i) amino acid positions 377, 625 and 638; (j) amino acid positions 377, 638, 698 and 747; (k) amino acid positions 377, 625, 638, 691 and 698; (l) amino acid positions 625, 638 and 698; (m) amino acid positions 377, 625, 691 and 698; (n) amino acid positions 377, 638 and 698; (o) amino acid positions 625, 691, 698 and 747; (p) amino acid positions 638 and 698; and (q) amino acid positions 377 and 691. The nucleic acid molecule as claimed in claim 15, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 1123; (b) an amino acid sequence as shown in SEQ ID NO: 1158; (c) an amino acid sequence as shown in SEQ ID NO: 1159; (d) an amino acid sequence as shown in SEQ ID NO: 1148; (e) an amino acid sequence as shown in SEQ ID NO: 1160; (f) an amino acid sequence as shown in SEQ ID NO: 1122; (g) an amino acid sequence as shown in SEQ ID NO: 1146; (h) an amino acid sequence as shown in SEQ ID NO: 1147; (i) an amino acid sequence as shown in SEQ ID NO: 1121; (j) an amino acid sequence as shown in SEQ ID NO: 1149; (k) an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 1156; (l) the amino acid sequence shown in SEQ ID NO: 1132; (m) the amino acid sequence shown in SEQ ID NO: 1144; (n) the amino acid sequence shown in SEQ ID NO: 1126; (o) the amino acid sequence shown in SEQ ID NO: 1154; (p) the amino acid sequence shown in SEQ ID NO: 1116; and (q) the amino acid sequence shown in SEQ ID NO: 1108. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4. The nucleic acid molecule as claimed in claim 17, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764 and 766 of the RGN polypeptide are positively charged amino acid residues. The nucleic acid molecule as described in claim 17 or claim 18, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide is an R or an ionine (K). The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein, at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 736, 743, 752, 753, 755, 757, 758 and 760; (b) amino acid positions 740, 747, 751, 753, 755, 760, 764 and 766; (c) amino acid positions 685, 691 and 698; and (d) amino acid positions 685, 692, 695, 702 and 707. The nucleic acid molecule as claimed in claim 20, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1168; (b) an amino acid sequence as shown in SEQ ID NO: 1171; (c) an amino acid sequence as shown in SEQ ID NO: 1169; and (d) an amino acid sequence as shown in SEQ ID NO: 1170. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the RGN polypeptide comprises a deletion of an amino acid residue corresponding to amino acid residues 751 to 769 of SEQ ID NO: 4. The nucleic acid molecule as described in claim 22, wherein the RGN polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1173. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more of the amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596 and 862 of the RGN polypeptide comprises substitution or insertion of one or more glycine (G) at the N-terminal position adjacent to the amino acid position, at the amino acid position, or at the C-terminal position adjacent to the amino acid position. The nucleic acid molecule as described in claim 24, wherein the substitution or insertion of one or more Gs comprises the substitution or insertion of one, two or three Gs. The nucleic acid molecule as described in claim 24 or claim 25, wherein the substitution or insertion of one or more Gs is selected from: (a) the insertion of three Gs between amino acid positions 293 and 294; (b) the insertion of two Gs between amino acid positions 359 and 360; (c) the substitution of G at amino acid position 361; (d) the insertion of one G between amino acid positions 402 and 403; (e) the substitution of G at amino acid position 401; (f) the substitution of G at amino acid position 596; and (g) the substitution of G at amino acid position 862. The nucleic acid molecule as claimed in claim 26, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 1304; (b) an amino acid sequence as shown in SEQ ID NO: 1322; (c) an amino acid sequence as shown in SEQ ID NO: 1324; (d) an amino acid sequence as shown in SEQ ID NO: 1328; (e) an amino acid sequence as shown in SEQ ID NO: 1330; (f) an amino acid sequence as shown in SEQ ID NO: 1361; and (g) an amino acid sequence as shown in SEQ ID NO: 1400. The nucleic acid molecule as claimed in claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more of the amino acid positions 566, 567, and 584 of the RGN polypeptide comprises one or more substitutions or insertions of G in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position. The nucleic acid molecule as described in claim 28, wherein the substitution or insertion of one or more Gs comprises the substitution or insertion of one, two or three Gs. The nucleic acid molecule as described in claim 28 or claim 29, wherein the substitution or insertion of one or more Gs is selected from: (a) substitution of G at amino acid position 566 and substitution of G at amino acid position 567; and (b) substitution of G at amino acid position 584. The nucleic acid molecule as claimed in claim 30, wherein the RGN polypeptide comprises an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1223; and (b) an amino acid sequence as shown in SEQ ID NO: 1228. The nucleic acid molecule as described in any of claims 1 to 31, wherein the polynucleotide encoding an RGN polypeptide is operatively linked to a promoter heterologous to the polynucleotide. The nucleic acid molecule described in any of claims 1 to 31, wherein the polynucleotide encoding an RGN polypeptide is an mRNA comprising a heterologous 5' untranslated region (UTR) and/or a heterologous 3' UTR. The nucleic acid molecule as described in any one of claims 1 to 31, wherein, when bound to a guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA-guided sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence. The nucleic acid molecule as described in claim 34, wherein the target sequence is disposed adjacent to and at its 3' of a protoseptum adjacent motif (PAM). The nucleic acid molecule as claimed in claim 35, wherein a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 recognizes a PAM having a common nucleotide sequence shown in AYG, wherein the PAM is adjacent to the target sequence and is located at its 5'; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 1933, or 1944 recognizes a PAM having a common nucleotide sequence shown in ATTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949 recognizes a PAM having a common nucleotide sequence shown in VTTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4. RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is located at its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: The RGN polypeptide of the amino acid sequence shown in SEQ ID NO: 1937 or 1950 identifies a PAM having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) the RGN polypeptide of the amino acid sequence shown in SEQ ID NO: 1951 identifies a PAM having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) the RGN polypeptide of the amino acid sequence shown in SEQ ID NO: 1953 identifies a PAM having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'. The nucleic acid molecule described in any of claims 34 to 36, wherein the gRNA does not include a trans-activated CRISPR RNA (tracrRNA). The nucleic acid molecule as described in any one of claims 34 to 37, wherein the gRNA has a total length of 35 to 250 nucleotides (nt), or 35 to 170 nt, or 35 to 125 nt, or 40 to 100 nt, or 40 to 70 nt, or 40 to 60 nt. The nucleic acid molecule as described in any one of claims 34 to 38, wherein the gRNA has a crRNA backbone having a total length of up to 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt or 40 nt. The nucleic acid molecule as described in any one of claims 1 to 39, wherein the nucleotide sequence encoding the RGN polypeptide includes any one of SEQ ID NO: 29 to 32 and 2659 to 2685. The nucleic acid molecule as described in any one of claims 34 to 40, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding. The nucleic acid molecule as described in claim 41, wherein the RGN polypeptide is capable of producing a double-strand break. The nucleic acid molecule as described in claim 1, wherein the RGN polypeptide is nuclease-inactivating. The nucleic acid molecule as claimed in claim 43, wherein the RGN polypeptide comprises a monoamino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 408, 597, 678, 827, 896, 979, 2686, and 2687. The nucleic acid molecule as described in any of claims 1 to 44, wherein the RGN polypeptide is operatively fused with a base-editing polypeptide. The nucleic acid molecule as described in claim 45, wherein the base-editing polypeptide includes a deaminerase. The nucleic acid molecule as described in any of claims 1 to 44, wherein the RGN polypeptide is operatively fused with a polymerase editing polypeptide. The nucleic acid molecule as described in claim 47, wherein the polymerase editing polypeptide comprises a DNA polymerase. The nucleic acid molecule as described in claim 47, wherein the polymerase-editing polypeptide includes a reverse transcriptase. The nucleic acid molecule as described in any of claims 1 to 44, wherein the RGN polypeptide is operatively fused with an effector domain. The nucleic acid molecule as described in claim 50, wherein the effector domain is operatively fused to the N-terminus or C-terminus of the RGN polypeptide. The nucleic acid molecule as described in claim 50, wherein the effector domain is operatively fused to an internal address of the RGN polypeptide. The nucleic acid molecule as described in claim 50, wherein the effector domain includes a cleavage domain, a deaminase domain, or a performance regulation domain. The nucleic acid molecule as described in claim 50, wherein the phenotype regulatory domain includes a transcriptional activation domain, a transcriptional repression domain, or an epigenetic modification domain. The nucleic acid molecule as described in claim 50, wherein the RGN polypeptide includes one or more nuclear localization signals (NLS). The nucleic acid molecule as claimed in claim 50, wherein the one or more NLS comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 33, 35, 407, and 1927. The nucleic acid molecule as described in any of claims 1 to 56, wherein the nucleotide sequence encoding the RGN polypeptide is codon-optimized for expression in a eukaryotic cell. A vector comprising the nucleic acid molecule described in any one of claims 1 to 57. The vector as described in claim 58, wherein the vector further comprises at least one nucleotide sequence encoding a guide RNA (gRNA). The vector as described in claim 59, wherein the at least one nucleotide sequence encoding a gRNA includes a nucleotide sequence encoding one or more gRNAs. The vector as described in claim 60, wherein the nucleotide sequence encoding one or more gRNAs is operatively linked to a single promoter. The vector as described in claim 59, wherein the at least one nucleotide sequence encoding a gRNA comprises two or more nucleotide sequences, each nucleotide sequence encoding a gRNA. The vector as described in claim 62, wherein each nucleotide sequence encoding a gRNA is operatively linked to a promoter. The vector as described in claim 61 or claim 63, wherein the promoter comprises an RNA polymerase III promoter. The vector as described in claim 64, wherein the RNA polymerase III promoter comprises a human U6 promoter or a human 7SK promoter. The vector as described in claim 65, wherein the human U6 promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1672. The vector as described in claim 65, wherein the human 7SK promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1673. The vector as described in claim 58 or claim 67, wherein the vector further includes a promoter operatively linked to a nucleic acid molecule comprising a writing-RGN polypeptide. The vector as described in claim 68, wherein the promoter comprises an RNA polymerase II promoter. The vector as described in claim 69, wherein the RNA polymerase II promoter comprises a CMV promoter or a CBh promoter. The carrier as described in claim 70, wherein the CMV initiator further includes a CMV upstream enhancer. The vector as described in claim 71, wherein the CMV initiator and the CMV upstream enhancer comprise a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1674. The vector as described in claim 70, wherein the CBh promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1675. The vector as described in any one of claims 58 to 73, wherein the vector further comprises a nucleotide sequence that encodes a peptide linker. The carrier as described in claim 74, wherein the peptide linker comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with an amino acid sequence as shown in SEQ ID NO: 34 or 1928. The carrier as described in any one of claims 58 to 75, wherein the carrier further comprises a polyadenylated (polyA) tail. The carrier as described in claim 76, wherein the polyA tail includes an SV40 polyA tail. The vector as described in claim 77, wherein the SV40 polyA tail comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1678. The vector as described in any of claims 59 to 78, wherein the gRNA comprises at least one CRISPR RNA (crRNA) backbone (bb) and at least one spacer. The vector as described in claim 79, wherein the gRNA comprises two crRNA bbs and a spacer, in the form of crRNA bb-spacer-crRNA bb from 5' to 3'. The vector as described in claim 79, wherein the gRNA comprises three crRNA bb, a first spacer and a second spacer, in the form of crRNA bb-first spacer-crRNA bb-second spacer-crRNA bb from 5' to 3'. The vector as described in claim 79, wherein the gRNA comprises a crRNA bb and a spacer, in the form of crRNA bb-spacer from 5' to 3'. The vector as described in any one of claims 59 to 82, wherein the gRNA is selected from the group consisting of: a) a gRNA comprising a CRISPR RNA having a CRISPR RNA (crRNA) backbone comprising a nucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; b) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; c) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1. The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence shown in SEQ ID NO: 7 or differs from SEQ ID NO: 7 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence shown in SEQ ID NO: 3; and d) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is at least 90% sequence identical to the amino acid sequence shown in SEQ ID NO: 8 or differs from SEQ ID NO: 8 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence shown in SEQ ID NO: 4; (e) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is at least 90% sequence identical to the amino acid sequence shown in SEQ ID NO: 7 or differs from SEQ ID NO: 1617 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence shown in SEQ ID NO: 7; (f) A gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1618 or differing from SEQ ID NO: 1618 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (g) A gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1619 or differing from SEQ ID NO: 1619 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (h) A gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1618 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; ID NO: 1620 has a nucleotide sequence that is 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 4; (i) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1631 or having a nucleotide sequence that is 1 to 5 nucleotides different from SEQ ID NO: 1631, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1; (j) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1957 or having a nucleotide sequence that is 1 to 5 nucleotides different from SEQ ID NO: 1957, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 4; The amino acid sequence of SEQ ID NO: 1930 has at least 90% sequence identity with a single amino acid sequence; (k) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1958 or differing from SEQ ID NO: 1958 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1931; (l) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1959 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1932; (m) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1930; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1933, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1960; (n) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1961, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1961, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1934; (o) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1962, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1962, wherein the RGN polypeptide comprises an amino acid sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1960; The amino acid sequence of SEQ ID NO: 1935 has at least 90% sequence identity with a single amino acid sequence; (p) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1963 or differing from SEQ ID NO: 1963 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1936; (q) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1964 or differing from SEQ ID NO: 1964 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937; (r) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1935; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1938, or differs from SEQ ID NO: 1965 by 1 to 5 nucleotides; (s) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1966, or differs from SEQ ID NO: 1966 by 1 to 5 nucleotides; (t) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1939, or differs from SEQ ID NO: 1967 by 1 to 5 nucleotides; wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1939, or differs from SEQ ID NO: 1965 by 1 to 5 nucleotides; The amino acid sequence of SEQ ID NO: 1940 has at least 90% sequence identity with a single amino acid sequence; (u) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1968 or differing from SEQ ID NO: 1968 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941; (v) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1969 or differing from SEQ ID NO: 1969 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1942; (w) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1940; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1970 or different from SEQ ID NO: 1970 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1943; (x) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1971 or different from SEQ ID NO: 1971 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1944; (y) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1970 or different from SEQ ID NO: 1972 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1970; The amino acid sequence of SEQ ID NO: 1945 has at least 90% sequence identity with a single amino acid sequence; (z) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1973 or differing from SEQ ID NO: 1973 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1946; (aa) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1974 or differing from SEQ ID NO: 1974 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947; (bb) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1945 or differing from SEQ ID NO: 1974 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1947; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1975 or differs from SEQ ID NO: 1975 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1948; (cc) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1976 or differs from SEQ ID NO: 1976 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1949; (dd) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1977 or differs from SEQ ID NO: 1977 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1975; The amino acid sequence of SEQ ID NO: 1950 has at least 90% sequence identity with a single amino acid sequence; (ee) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1978 or differing from SEQ ID NO: 1978 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951; (ff) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1979 or differing from SEQ ID NO: 1979 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952; (gg) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1952; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1953, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1980; (ii) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1981, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1981, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1954; (iii) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1982, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1982, wherein the RGN polypeptide comprises an amino acid sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1980; The amino acid sequence of 1955 has at least 90% sequence identity with an amino acid sequence; and (jj)-gRNA, including a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1983 or differing from SEQ ID NO: 1983 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1956. The vector as described in claim 83, wherein the gRNA is selected from the group consisting of: a) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 5, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1; b) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 6, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2; c) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 7, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3; and d) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 5. The nucleotide sequence shown in SEQ ID NO: 8, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; (e) a gRNA, comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1617, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; (f) a gRNA, comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1618, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; (g) a gRNA, comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1619, wherein the RGN polypeptide includes the amino acid sequence shown in SEQ ID NO: 4; (h) a gRNA, comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 8. The nucleotide sequence shown in SEQ ID NO: 1620, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4; (i) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1631, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1; (j) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1957, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1930; (k) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1958, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1931; (l) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1931; The RNA comprises a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1959, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1932; (m) a gRNA comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1960, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1933; (n) a gRNA comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1961, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1934; (o) a gRNA comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1962, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1934. The following are the amino acid sequences shown in SEQ ID NO: 1935; (p) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1963, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1936; (q) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1964, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1937; (r) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1965, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1938; (s) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the amino acid sequence shown in SEQ ID NO: 1938. The nucleotide sequence shown in SEQ ID NO: 1966, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1939; (t) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1967, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1940; (u) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1968, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1941; (v) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1969, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1942; (w) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1969, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1942; The RNA comprises a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1970, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1943; (x) a gRNA comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1971, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1944; (y) a gRNA comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1972, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1945; (z) a gRNA comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1973, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1945. The following are listed: (aa) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1974, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1947; (bb) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1975, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1948; (cc) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1976, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1949; (dd) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising the amino acid sequence shown in SEQ ID NO: 1949. The nucleotide sequence shown in SEQ ID NO: 1977, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1950; (ee) gRNA, comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1978, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1951; (ff) gRNA, comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1979, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1952; (gg) gRNA, comprising a CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1980, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1953; (hh) gRNA, comprising a CRISPR RNA having a crRNA backbone comprising the CRISPR RNA having a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1977, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1953; The RNA comprises a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1981, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1954; (ii) a gRNA comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1982, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1955; and (jj) a gRNA comprising a CRISPR RNA having a crRNA backbone including the nucleotide sequence shown in SEQ ID NO: 1983, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1956. The vector as described in any one of claims 59 to 84, wherein the gRNA comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequences of any one of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658. The vector as described in any of claims 58 to 85, wherein the vector is an adeno-associated virus (AAV) vector. The vector as described in claim 86, wherein the vector comprises a nucleotide sequence as shown in any one of SEQ ID NO: 1679 to 1683. A cell comprising a nucleic acid molecule as described in any of claims 1 to 57 or a vector as described in any of claims 58 to 87. A method for producing an RGN polypeptide, comprising culturing cells as described in claim 88 under conditions in which the RGN polypeptide is expressed. A method for manufacturing an RGN polypeptide includes: introducing a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide into a cell, the RNA-guided nuclease (RGN) polypeptide comprising, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 10 The amino acid sequence represented by any one of 87, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has a monoamino acid sequence with at least 90% sequence identity; and the cells are cultured under the conditions under which the RGN polypeptide is expressed. The method as described in claim 90 further includes purifying the RGN polypeptide. The method as described in claim 90 or claim 91, wherein the cell further expresses one or more guide RNAs capable of binding to the RGN polypeptide to form an RGN ribonucleoprotein complex. The method as described in claim 92 further includes purifying the RGN ribonucleoprotein complex. An RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises the same as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1 The amino acid sequence represented by any one of 083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has a monoamino acid sequence with at least 90% sequence identity. The RGN polypeptide as described in claim 94, wherein the RGN polypeptide comprises, as in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 to 2687. The RGN polypeptide as described in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein the amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 1. The RGN polypeptide as described in claim 96, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are positively charged amino acid residues. The RGN polypeptide as described in claim 96 or claim 97, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more amino acid residues at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790 and 801 of the RGN polypeptide are arginine (R). The RGN polypeptide as described in claim 98, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 450; (b) an amino acid sequence as shown in SEQ ID NO: 463; (c) an amino acid sequence as shown in SEQ ID NO: 471; (d) an amino acid sequence as shown in SEQ ID NO: 481; (e) an amino acid sequence as shown in SEQ ID NO: 484; (f) an amino acid sequence as shown in SEQ ID NO: 485; (g) an amino acid sequence as shown in SEQ ID NO: 486; (h) an amino acid sequence as shown in SEQ ID NO: 500; (i) an amino acid sequence as shown in SEQ ID NO: 502; (j) an amino acid sequence as shown in SEQ ID NO: 505; (k) an amino acid sequence as shown in SEQ ID NO: 508; (l) an amino acid sequence as shown in SEQ ID NO: 509; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v ...0; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v) an amino acid sequence as shown in SEQ ID NO: 500; (v) an amino acid sequence as shown in The amino acid sequence shown in SEQ ID NO: 539; (m) the amino acid sequence shown in SEQ ID NO: 602; (n) the amino acid sequence shown in SEQ ID NO: 707; (o) the amino acid sequence shown in SEQ ID NO: 708; and (p) the amino acid sequence shown in SEQ ID NO: 717. The RGN polypeptide as described in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein, at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; (c) amino acid positions 530, 552, 677, and 801; (d) amino acid positions 530, 577, and 790; (e) amino acid positions 517, 530, 538, 552, and 677; (f) amino acid positions 530, 538, and 677. 677 and 801; (g) amino acid positions 538, 552, 677 and 801; (h) amino acid positions 517, 530, 538 and 552; (i) amino acid positions 517 and 530; (j) amino acid positions 530, 538 and 552; (k) amino acid positions 517, 530, 538 and 677; (l) amino acid positions 517, 552, 677 and 801; (m) amino acid positions 530, 677 and 801; (n) amino acid positions 517, 530, 677 and 801; (o) amino acid positions 517, 530 and 677; (p) amino acid positions 530, 538, 552 and 677; (q) amino acid position 517, 552 and 677; and (r) amino acid positions 517, 530, 552, 677 and 801. The RGN polypeptide as described in claim 100, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 1072; (b) an amino acid sequence as shown in SEQ ID NO: 1080; (c) an amino acid sequence as shown in SEQ ID NO: 1082; (d) an amino acid sequence as shown in SEQ ID NO: 1105; (e) an amino acid sequence as shown in SEQ ID NO: 1084; (f) an amino acid sequence as shown in SEQ ID NO: 1081; (g) an amino acid sequence as shown in SEQ ID NO: 1083; (h) an amino acid sequence as shown in SEQ ID NO: 1069; (i) an amino acid sequence as shown in SEQ ID NO: 1034; (j) an amino acid sequence as shown in SEQ ID NO: 1059; (k) an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 1070; (l) the amino acid sequence shown in SEQ ID NO: 1078; (m) the amino acid sequence shown in SEQ ID NO: 1064; (n) the amino acid sequence shown in SEQ ID NO: 1074; (o) the amino acid sequence shown in SEQ ID NO: 1051; (p) the amino acid sequence shown in SEQ ID NO: 1079; (q) the amino acid sequence shown in SEQ ID NO: 1056; and (r) the amino acid sequence shown in SEQ ID NO: 1087. The RGN polypeptide as described in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4. The RGN polypeptide as claimed in claim 102, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are positively charged amino acid residues. The RGN polypeptide as described in claim 102 or claim 103, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747 and 755 of the RGN polypeptide are an R. The RGN polypeptide as described in claim 104, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 809; (b) an amino acid sequence as shown in SEQ ID NO: 810; (c) an amino acid sequence as shown in SEQ ID NO: 866; (d) an amino acid sequence as shown in SEQ ID NO: 898; (e) an amino acid sequence as shown in SEQ ID NO: 909; (f) an amino acid sequence as shown in SEQ ID NO: 912; (g) an amino acid sequence as shown in SEQ ID NO: 916; (h) an amino acid sequence as shown in SEQ ID NO: 960; (i) an amino acid sequence as shown in SEQ ID NO: 961; (j) an amino acid sequence as shown in SEQ ID NO: 963; (k) an amino acid sequence as shown in SEQ ID NO: 964; (v) an amino acid sequence as shown in SEQ ID NO: 965; (v) an amino acid sequence as shown in SEQ ID NO: 96 ... The amino acid sequence shown in SEQ ID NO: 966; (l) the amino acid sequence shown in SEQ ID NO: 970; (m) the amino acid sequence shown in SEQ ID NO: 975; (n) the amino acid sequence shown in SEQ ID NO: 1012; and (o) the amino acid sequence shown in SEQ ID NO: 1019. The RGN polypeptide as claimed in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein, at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid positions 377, 625, and 691; (g) amino acid Positions 377, 625, 698 and 747; (h) amino acid positions 377, 638, 691 and 698; (i) amino acid positions 377, 625 and 638; (j) amino acid positions 377, 638, 698 and 747; (k) amino acid positions 377, 625, 638, 691 and 698; (l) amino acid positions 625, 638 and 698; (m) amino acid positions 377, 625, 691 and 698; (n) amino acid positions 377, 638 and 698; (o) amino acid positions 625, 691, 698 and 747; (p) amino acid positions 638 and 698; and (q) amino acid positions 377 and 691. The RGN polypeptide as described in claim 106, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 1123; (b) an amino acid sequence as shown in SEQ ID NO: 1158; (c) an amino acid sequence as shown in SEQ ID NO: 1159; (d) an amino acid sequence as shown in SEQ ID NO: 1148; (e) an amino acid sequence as shown in SEQ ID NO: 1160; (f) an amino acid sequence as shown in SEQ ID NO: 1122; (g) an amino acid sequence as shown in SEQ ID NO: 1146; (h) an amino acid sequence as shown in SEQ ID NO: 1147; (i) an amino acid sequence as shown in SEQ ID NO: 1121; (j) an amino acid sequence as shown in SEQ ID NO: 1149; (k) an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 1156; (l) the amino acid sequence shown in SEQ ID NO: 1132; (m) the amino acid sequence shown in SEQ ID NO: 1144; (n) the amino acid sequence shown in SEQ ID NO: 1126; (o) the amino acid sequence shown in SEQ ID NO: 1154; (p) the amino acid sequence shown in SEQ ID NO: 1116; and (q) the amino acid sequence shown in SEQ ID NO: 1108. The RGN polypeptide as claimed in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are different from the corresponding amino acid residues of SEQ ID NO: 4. The RGN polypeptide as described in claim 108, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein one or more amino acid residues at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide are positively charged amino acid residues. The RGN polypeptide as described in claim 108 or claim 109, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein the amino acid residue at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of the RGN polypeptide is an R or an ionized amino acid (K). The RGN polypeptide as claimed in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and wherein, at an amino acid position combination selected from the following amino acid position combinations of the RGN polypeptide, the amino acid residue is arginine (R): (a) amino acid positions 736, 743, 752, 753, 755, 757, 758 and 760; (b) amino acid positions 740, 747, 751, 753, 755, 760, 764 and 766; (c) amino acid positions 685, 691 and 698; and (d) amino acid positions 685, 692, 695, 702 and 707. The RGN polypeptide as claimed in claim 111, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 1168; (b) an amino acid sequence as shown in SEQ ID NO: 1171; (c) an amino acid sequence as shown in SEQ ID NO: 1169; and (d) an amino acid sequence as shown in SEQ ID NO: 1170. The RGN polypeptide as claimed in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein the RGN polypeptide comprises a deletion of an amino acid residue corresponding to amino acid residues 751 to 769 of SEQ ID NO: 4. The RGN polypeptide as described in claim 113, wherein the RGN polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1173. The RGN polypeptide as claimed in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and wherein one or more of the amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596 and 862 of the RGN polypeptide comprises substitution or insertion of one or more glycine (G) in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position. The RGN polypeptide as described in claim 115, wherein the substitution or insertion of one or more Gs comprises the substitution or insertion of one, two or three Gs. The RGN polypeptide as claimed in claim 115, wherein the substitution or insertion of one or more Gs is selected from: (a) the insertion of three Gs between amino acid positions 293 and 294; (b) the insertion of two Gs between amino acid positions 359 and 360; (c) the substitution of G at amino acid position 361; (d) the insertion of one G between amino acid positions 402 and 403; (e) the substitution of G at amino acid position 401; (f) the substitution of G at amino acid position 596; and (g) the substitution of G at amino acid position 862. The RGN polypeptide as claimed in claim 117, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the following: (a) an amino acid sequence as shown in SEQ ID NO: 1304; (b) an amino acid sequence as shown in SEQ ID NO: 1322; (c) an amino acid sequence as shown in SEQ ID NO: 1324; (d) an amino acid sequence as shown in SEQ ID NO: 1328; (e) an amino acid sequence as shown in SEQ ID NO: 1330; (f) an amino acid sequence as shown in SEQ ID NO: 1361; and (g) an amino acid sequence as shown in SEQ ID NO: 1400. The RGN polypeptide as claimed in claim 94, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 4, and wherein one or more of the amino acid positions 566, 567, and 584 of the RGN polypeptide comprises one or more substitutions or insertions of G in the N-terminal direction adjacent to the amino acid position, at the amino acid position, or in the C-terminal direction adjacent to the amino acid position. The RGN polypeptide as described in claim 119, wherein the substitution or insertion of one or more Gs comprises the substitution or insertion of one, two or three Gs. The RGN polypeptide as described in claim 119 or claim 121, wherein the substitution or insertion of one or more Gs is selected from: (a) substitution of G at amino acid position 566 and substitution of G at amino acid position 567; and (b) substitution of G at amino acid position 584. The RGN polypeptide as claimed in claim 121, wherein the RGN polypeptide comprises an amino acid sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO: 1223; and (b) an amino acid sequence as shown in SEQ ID NO: 1228. The RGN polypeptide as described in any of claims 94 to 122, wherein the RGN polypeptide is operatively fused with a heterologous polypeptide. The RGN polypeptide as described in claim 123, wherein the heterologous polypeptide includes a base-editing polypeptide, a polymerase-editing polypeptide, an effector domain, an expression regulator, a detectable tracer, or a purified tracer. The RGN polypeptide as described in claim 124, wherein the base-editing polypeptide includes a deaminerase. The RGN polypeptide as described in claim 124, wherein the polymerase editing polypeptide includes a DNA polymerase. The RGN polypeptide as described in claim 124, wherein the polymerase editing polypeptide includes a reverse transcriptase. The RGN polypeptide as described in claim 124, wherein the heterologous polypeptide includes an effector domain. The RGN polypeptide as described in claim 128, wherein the effector domain is operatively fused to the N-terminus or C-terminus of the RGN polypeptide. The RGN polypeptide as described in claim 128, wherein the effector subdomain is operatively fused to an internal address of the RGN polypeptide. The RGN polypeptide as described in claim 128, wherein the effector domain includes a cleavage domain, a deaminase domain, or a performance regulation domain. The RGN polypeptide as described in claim 131, wherein the expression regulatory domain includes a transcription activation domain, a transcription repression domain, or an epigenetic modification domain. The RGN polypeptide as described in any of claims 94 to 132, wherein, when bound to a guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA guide sequence specific manner, wherein the target sequence comprises a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence. The RGN polypeptide as described in claim 133, wherein the target sequence is disposed adjacent to and at its 3' of a protoseptum adjacent motif (PAM). The RGN polypeptide as described in claim 134, wherein a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 recognizes a PAM having a common nucleotide sequence shown in AYG, wherein the PAM is adjacent to the target sequence and is located at its 5'; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 1933, or 1944 recognizes a PAM having a common nucleotide sequence shown in ATTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949 recognizes a PAM having a common nucleotide sequence shown in VTTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4. RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is located at its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1937 The RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1950 identifies a PAM having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1951 identifies a PAM having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1953 identifies a PAM having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'. The RGN polypeptide as described in any one of claims 133 to 135, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding. The RGN polypeptide as described in claim 136, wherein cleavage of the RGN polypeptide produces a double-strand break. The RGN polypeptide as described in claim 94, wherein the RGN polypeptide is nuclease-inactivating. The RGN polypeptide as described in claim 138, wherein the RGN polypeptide comprises a monoamino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 408, 597, 678, 827, 896, 979, 2686, and 2687. The RGN polypeptide as described in any of claims 94 to 139, wherein the RGN polypeptide is operatively fused with a base-editing polypeptide. The RGN polypeptide as described in claim 140, wherein the base-editing polypeptide includes a deaminerase. The RGN polypeptide as described in any of claims 94 to 139, wherein the RGN polypeptide is operatively fused with a polymerase-editing polypeptide. The RGN polypeptide as described in claim 142, wherein the polymerase editing polypeptide comprises a DNA polymerase. The RGN polypeptide as described in claim 142, wherein the polymerase editing polypeptide comprises a reverse transcriptase. The RGN polypeptide as described in any of claims 94 to 144, wherein the RGN polypeptide includes one or more nuclear localization signals (NLS). The RGN polypeptide as described in claim 145, wherein the one or more NLS comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 33, 35, 407, and 1927. A ribonucleoprotein (RNP) complex comprising an RGN polypeptide as described in any one of claims 94 to 146 and a guide RNA bound to the RGN polypeptide. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA), wherein the crRNA comprises a crRNA backbone and a spacer, wherein the crRNA backbone is selected from the group consisting of: a) a crRNA backbone comprising a mononucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides; b) a crRNA backbone comprising a mononucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides; c) a crRNA backbone comprising a mononucleotide sequence as shown in SEQ ID NO: 7 or differing from SEQ ID NO: 7 by 1 to 5 nucleotides; d) a crRNA backbone comprising a mononucleotide sequence as shown in SEQ ID NO: 8 or differing from SEQ ID NO: 8 by 1 to 5 nucleotides; (e) a crRNA backbone comprising a mononucleotide sequence as shown in SEQ ID NO: 1617 or differing from SEQ ID NO: 1617 by 1 to 5 nucleotides; 1617 has a nucleotide sequence that differs from SEQ ID NO: 1618 by 1 to 5 nucleotides; (f) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1618 or differing from SEQ ID NO: 1618 by 1 to 5 nucleotides; (g) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1619 or differing from SEQ ID NO: 1619 by 1 to 5 nucleotides; (h) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1620 by 1 to 5 nucleotides; (i) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1631 or differing from SEQ ID NO: 1631 by 1 to 5 nucleotides; (j) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1957 or differing from SEQ ID NO: 1618 by 1 to 5 nucleotides; 1957 has a nucleotide sequence that differs from SEQ ID NO: 1958 by 1 to 5 nucleotides; (k) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1958 or differing from SEQ ID NO: 1958 by 1 to 5 nucleotides; (l) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1959 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides; (m) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1960 or differing from SEQ ID NO: 1960 by 1 to 5 nucleotides; (n) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1961 or differing from SEQ ID NO: 1961 by 1 to 5 nucleotides; (o) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1962 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides; 1962 has a nucleotide sequence that differs from SEQ ID NO: 1963 by 1 to 5 nucleotides; (p) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1963 or differing from SEQ ID NO: 1963 by 1 to 5 nucleotides; (q) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1964 or differing from SEQ ID NO: 1964 by 1 to 5 nucleotides; (r) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1965 or differing from SEQ ID NO: 1965 by 1 to 5 nucleotides; (s) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1966 or differing from SEQ ID NO: 1966 by 1 to 5 nucleotides; (t) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1967 or differing from SEQ ID NO: 1968 by 1 to 5 nucleotides; 1967 has a nucleotide sequence that differs from SEQ ID NO: 1968 by 1 to 5 nucleotides; (u) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1968 or differing from SEQ ID NO: 1968 by 1 to 5 nucleotides; (v) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1969 or differing from SEQ ID NO: 1969 by 1 to 5 nucleotides; (w) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1970 or differing from SEQ ID NO: 1970 by 1 to 5 nucleotides; (x) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1971 or differing from SEQ ID NO: 1971 by 1 to 5 nucleotides; (y) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1972 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides; 1972 has a nucleotide sequence that differs from SEQ ID NO: 1973 by 1 to 5 nucleotides; (z) a crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1973 by 1 to 5 nucleotides; (aa) a crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1974 by 1 to 5 nucleotides; (bb) a crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1975 by 1 to 5 nucleotides; (cc) a crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1976 by 1 to 5 nucleotides; (dd) a crRNA backbone, including a nucleotide sequence that differs from SEQ ID NO: 1977 by 1 to 5 nucleotides; 1977 has a nucleotide sequence that differs from SEQ ID NO: 1978 by 1 to 5 nucleotides; (ee) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1978 or differing from SEQ ID NO: 1978 by 1 to 5 nucleotides; (ff) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1979 or differing from SEQ ID NO: 1979 by 1 to 5 nucleotides; (gg) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1980 or differing from SEQ ID NO: 1980 by 1 to 5 nucleotides; (hh) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1981 or differing from SEQ ID NO: 1981 by 1 to 5 nucleotides; (ii) a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1982 or differing from SEQ ID NO: 1983. 1982 has a nucleotide sequence that is 1 to 5 nucleotides different; and (jj)-crRNA backbone, including a nucleotide sequence that is as shown in SEQ ID NO: 1983 or that is 1 to 5 nucleotides different from SEQ ID NO: 1983. The gRNA as described in claim 148, wherein the crRNA backbone is selected from the group consisting of: (a) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 5; (b) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 6; (c) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 7; (d) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 8; (e) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1617; (f) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1618; (g) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1619; (h) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1620; (i) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1620; and (ii) a crRNA backbone comprising the nucleotide sequence shown in SEQ ID NO: 1620. The nucleotide sequence shown in SEQ ID NO: 1931; (j) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1957; (k) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1958; (l) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1959; (m) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1960; (n) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1961; (o) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1962; (p) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1963; (q) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1964; (r) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1964; The nucleotide sequence shown in SEQ ID NO: 1965; (s) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1966; (t) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1967; (u) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1968; (v) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1969; (w) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1970; (x) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1971; (y) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1972; (z) a crRNA main chain, including the nucleotide sequence shown in SEQ ID NO: 1973; (aa) a crRNA main chain, including the ...v) a crRNA The nucleotide sequence shown in SEQ ID NO: 1974; (bb)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1975; (cc)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1976; (dd)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1977; (ee)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1978; (ff)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1979; (gg)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1980; (hh)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1981; (ii)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1982; and (jj)-crRNA backbone, including the nucleotide sequence shown in SEQ ID NO: 1983. The gRNA as described in claim 148 or claim 149, wherein the gRNA includes a tracrRNA. The gRNA as described in claim 148 or claim 149, wherein the gRNA is composed of cRNA. The gRNA described in any one of claims 148 to 151, wherein the gRNA has a total length of 35 to 250 nucleotides (nt), or 35 to 170 nt, or 35 to 125 nt, or 40 to 100 nt, or 40 to 70 nt, or 40 to 60 nt. The gRNA described in any one of claims 148 to 151, wherein the gRNA has a crRNA backbone having a total length of up to 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt or 40 nt. The gRNA as described in any one of claims 148 to 153, wherein the spacer comprises a nucleotide sequence as shown in any one of SEQ ID NO: 294 to 396, 1789 to 1796, 1798 to 1880, 1890 to 1893 and 2082 to 2125. The gRNA as described in any one of claims 148 to 154, wherein the gRNA comprises the nucleotide sequence shown in any one of SEQ ID NO: 39 to 182, 1405 to 1412, 1414 to 1501, 1556, 1567 to 1616, 1662 to 1671 and 2126 to 2658. The gRNA described in any one of claims 148 to 155, wherein, when the gRNA binds to an RNA-guided nuclease (RGN) polypeptide, the gRNA is capable of target hybridizing with a target strand of a target sequence in a target nucleic acid molecule in a sequence-specific manner via a spacer of the crRNA. The gRNA as described in claim 156, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides; b) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides; c) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 7 or differing from SEQ ID NO: 8 by 1 to 5 nucleotides; 7. A nucleotide sequence differing by 1 to 5 nucleotides; d) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing by 1 to 5 nucleotides as shown in SEQ ID NO: 8; (e) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing by 1 to 5 nucleotides as shown in SEQ ID NO: 1617; (f) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing by 1 to 5 nucleotides as shown in SEQ ID NO: 1618; (g) An RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence differing by 1 to 5 nucleotides as shown in SEQ ID NO: 1618; The amino acid sequence of SEQ ID NO: 4 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1619 or differing from SEQ ID NO: 1619 by 1 to 5 nucleotides; (h) an RGN polypeptide comprising an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1620 by 1 to 5 nucleotides; (i) an RGN polypeptide comprising an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1631 or differing from SEQ ID NO: 1631 by 1 to 5 nucleotides; (j) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1631 by 1 to 5 nucleotides; The amino acid sequence of SEQ ID NO: 1930 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1957 or differing from SEQ ID NO: 1957 by 1 to 5 nucleotides; (k) an RGN polypeptide comprising a single amino acid sequence as shown in SEQ ID NO: 1931 with at least 90% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1958 or differing from SEQ ID NO: 1958 by 1 to 5 nucleotides; (l) an RGN polypeptide comprising a single amino acid sequence as shown in SEQ ID NO: 1932 with at least 90% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1959 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides; (m) an RGN polypeptide comprising a single amino acid sequence as shown in SEQ ID NO: 1932 with at least 90% sequence identity, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1932 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides; The amino acid sequence of SEQ ID NO: 1933 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1960 or differing from SEQ ID NO: 1960 by 1 to 5 nucleotides; (n) an RGN polypeptide comprising a single amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1961 or differing from SEQ ID NO: 1961 by 1 to 5 nucleotides; (o) an RGN polypeptide comprising a single amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1935, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1962 or differing from SEQ ID NO: 1962 by 1 to 5 nucleotides; (p) an RGN polypeptide comprising a single amino acid sequence as shown in SEQ ID NO: 1934 or differing from SEQ ID NO: 1935 by 1 to 5 nucleotides; The amino acid sequence of SEQ ID NO: 1936 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1963 or differing from SEQ ID NO: 1963 by 1 to 5 nucleotides; (q) an RGN polypeptide comprising a single amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1937, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1964 or differing from SEQ ID NO: 1964 by 1 to 5 nucleotides; (r) an RGN polypeptide comprising a single amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1965 or differing from SEQ ID NO: 1965 by 1 to 5 nucleotides; (s) an RGN polypeptide comprising a single amino acid sequence as shown in SEQ ID NO: 1937 or differing from SEQ ID NO: 1938 by 1 to 5 nucleotides; The amino acid sequence of SEQ ID NO: 1939 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1966 or differing from SEQ ID NO: 1966 by 1 to 5 nucleotides; (t) an RGN polypeptide comprising a single amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1940, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1967 or differing from SEQ ID NO: 1967 by 1 to 5 nucleotides; (u) an RGN polypeptide comprising a single amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1941, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1968 or differing from SEQ ID NO: 1968 by 1 to 5 nucleotides; (v) an RGN polypeptide comprising a single amino acid sequence as shown in SEQ ID NO: 1939 or differing from SEQ ID NO: 1940 by 1 to 5 nucleotides; The amino acid sequence of SEQ ID NO: 1942 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1969 or differing from SEQ ID NO: 1969 by 1 to 5 nucleotides; (w) an RGN polypeptide comprising a single amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1970 or differing from SEQ ID NO: 1970 by 1 to 5 nucleotides; (x) an RGN polypeptide comprising a single amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1971 or differing from SEQ ID NO: 1971 by 1 to 5 nucleotides; (y) an RGN polypeptide comprising a single amino acid sequence as shown in SEQ ID NO: 1942 or differing from SEQ ID NO: 1943 by 1 to 5 nucleotides; The amino acid sequence of SEQ ID NO: 1945 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1972 or differing from SEQ ID NO: 1972 by 1 to 5 nucleotides; (z) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1945 or differing from SEQ ID NO: 1973 by 1 to 5 nucleotides; (aa) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1945 or differing from SEQ ID NO: 1974 by 1 to 5 nucleotides; (bb) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1945 or differing from SEQ ID NO: 1974 by 1 to 5 nucleotides; The amino acid sequence of SEQ ID NO: 1948 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1975 or differing from SEQ ID NO: 1975 by 1 to 5 nucleotides; (cc)-RGN polypeptide comprises a single amino acid sequence as shown in SEQ ID NO: 1949, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1976 or differing from SEQ ID NO: 1976 by 1 to 5 nucleotides; (dd)-RGN polypeptide comprises a single amino acid sequence as shown in SEQ ID NO: 1949, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1950, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1977 or differing from SEQ ID NO: 1977 by 1 to 5 nucleotides; (ee)-RGN polypeptide comprises a single amino acid sequence as shown in SEQ ID NO: 1949, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1949, having at least 90% sequence identity with the amino acid sequence of ... The following amino acid sequences are described: (1) a monoamino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1951, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1978 or differing from SEQ ID NO: 1978 by 1 to 5 nucleotides; (ff) a-RGN polypeptide comprising a monoamino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1952, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1979 or differing from SEQ ID NO: 1979 by 1 to 5 nucleotides; (gg) a-RGN polypeptide comprising a monoamino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1980 or differing from SEQ ID NO: 1980 by 1 to 5 nucleotides; and (hh) a-RGN polypeptide comprising a monoamino acid sequence as shown in SEQ ID NO: 1951 or differing from SEQ ID NO: 1952 by 1 to 5 nucleotides; and (hh) a-RGN polypeptide comprising a monoamino acid sequence as shown in SEQ ID NO: 1953 or differing from SEQ ID NO: 1954 by 1 to 5 nucleotides. The amino acid sequence of 1954 has at least 90% sequence identity with a single amino acid sequence, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1981 or differing from SEQ ID NO: 1981 by 1 to 5 nucleotides; (ii) an RGN polypeptide comprising an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1955, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1982 or differing from SEQ ID NO: 1982 by 1 to 5 nucleotides; and (jj) an RGN polypeptide comprising an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1956, wherein the crRNA backbone comprises a nucleotide sequence as shown in SEQ ID NO: 1983 or differing from SEQ ID NO: 1983 by 1 to 5 nucleotides. The gRNA as described in claim 156 or claim 157, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 5; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 6; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 7; d) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 8; (e) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 8. (f) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1618; (g) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1619; (h) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1620; (i) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1631; (j) An RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1930, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1631; The following are listed: (k) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1931, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1958; (l) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1932, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1959; (m) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1933, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1960; (n) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1934, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1961; (o) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1935 ... The following are listed: (p) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1936, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1963; (q) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1937, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1964; (r) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1938, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1965; (s) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1939, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1966; (t) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1940, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1966; The following are listed: (u) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1941, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1968; (v) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1942, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1969; (w) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1943, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1970; (x) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1944, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1971; (y) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1945, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1967; The following are listed: (z) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1946, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1973; (aa) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1947, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1974; (bb) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1948, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1975; (cc) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1949, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1976; (dd) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1950, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1976; The following are listed: (i) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1977; (ii) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1951, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1978; (f) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1952, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1979; (g) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1953, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1980; (hh) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1954, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1981; (ii) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1955, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1977; (f) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1952, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1979; (g) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1953, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1980; (hh) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1954, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1981; (hh) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1955, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1977; (f) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1955; (hh) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: The nucleotide sequence shown in SEQ ID NO: 1982; and the (jj)-RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1956, wherein the crRNA backbone comprises the nucleotide sequence shown in SEQ ID NO: 1983. The gRNA as described in any of claims 156 to 158, wherein the target sequence is disposed adjacent to and at its 3' of a protoseptum adjacent motif (PAM). The gRNA as described in claim 159, wherein a) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 recognizes a PAM having a common nucleotide sequence shown in AYG, wherein the PAM is adjacent to the target sequence and is located at its 5'; b) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 1933, or 1944 recognizes a PAM having a common nucleotide sequence shown in ATTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; c) an RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949 recognizes a PAM having a common nucleotide sequence shown in VTTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; and d) a gRNA comprising the amino acid sequence shown in SEQ ID NO: 4. RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is located at its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: The RGN polypeptide of the amino acid sequence shown in SEQ ID NO: 1937 or 1950 identifies a PAM having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) the RGN polypeptide of the amino acid sequence shown in SEQ ID NO: 1951 identifies a PAM having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) the RGN polypeptide of the amino acid sequence shown in SEQ ID NO: 1953 identifies a PAM having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'. A nucleic acid molecule comprising at least one polynucleotide that encodes a gRNA as described in any one of claims 148 to 160. A vector comprising the nucleic acid molecule described in claim 161. A cell comprising an RGN polypeptide as described in any of claims 94 to 146, an RNP complex as described in claim 147, a gRNA as described in any of claims 148 to 160, a nucleic acid molecule as described in claim 161, or a vector as described in claim 162. The cell as described in claim 163, wherein the cell is a eukaryotic cell. An RNA-guided nuclease (RGN) system for binding to one or more target sequences, the system comprising: a) one or more guide RNAs (gRNAs), or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more gRNAs, wherein the one or more gRNAs include a CRISPR RNA (crRNA) comprising a crRNA backbone and a spacer; and b) an RGN polypeptide or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide, the RGN polypeptide comprising, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 108 1. The amino acid sequence represented by any one of 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has a monoamino acid sequence with at least 90% sequence identity. The RGN system as described in claim 165, wherein the RGN polypeptide comprises, as in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1 The amino acid sequence represented by any one of 081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687. The RGN system as described in claim 165 or claim 166, wherein each of the one or more gRNAs is capable of hybridizing with a target strand of a corresponding target sequence and forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target sequence. An RGN system as described in any of claims 165 to 167, wherein at least one of the one or more nucleotide sequences encoding the one or more gRNAs and the nucleotide sequences encoding the RGN polypeptide is operatively linked to a promoter heterologous to the nucleotide sequences. The RGN system as described in any of claims 165 to 168, wherein the one or more nucleotide sequences encoding the one or more gRNAs comprise: (i) a nucleotide sequence encoding one or more gRNAs; or (ii) two or more nucleotide sequences, each encoding a gRNA. The RGN system as described in claim 169, wherein (i) the nucleotide sequence of a gRNA that writes to one or more gRNAs is operatively linked to a single promoter. The RGN system as described in claim 169, wherein (ii) each nucleotide sequence of the write-coding gRNA is operatively linked to a promoter. The RGN system as described in any of claims 165 to 167, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is an mRNA comprising a heterologous 5' untranslated region (UTR) and/or a heterologous 3' UTR. The RGN system as described in any one of claims 165 to 172, wherein the one or more gRNAs are selected from the group consisting of: a) a gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 5 or differing from SEQ ID NO: 5 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 1; b) a gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 6 or differing from SEQ ID NO: 6 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in SEQ ID NO: 2; c) a gRNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 7 or differing from SEQ ID NO: 8 by 1 to 5 nucleotides. 7. A mononucleotide sequence differing by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; d) a gRNA having a crRNA backbone comprising a mononucleotide sequence differing by 1 to 5 nucleotides as shown in SEQ ID NO: 8 or differing from SEQ ID NO: 8, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (e) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a mononucleotide sequence differing by 1 to 5 nucleotides as shown in SEQ ID NO: 1617 or differing from SEQ ID NO: 1617, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (e) a gRNA comprising a CRISPR RNA having a crRNA backbone having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; The RNA comprises a crRNA backbone including a nucleotide sequence as shown in SEQ ID NO: 1617 or differing from SEQ ID NO: 1617 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (f) a gRNA comprising a CRISPR RNA having a crRNA backbone including a nucleotide sequence as shown in SEQ ID NO: 1618 or differing from SEQ ID NO: 1618 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (g) a gRNA comprising a CRISPR RNA having a crRNA backbone including a nucleotide sequence as shown in SEQ ID NO: 1619 or differing from SEQ ID NO: 1619 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; The amino acid sequence of 4 has at least 90% sequence identity with a single amino acid sequence; (h) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1620 or differing from SEQ ID NO: 1620 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (i) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1631 or differing from SEQ ID NO: 1631 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1; (j) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1957 or differing from SEQ ID NO: 1620 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1; ID NO: 1957 has a nucleotide sequence that is 1 to 5 nucleotides different, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1930; (k) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1958 or differing from SEQ ID NO: 1958 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1931; (l) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1959 or differing from SEQ ID NO: 1959 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1930; The amino acid sequence of SEQ ID NO: 1932 has at least 90% sequence identity with a single amino acid sequence; (m) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1960 or differing from SEQ ID NO: 1960 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1933; (n) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1961 or differing from SEQ ID NO: 1961 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1934; (o) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1932; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1935, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1962; (p) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1963, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1963, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1936; (q) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is 1 to 5 nucleotides different from the amino acid sequence shown in SEQ ID NO: 1964, or a nucleotide sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1964, wherein the RGN polypeptide comprises an amino acid sequence that is 1 to 5 nucleotides different from the amino acid sequence of SEQ ID NO: 1962; The amino acid sequence of SEQ ID NO: 1937 has at least 90% sequence identity with a single amino acid sequence; (r) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1965 or differing from SEQ ID NO: 1965 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1938; (s) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1966 or differing from SEQ ID NO: 1966 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1939; (t) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1937; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1967 or different from SEQ ID NO: 1967 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1940; (u) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1968 or different from SEQ ID NO: 1968 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1941; (v) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1969 or different from SEQ ID NO: 1969 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1967; The amino acid sequence of SEQ ID NO: 1942 has at least 90% sequence identity with a single amino acid sequence; (w) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1970 or differing from SEQ ID NO: 1970 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1943; (x) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1971 or differing from SEQ ID NO: 1971 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1944; (y) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1942; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1972 or different from SEQ ID NO: 1972 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1945; (z) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1973 or different from SEQ ID NO: 1973 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1946; (aa) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1974 or different from SEQ ID NO: 1974 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1972; The amino acid sequence of SEQ ID NO: 1947 has at least 90% sequence identity with a single amino acid sequence; (bb) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1975 or differing from SEQ ID NO: 1975 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1948; (cc) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1976 or differing from SEQ ID NO: 1976 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1949; (dd) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1947. The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1977 or different from SEQ ID NO: 1977 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1950; (ee) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1978 or different from SEQ ID NO: 1978 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1951; (ff) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1979 or different from SEQ ID NO: 1979 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1977; The amino acid sequence of 1952 has at least 90% sequence identity with a single amino acid sequence; (gg) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1980 or differing from SEQ ID NO: 1980 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1953; (hh) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1981 or differing from SEQ ID NO: 1981 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954; (ii) a gRNA comprising a CRISPR RNA having a crRNA backbone comprising a nucleotide sequence as shown in SEQ ID NO: 1952 or differing from SEQ ID NO: 1980 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1954; The RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1982 or SEQ ID NO: 1982, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1955; and (jj)-gRNA, comprising a CRISPR RNA having a crRNA backbone comprising an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1983 or SEQ ID NO: 1983, wherein the RGN polypeptide comprises an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of SEQ ID NO: 1956. The RGN system as described in any of claims 165 to 173, wherein the target sequence is disposed adjacent to and at its 3' of a primary spacer adjacent modulus (PAM). The RGN system as described in claim 174, wherein: a) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1 identifies a PAM having a common nucleotide sequence as shown in AYG, wherein the PAM is adjacent to the target sequence and is located at its 5'; b) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2, 1933, or 1944 identifies a PAM having a common nucleotide sequence as shown in ATTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; c) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949 identifies a PAM having a common nucleotide sequence as shown in VTTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; d) an RGN polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 4. RGN polypeptides comprising the amino acid sequences shown in SEQ ID NO: 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956 identify PAMs having a common nucleotide sequence as shown in TTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; e) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1931 identify PAMs having a common nucleotide sequence as shown in STTN, wherein the PAM is adjacent to the target sequence and is located at its 5'; f) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1935 identify PAMs having a common nucleotide sequence as shown in TTH, wherein the PAM is adjacent to the target sequence and is located at its 5'; g) RGN polypeptides comprising the amino acid sequence shown in SEQ ID NO: 1937 The RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1950 identifies a PAM having a common nucleotide sequence as shown in RTTN, wherein the PAM is adjacent to the target sequence and is in its 5'; h) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1951 identifies a PAM having a common nucleotide sequence as shown in RTYN, wherein the PAM is adjacent to the target sequence and is in its 5'; and i) the RGN polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1953 identifies a PAM having a common nucleotide sequence as shown in ATG, wherein the PAM is adjacent to the target sequence and is in its 5'. The RGN system as described in any of claims 165 to 175, wherein the target sequence is within a cell. The RGN system as described in claim 176, wherein the cell is a eukaryotic cell. The RGN system described in any of claims 165 to 177, wherein the system guides the cleavage of the target nucleic acid molecule. The RGN system as described in claim 178, wherein the shearing produces a double-strand fracture. The RGN system as described in claim 179, wherein the double-strand break includes (i) a 5' protrusion of 5 to 12 nt, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; or (ii) a 3' protrusion of 1 nt, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The RGN system as described in any one of claims 165 to 177, wherein the RGN polypeptide is nuclease-inactivating. The RGN system as described in claim 181, wherein the RGN polypeptide comprises a monoamino acid sequence having at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 408, 597, 678, 827, 896, 979, 2686, and 2687. The RGN system as described in any of claims 165 to 182, wherein the RGN polypeptide is operatively fused with a base-editing polypeptide or a polymerase-editing polypeptide. The RGN system as described in claim 183, wherein the base-editing polypeptide includes a deaminerase. The RGN system as described in claim 183, wherein the polymerase editing polypeptide comprises a DNA polymerase or a reverse transcriptase. The RGN system as described in any of claims 165 to 185, wherein the RGN polypeptide includes one or more nuclear localization signals (NLS). The RGN system as described in any of claims 165 to 186, wherein the system further comprises one or more donor polynucleotides. A cell comprising an RGN system as described in any of claims 165 to 187. The cell as described in claim 192, wherein the cell is a eukaryotic cell. A pharmaceutical composition comprising a nucleic acid molecule as described in any of claims 1 to 57 and 161; a vector as described in any of claims 58 to 87 and 162; an RGN polypeptide as described in any of claims 94 to 146; an RNP complex as described in claim 147; a gRNA as described in any of claims 148 to 160; a cell as described in any of claims 88, 163, 164, 188 and 189; or an RGN system as described in any of claims 165 to 187; and a pharmaceutically acceptable delivery method. A method for binding to a target sequence in a target nucleic acid molecule, comprising delivering an RGN system according to any one of claims 165 to 187 to the target sequence or a cell including the target sequence. A method for cleaving and/or modifying a target nucleic acid molecule comprising a target sequence, the method comprising delivering a system according to any one of claims 165 to 187 to the target sequence or a cell comprising the target sequence, wherein cleaving or modification of the target nucleic acid molecule occurs. A method for binding one or more target sequences in a target nucleic acid molecule to an RNA-guided nuclease (RGN) polypeptide, the method comprising: a) assembling a ribonucleoprotein (RNP) complex under conditions suitable for forming the RNP complex by combining: i) a guide RNA (gRNA); and ii) an RGN polypeptide, including as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 108 1. The amino acid sequence represented by any one of 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686, and 2687 has at least A) A monoamino acid sequence with 90% sequence identity; and b) Contacting the target nucleic acid molecule or a cell including the target nucleic acid molecule with the assembled RNP complex; thereby binding the one or more target sequences to the RGN polypeptide. A method for binding one or more target sequences in a target nucleic acid molecule to an RNA-guided nuclease (RGN) polypeptide, the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with: i) a guide RNA (gRNA); and ii) an RGN polypeptide or a polynucleotide encoding the RGN polypeptide, the RGN polypeptide comprising, as shown in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084 The amino acid sequence represented by any one of the following numbers has at least 90% sequence identity, thereby enabling the target sequence to bind to an RGN. The method described in claim 194, wherein the polynucleotide encoding the RGN polypeptide is an mRNA comprising a heterologous 5' untranslated region (UTR) and/or a heterologous 3' UTR. The method described in any one of claims 193 to 195, wherein the method is performed in vitro, in vivo or ex vivo. The method described in any of claims 193 to 196, wherein the cell is a eukaryotic cell. The method as described in any one of claims 193 to 197, wherein the RGN polypeptide further comprises a base-editing polypeptide or a polymerase-editing polypeptide, thereby allowing modification of the target nucleic acid molecule. The method as described in claim 198, wherein the base-editing polypeptide includes a deaminerase. The method as described in claim 198, wherein the polymerase editing polypeptide comprises a DNA polymerase or a reverse transcriptase. The method as described in any one of claims 193 to 197, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule, thereby allowing cleavage and/or modification of the target nucleic acid molecule. The method as described in claim 201, wherein the cleavage produces a double-stranded break in the target nucleic acid molecule, wherein the double-stranded break comprises: (i) a 5' protrusion of 5 to 12 nt, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; and (ii) a 3' protrusion of 1 nt, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. A method for cleaving and/or modifying a target nucleic acid molecule comprising one or more target sequences, the method comprising contacting the target nucleic acid molecule with: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises, as in SEQ ID NO: 1 to 4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116 The amino acid sequence represented by any one of 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930 to 1956, 2686 and 2687 has an amino acid sequence with at least 90% sequence identity; and b) a guide RNA (gRNA) capable of targeting the RGN polypeptide of (a) to a target sequence; wherein splicing and/or modification of the target nucleic acid molecule occurs. The method as described in claim 203, wherein the cleavage of the RGN polypeptide produces a double-strand break in the target DNA molecule, and wherein the double-strand break comprises: (i) a 5' protrusion of 5 to 12 nt, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; and (ii) a 3' protrusion of 1 nt, wherein the RGN polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The method as described in claim 203, wherein the RGN polypeptide is nuclease-inactivating and operatively fused to a base-editing polypeptide or a polymerase-editing polypeptide. The method as described in claim 205, wherein the base-editing polypeptide includes a deaminerase. The method as described in claim 205, wherein the polymerase editing polypeptide comprises a DNA polymerase or a reverse transcriptase. The method as described in claim 203, wherein the modified target nucleic acid molecule comprises: (i) inserting a heterologous DNA into the target nucleic acid molecule; (ii) deleting at least one nucleotide from the target nucleic acid molecule; or (iii) a mutation of at least one nucleotide in the target nucleic acid molecule. The method as described in any of claims 203 to 208, wherein the target sequence is in a cell. The method as described in claim 209 further includes: culturing the cell under conditions in which the RGN polypeptide is expressed and the target nucleic acid molecule is cleaved and/or modified to generate a modified target nucleic acid molecule; and selecting a cell comprising the modified target nucleic acid molecule. A cell generated according to the method of claim 210, wherein the target nucleic acid molecule has been cleaved and/or modified at the target sequence. The cell as described in claim 211, wherein the cell is a eukaryotic cell. A pharmaceutical composition comprising cells as described in claim 211 or claim 212 and a pharmaceutically acceptable delivery vehicle.