WO2025171210A1 - Compositions and methods for gene editing via homology-mediated end joining - Google Patents

Abstract

A gene editing system for inserting a nucleic acid into a genomic site, comprising: (a) a CRISPR nucleases or an encoding nucleic acid; (b) a guide RNA (gRNA) or an encoding nucleic acid, the gRNA being specific to a target sequence in a genomic site of interest; and (c) a donor DNA template comprising (i) a transgene; (ii) a left homology arm upstream to the transgene and a right homology arm downstream to the transgene; and (iii) two copies of a target sequence, one being upstream to the left homology arm and the other being downstream to the right homology arm. The left homology arm and the right homology arm are homologous to the upstream and downstream sequences flanking the target sequence in the genomic site.

Description

COMPOSITIONS AND METHODS FOR GENE EDITING VIA HOMOLOGY- MEDIATED END JOINING

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 63/551,831, filed February 9, 2024, the entire contents of which is incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 14, 2025, is named 063586-538001WO_Seq- Listing_ST26.xml and is 30,209 bytes in size.

BACKGROUND OF THE INVENTION

Many genetic diseases, while involving a single gene, have large numbers of mutations distributed across the whole gene. Patients having such a genetic disease would not benefit from repairing a single mutation or a cluster of mutations within a small region of the gene.

Ectopic expression, such as AAV gene replacement, has been used in some cases; however, such approaches are limited to only genes that are below the packaging capacity of AAV. Homologous recombination (HR)-mediated gene editing methods could be used to replace a large region in an endogenous gene with a desired sequence. However, such an approach is generally inefficient in non-dividing cells since HR is active only during the late S/G2 phrase. Yao et al., Cell Research (2017) 27:801-814.

It is therefore of great importance to develop efficient gene editing methods that are allows for insertion of large DNA fragments into endogenous locus to restore normal function in both dividing and non-dividing cells.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on a gene editing system capable of precise insertion of a transgene into a genomic site of interest. By specific designs of a donor template that carries the transgene, the gene editing system facilitates transgene insertion at the genomic site via homology-mediated end joining. As such, the gene editing system can be used to insert an exogenous nucleic acid (e.g., a large transgene) at a genomic site of interest in both dividing and non-dividing host cells.

Accordingly, one aspect of the present disclosure features a gene editing system for inserting a nucleic acid into a genomic site, comprising: (a) a CRISPR nucleases or a nucleic acid encoding the CRISPR nuclease; (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA; and (c) a donor DNA template comprising (i) a transgene; (ii) a left homology arm upstream to the transgene and a right homology arm downstream to the transgene. The gRNA comprising (i) a spacer sequence specific to a target sequence in the genomic site and (ii) a direct repeat sequence recognizable by the CRISPR nuclease. The donor DNA template also contains two copies of the target sequence, one being upstream to the left homology arm and the other being downstream to the right homology arm. The left homology arm and the right homology arm in the donor template are homologous to the upstream and downstream sequences flanking the target sequence in the genomic site.

In some embodiments, the CRISPR nuclease is a Type V nuclease. In some examples, the Type V nuclease comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. In specific examples, the Type V nuclease comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The direct repeat sequence in the gRNA that is recognizable by such Type V nucleases may comprise a nucleotide sequence at least 90% identical to SEQ ID NO: 5. In some examples, the direct repeat sequence is SEQ ID NO: 5.

In other examples, the Type V nuclease comprises an amino acid sequence at least 90% identical to SEQ ID NO : 3. In specific examples, the Type V nuclease comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. The direct repeat sequence in the gRNA that is recognizable by such Type V nucleases may comprise a nucleotide sequence at least 90% identical to SEQ ID NO: 6. In some examples, the direct repeat sequence is SEQ ID NO: 6.

In some embodiments, the left and right homology arms may be 20-1,000-bp in length. In some examples, the left and right homology arms may be about 600-800-bp in length. In some specific examples, the left and right homology arms may be about 600-bp in length. In some specific examples, the left and right homology arms may be about 700-bp in length. In other specific examples, the left and right homology arms may be about 800-bp in length.

In some instances, the donor DNA template can be located on a vector, for a viral vector. In some examples, the donor DNA template may be located on an adeno-associated viral (AAV) vector. In some embodiments, the transgene in the donor DNA template may be at least 2-kb in length, for example, up to 100-kb in length. In some instances, the transgene may be about 2.0-kb to about 4.5-kb in length (e.g., about 2-kb, about 2.5-kb, about 3-kb, about 3.5-kb, about 4-kb, or about 4.5-kb in length), for example, when the transgenecontaining donor DNA template is located on an AAV vector.

In some embodiments, the gene editing system disclosed herein may comprise a nucleic acid encoding the CRISPR nuclease as disclosed herein. In some examples, the nucleic acid can be an expression vector, which comprises the nuclease-encoding nucleotide sequence. Alternatively or in addition, the gene editing system disclosed herein may comprise nucleic acid encoding the gRNA. In some instances, the gene editing system may comprise an expression vector comprising coding sequences for both the nuclease and the gRNA. In some examples, each of the nuclease-coding sequence and the gRNA-coding sequence may be in operable linkage to a suitable promoter.

Alternatively, the nuclease-encoding nucleic acid may be a messenger RNA (mRNA). In some examples, the gene editing system comprises lipid nanoparticles (LNPs), which are associated with the mRNA and optionally the gRNA.

In other embodiments, the gene editing system disclosed herein may comprise the CRISPR nuclease. In some examples, the CRISPR and the gRNA may form a ribonucleoprotein (RNP) complex.

In some embodiments, the genomic site targeted by the gene editing system disclosed herein may be in a safe harbor locus. Examples include, but are not limited to, the AAVS1 locus, the chemokine (C-C motif) receptor 5 (CCR5) locus, the human orthologue of the mouse Rosa26 (hROSA26) locus, or a region within intron 2 of the Citrate Lyase Beta-Like (CLYBY) gene. In other embodiments, the genomic site can be in a gene associated with a disease.

In other aspects, the present disclosure features a method for inserting a nucleic acid into a genomic site, the method comprising delivering into a mammalian host cell a gene editing system, such as any of the gene editing systems provided herein. When delivered to a host cell, the gene editing system cleaves at the target sequence in the genomic site and at the target sequences in the donor DNA template. The cleaved donor DNA template, which comprises the transgene, can insert into the genomic site via, for example, homology- mediated end joining (HMEJ).

In some embodiments, the mammalian host cell can be a human mammalian cell. In some examples, the mammalian cell can be a non-dividing mammalian cell. In some examples, the mammalian cell can be a human primary cell. In some examples, the mammalian cell can be a terminally differentiated cell. In some instances, the mammalian cell is collected from a subject, for example, a human subject. Alternatively, the mammalian cell is in a cell culture.

In some embodiments, the gene editing method provided herein may comprise delivering the gene editing system to a subject in need thereof. In some examples, the subject is a human patient.

Also within the scope of the present disclosure is any of the gene editing systems disclosed herein for use in inserting a transgene into to a genomic site of a subject in need of the treatment and for treating a disease targeted by the transgene. Further, the instant disclosure provides uses of any of the gene editing system for manufacturing a medicament for use in genetic editing a genomic site (e.g., inserting a transgene) and for treating a disease targeted by the transgene.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

FIGs. 1A-1C include schematic illustrations for various designs of donor constructs as indicated and method of integration into a genomic target site. FIG. 1A: donor plasmid vectors. FIG. IB: plasmid vectors after nuclease cutting. FIG. 1C: integration of the transgene cassette carried by the donor plasmid vectors into a genomic target site.

FIG. 2 is a diagram showing the knock-in efficiency (%) at the EMX1 target site as quantified by ddPCR in HEK293. The HMEJ plasmid donor exhibits higher knock-in efficiency compared to an HR donor with the same homology arm lengths. HMEJ knock-in efficiency increases with increasing homology arm length. Bars represent mean of 3-6 technical replicates, error bars represent standard deviation.

FIG. 3 is a diagram showing the knock-in efficiency (%) in non-dividing iPSC- derived hepatocytes at the EMX1 target site as quantified by ddPCR. The HMEJ plasmid donors result in the highest knock-in efficiency compared to HITI, MITI, and HR knock-in approaches with a Type V nuclease.

FIGs. 4A and 4B include diagrams illustrating exemplary genomic knock-in strategy in an animal model. FIG. 4A: AAV donor construct and HMEJ editing strategy. FIG. 4B: exemplary experimental approach.

FIGs. 5A and 5B include diagrams showing immunostaining of mouse liver in mice having a reporter gene expressing donor inserted into a genomic site (target) via the knock-in strategy illustrated in FIGs. 4A and 4B. FIG. 5A: representative images from immunostaining of mouse liver, 7 days post-LNP delivery and 8 days post- AAV delivery. FIG. 5B: quantification of the percentages of mCherry-positive cells (at least 80,000 nuclei assessed per mouse per condition). Points represent individual mice, bars represent the mean value for experimental groups 1-6 (Table 5), and error bars represent standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are gene editing systems and methods of using such to insert transgenes into an endogenous genetic locus. The gene editing system provided herein comprises (a) a CRISPR nuclease or its encoding nucleic acid, (b) a guide RNA (gRNA) or its encoding nucleic acid, and (c) a donor DNA template comprising a transgene flanked by a left homology arm and a right homology arm. By design of the donor DNA template, i.e., comprising two copies of a target sequence, which is also located in the genomic site where insertion is desired, flanking the left homology arm and the right homology arm (see, e.g., FIGs. 1A-1C), the gene editing system provided herein facilitates insertion of the transgene into the endogenous genomic site via homology-mediated end joining (HMEJ).

Without being bound by theory, the gene editing system provided herein may confer at least the following advantages: (a) allow for precise insertion of large nucleic acid fragments (e.g., about 10-kb) into an endogenous genomic site; (b) applicable to both dividing and non-dividing cells; (c) certain CRISPR nucleases such as Type V nucleases exhibit higher gene editing efficiencies than the spCas9 enzyme.

I. Homology-Mediated End Joining (HMEJ)-Mediated Gene Editing Systems

In some aspects, the present disclosure provides gene editing systems that can facilitate insertion of transgenes, including transgenes of large size, into an endogenous genomic site in host cells. As described herein, the term “transgene” disclosed herein refer to any exogenous nucleic acid to be introduced into host cells. The gene editing system provided herein comprises (a) a CRISPR nuclease or a nucleic acid encoding such; (b) a guide RNA (gRNA) specific to a target sequence at a genomic site or a nucleic acid encoding such; and (c) a donor DNA template comprising a transgene flanked by a left homology arm and a right homology arm, which are homologous to the regions flanking the target sequence at the genomic site. The donor DNA template further comprises the same target sequence as in the genomic site located both upstream to the left homology arm and downstream to the right homology arm. This specific design allows for nuclease cleavage at the genomic site, as well as at sites flanking the left homology arm and the right homology arm. Strands of the cleaved donor DNA template and the genomic site can recombine via the homologous sequences, resulting in insertion of the transgene in the donor DNA template into the genomic site via HMEJ. An exemplary illustration of the donor DNA template is provided in FIG. 1.

A. CRISPR Nucleases

Any of the gene editing systems disclosed herein may comprises a CRISPR nuclease. Mediated by the gRNA also contained in the gene editing system, the CRISPR nuclease cleaves DNA at a target sequence, which is located both at a genomic site of interest and at a donor DNA template of the gene editing system. See, e.g., FIGs. 1A-1C.

In some instances, the gene editing system provided herein is a CRISPR/Cas gene editing system, which involves a CRISPR/Cas nuclease. Non-limiting CRISPR nucleases include Cas9 (e.g., Cas9 and nCas9), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, and Casl2j/ CasPhi). In some instances, the CRISPR nuclease can be a Type II CRISPR nuclease. In other instances, the CRISPR nuclease can be a Type V CRISPR nuclease. Other CRISPR nucleases are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPRJ. l(5):325-36 (2018).

(i) Type V Nucleases

In some embodiments, the gene editing system provided herein comprises a Type V nuclease. As used herein, the terms “Type V” and “Type V nuclease” refer to an RNA-guided CRISPR nuclease with a RuvC domain. In some embodiments, a Type V nuclease does not require a tracrRNA. In some embodiments, a Type V nuclease requires a tracrRNA. In some embodiments, the Type V nuclease is a Casl2 polypeptide, such as a Casl2a (Cpfl), Casl2b (C2cl), Casl2c, Casl2d, Casl2e, Casl2f, Casl2h, Casl2i, or Casl2j (CasPhi) polypeptide.

(a) Casl2i Nucleases

“Casl2i nucleases” (also referred to herein as Casl2i) refers to a polypeptide that binds to a target sequence on a target nucleic acid specified by an RNA guide, wherein the polypeptide has at least some amino acid sequence homology to a wild-type Casl2i nuclease. Such Casl2i polypeptides are known in the art or disclosed herein. See, e.g., WO/2021/202800 and WO2022256440, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose disclosed herein. In some embodiments, the Casl2i nuclease is a Casl2i2 polypeptide. Examples include Casl2i2 A and Casl2i2 B as provided in Table 1 below.

Table 1. Exemplary Type V Nucleases

In some instances, the Casl2i2 polypeptide may comprise an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, or at least 99% identical to SEQ ID NO: 1. In one example, the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO:1. In another example, the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

The “percent identity” (a.k.a., sequence identity) of two nucleic acids or of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength- 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

In some instances, the Casl2i2 polypeptide may comprise one or more conservative amino acid substitutions as compared with SEQ ID NO: 1. In some instances, the Casl2i2 polypeptide may comprise one or more conservative amino acid substitutions as compared with SEQ ID NO: 2. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

(b) Other Type V Nucleases

Other Type V nucleases can also be used in the gene editing systems and methods disclosed herein. Exemplary suitable Type V nucleases can be found in WO2019178427, WO202 1202800, WO2021050534, WO2022192391, W02024020567, WO2022192381, W02024020557, W02020018142, and WO2023039472, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein. Specific examples of Type V nucleases are provided in Table 1 above, all of which are within the scope of the present disclosure.

In some instances, the Type V nuclease may comprise an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, or at least 99% identical to SEQ ID NO: 3. Alternatively or in addition, the Type V nuclease may comprise one or more conservative amino acid substitutions relative to SEQ ID NO: 4. In specific examples, the Type V nuclease comprises the amino acid sequence of SEQ ID NO: 3 (Nuclease C listed in Table 1 below). In specific examples, the Type V nuclease comprises the amino acid sequence of SEQ ID NO: 4 (Nuclease D listed in Table 1 above).

Any of the Type V nuclease polypeptides provided herein may comprise one or more nuclear localization signals (NLS), for example, at the N-terminus, at the C-terminus, or both.

(ii) Preparation of CRISPR Nucleases

In some embodiments, any suitable nucleases (e.g., CRISPR nucleases) for use in gene editing as known in the art or disclosed herein such as a Type V nuclease (e.g., a Casl2i2 polypeptide or other Type V nucleases as disclosed herein) can be prepared by (a) culturing host cells such as bacteria cells or mammalian cells, capable of producing the proteins, isolating the proteins thus produced, and optionally, purifying the proteins. The nucleases can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the nuclease from bacteria, constructing a recombinant expression vector, and then transferring the vector into an appropriate host cell that expresses guide RNAs that complexes with the nucleases in the host cell. Alternatively, the nuclease can be prepared by (c) an in vitro coupled transcription-translation system and then complexes with guide RNAs.

Unless otherwise noted, all compositions and complexes and polypeptides provided herein are made in reference to the active level of that composition or complex or polypeptide, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources. Enzymatic component weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated. In the exemplified composition, the enzymatic levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the ingredients are expressed by weight of the total compositions.

In some embodiments, a host cell described herein is used to express the Type V nuclease. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used.

After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production of the Type V nuclease. After expression of the nuclease and/or the guide RNA, the host cells can be collected and the nuclease purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).

A variety of methods can be used to determine the level of production of a mature Type V nuclease in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the proteins or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).

B. Guide RNAs

Any of the gene editing systems disclosed herein comprises a guide RNA (gRNA) targeting a genomic site of interest, leading to gene editing at the desired genomic site. The gRNA mediate cleavage of a target sequence at the genomic site via the CRISPR nuclease also contained in the gene editing system. The gRNA comprises a nuclease binding sequence (a direct repeat sequence) and a DNA-binding sequence (a spacer). The nuclease binding sequence may comprise one or more binding sites that can be recognized by the CRISPR nuclease for binding. In some instances, the gRNA is a single RNA molecule comprising both the nuclease binding sequence and a spacer. In some instances, the RNA guide disclosed herein may further comprise a linker sequence, a 5’ end and/or 3’ end protection fragment (see disclosures herein), or a combination thereof.

As used herein, the terms “RNA guide” or “RNA guide sequence” refer to an RNA molecule that facilitates the targeting of a CRISPR nuclease described herein to a genomic site of interest. For example, an RNA guide can be a molecule that recognizes (e.g., binds to) a site in a non-PAM strand that is complementary to a target sequence in the PAM strand, e.g., designed to be complementary to a specific nucleic acid sequence. An RNA guide comprises a spacer and a nuclease binding sequence (e.g., a direct repeat (DR) sequence). The terms CRISPR RNA (crRNA), pre-crRNA and mature crRNA are also used herein to refer to an RNA guide. The 5’ end or 3’ end of an RNA guide may be fused to an RT donor RNA as disclosed herein.

As used herein, the term “protospacer adjacent motif’ or “PAM sequence” refers to a DNA sequence adjacent to a target sequence. In some embodiments, a PAM sequence is required for enzyme activity. In a double-stranded DNA molecule, the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non- PAM strand.” The RNA guide binds to a site in the non-PAM strand that is complementary to a target sequence disclosed herein, and the PAM sequence as described herein is present in the PAM-strand.

As used herein, the term “PAM strand” refers to the strand of a target nucleic acid (double-stranded) that comprises a PAM motif. In some embodiments, the PAM strand is a coding (e.g., sense) strand. In other embodiments, the PAM strand is a non-coding (e.g., antisense strand). The term “non-PAM strand” refers to the complementary strand of the PAM strand.

A guide RNA typically comprises a spacer sequence and a scaffold sequence. The spacer sequence (a.k.a., a DNA-binding sequence) is the RNA equivalent of the target sequence (a DNA sequence). The spacer contains a sequence capable of binding to the non- PAM strand via base-pairing at the site complementary to the target sequence (in the PAM strand). Such a spacer is also known as specific to the target sequence. In some instances, the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequence difference. In some instances, the spacer may be 100% identical to the target sequence except for the RNA-DNA sequence difference. The scaffold sequence comprises a motif recognizable by a nuclease (e.g., a Type V nuclease as disclosed herein). As used herein, the term “target sequence” refers to a DNA fragment adjacent to a PAM motif (on the PAM strand). The complementary region of the target sequence is on the non-PAM strand. A target sequence may be immediately adjacent to the PAM motif. Alternatively, the target sequence and the PAM may be separated by a small sequence segment (e.g., up to 5 nucleotides, for example, up to 4, 3, 2, or 1 nucleotide). A target sequence may be located at the 3’ end of the PAM motif or at the 5’ end of the PAM motif, depending upon the CRISPR nuclease that recognizes the PAM motif, which is known in the art. For example, a target sequence is located at the 3’ end of a PAM motif for a Casl2i polypeptide (e.g., a Casl2i2 polypeptide such as those disclosed herein) or the other Type V nucleases such as those disclosed herein.

As used herein, the term “complementary” refers to a first polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a second polynucleotide (e.g., the complementary sequence of a target sequence) such that the first and second polynucleotides can form a double-stranded complex via base-pairing to permit an effector polypeptide (e.g., a Type V nuclease or a variant thereof) that is complexed with the first polynucleotide to act on (e.g., cleave) the second polynucleotide. In some embodiments, the first polynucleotide may be substantially complementary to the second polynucleotide, i.e., having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second polynucleotide. In some embodiments, the first polynucleotide is completely complementary to the second polynucleotide, i.e., having 100% complementarity to the second polynucleotide.

(i) DNA-Binding Sequence (Spacer)

The RNA guide comprises a DNA-binding sequence (also known as a spacer). A spacer may have a length of from about 10 nucleotides to about 30 nucleotides. For example, the spacer can have a length of from about 10 nucleotides to about 25 nucleotides, from about 10 nucleotides to about 20 nucleotides. In some examples, the spacer may range from 15-25 nucleotides, for example 18-20 nucleotides.

In some examples, the spacer in the RNA guide may be generally designed to have a length of between 15 and 30 nucleotides or between 15 and 25 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and be complementary to a specific target sequence.

In some embodiments, the DNA-binding sequence has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a target sequence as described herein and is capable of binding to the complementary region of the target sequence via basepairing.

In some embodiments, the DNA-binding sequence comprises only RNA bases. In some embodiments, the DNA-binding sequence comprises a DNA base (e.g., the spacer comprises at least one thymine). In some embodiments, the DNA-binding sequence comprises RNA bases and DNA bases (e.g., the DNA-binding sequence comprises at least one thymine and at least one uracil).

The spacer in any of the RNA guides disclosed herein can be specific to a target sequence, i.e., capable of binding to the complementary region of the target sequence via base-pairing. In some instances, the target sequence may be within a genomic site of interest, e.g., where gene editing is needed. In some instances, the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequence difference. In some instances, the spacer may be 100% identical to the target sequence except for the RNA-DNA sequence difference.

In some embodiments, the target sequence is adjacent to a PAM sequence. PAM sequences are known in the art. In some embodiments, PAM sequences capable of being recognized by a CRISPR nuclease are disclosed in WO2021055874, W02020206036, W02020191102, WO2020186213, W02020028555, W02020033601, WO2019126762, WO2019126774, W02019071048, WO2019018423, W02019005866, WO2018191388, WO2018170333, WO2018035388, WO2018035387, WO2017219027, WO2017189308, WO2017184768, WO2017106657, WO2016205749, W02017070605, WO2016205764, W02016205711, WO2016028682, WO2015089473, WO2014093595, WO2015089427, WO2014204725, W02015070083, WO2014093655, WO2014093694, WO2014093712, WO2014093635, WO2021133829, W02021007177, WO2020197934, W02020181102, W02020181101, W02020041456, W02020023529, W02020005980, W02019104058, W02019089820, W02019089808, W02019089804, WO2019089796, WO2019036185, WO2018226855, WO2018213351, WO2018089664, WO2018064371, WO2018064352, WO2017106569, WO2017048969, WO2016196655, WO2016106239, WO2016036754, W02015103153, WO2015089277, WO2014150624, WO2013176772, WO2021119563, WO2021118626, WO2020247883, WO2020247882, WO2020223634, WO2020142754, W02020086475, W02020028729, WO2019241452, WO2019173248, WO2018236548, WO2018183403, WO2017027423, WO2018106727, WO2018071672, WO2017096328, W02017070598, W02016201155, WO2014150624, WO2013098244, WO2021113522, W02021050534, WO2021046442, WO2021041569, W02021007563, WO2020252378, W02020180699, W02020018142, WO2019222555, WO2019178428, WO2019178427, or W02019006471, the relevant disclosures of each of which are incorporated for the subject matter and purpose referenced herein.

When the gene editing system comprises a Casl2i polypeptide, the PAM sequence may comprise 5’-NTTN-3’ (or 5’-TTN-3’) wherein N is any nucleotide (e.g., A, G, T, or C). The PAM sequence is upstream to the target sequence. The PAM sequence in association with other CRISPR nucleases may comprises the sequence 5’-TTY-3’ or 5’-TTB-3’, wherein Y is C or T, and B is G, T, or C. The PAM sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence.

When the gene editing system comprises the other Type V nuclease as disclosed herein, the PAM comprises the motif of 5’-NTTR-3’, in which N is any of A, T, G, and C; and R is A or G. The PAM motif is located 5’ to the target sequence.

(ii) Nuclease Binding Sequence (Direct Repeat)

In some embodiments, the nuclease binding sequence in a gRNA disclosed herein is a CRISPR nuclease binding sequence (e.g., the nuclease binding sequence is capable of binding to a Type V nuclease). In some embodiments, the nuclease binding sequence comprises a direct repeat sequence. In certain embodiments, the nuclease binding sequence includes a direct repeat sequence linked to a DNA-binding sequence (e.g., a DNA-targeting sequence or spacer). In some embodiments, the nuclease binding sequence includes a direct repeat sequence and a DNA-binding sequence or a direct repeat- DNA-binding sequence - direct repeat sequence. In some embodiments, the nuclease binding sequence includes a truncated direct repeat sequence and a DNA-binding sequence, which is typical of processed or mature crRNA.

In the embodiments where the nuclease binding sequence is a direct repeat for a publicly available CRISPR nuclease, those direct repeat sequences are known in the art. In some embodiments, direct repeat sequences capable of binding a CRISPR nuclease are any of those disclosed in WO2021055874, W02020206036, W02020191102, WO2020186213, W02020028555, W02020033601, WO2019126762, WO2019126774, W02019071048, WO2019018423, W02019005866, WO2018191388, WO2018170333, WO2018035388, WO2018035387, WO2017219027, WO2017189308, WO2017184768, WO2017106657, WO2016205749, W02017070605, WO2016205764, W02016205711, WO2016028682, WO2015089473, WO2014093595, WO2015089427, WO2014204725, WO2015070083, WO2014093655, WO2014093694, WO2014093712, WO2014093635, WO2021133829, W02021007177, WO2020197934, W02020181102, W02020181101, W02020041456, W02020023529, W02020005980, W02019104058, W02019089820, W02019089808, W02019089804, WO2019089796, WO2019036185, WO2018226855, WO2018213351, WO2018089664, WO2018064371, WO2018064352, WO2017106569, WO2017048969, WO2016196655, WO2016106239, WO2016036754, W02015103153, WO2015089277, WO2014150624, WO2013176772, WO2021119563, WO2021118626, WO2020247883, WO2020247882, WO2020223634, WO2020142754, W02020086475, W02020028729, WO2019241452, WO2019173248, WO2018236548, WO2018183403, WO2017027423, WO2018106727, WO2018071672, WO2017096328, W02017070598, W02016201155, WO2014150624, WO2013098244, WO2021113522, W02021050534, WO2021046442, WO2021041569, W02021007563, WO2020252378, W02020180699, W02020018142, WO2019222555, WO2019178428, WO2019178427, or W02019006471, which are incorporated by reference for the subject matter and purpose referenced herein.

In some embodiments, the direct repeat sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-40 nucleotides (e.g., 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, or 40 nucleotides).

Example direct repeat sequences and their corresponding CRISPR nucleases are provided in Table 2 below.

Table 2. Exemplary Direct Repeat Sequences (iu)Modification of Nucleic Acids

Any of the gRNAs in the gene editing systems disclosed herein may include one or more modifications.

Exemplary modifications can include any modification to the sugar, the nucleobase, the intemucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.

The gRNAs may include any useful modification, such as to the sugar, the nucleobase, or the intemucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the intemucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.

In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

Different sugar modifications, nucleotide modifications, and/or intemucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the fimction of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

In some embodiments, sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural intemucleoside linkages such as intemucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its intemucleoside backbone.

Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3 ’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3 ’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 ’-5’ linkages, 2 ’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged.

The modified nucleotides, which may be incorporated into the sequence, can be modified on the intemucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another intemucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfiir (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.

In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5’-O-(l-thiophosphate)-adenosine, 5 ’-O-(l -thiophosphate) -cytidine (a-thio-cytidine), 5’-O-(l-thiophosphate)-guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’-O-(l- thiophosphate)-pseudouridine).

Other intemucleoside linkages that may be employed according to the present invention, including intemucleoside linkages which do not contain a phosphorous atom, are described herein.

In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5 -azacytidine, 4’-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, l-(2-C-cyano-2-deoxy-beta-D-arabino- pentofiiranosyl)-cytosine, decitabine, 5 -fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafiir ((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine- 2,4(1 H,3H)-dione), troxacitabine, tezacitabine, 2’-deoxy-2’-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-l- beta-D-arabinofuranosylcytosine, N4-octadecyl- 1 -beta-D-arabinofuranosylcytosine, N4- palmitoyl-l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofiiranosyl) cytosine, and P-4055 (cytarabine 5 ’-elaidic acid ester).

In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly- A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5- aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethylpseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2 -thio-uridine, 1 -taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l -methylpseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2 -methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2- thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5 -aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5 -hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l- methyl-l-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l- methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6-diaminopurine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2 -methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, 1 -methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine. The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally -occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self’. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.

In some embodiments, any gRNAs described herein may comprise an end modification (e.g., a 5’ end modification or a 3’ end modification). In some embodiments, the end modification is a chemical modification. In some embodiments, the end modification is a structural modification. See disclosures herein. In specific examples, the gRNAs may comprise 2’-o-methylation and phosphorothioate linkages, for example, at the 5’ and/or the 3’ end.

C. Donor DNA Template

The gene editing system further comprises a donor DNA template, which comprises a transgene flanked by a left homology arm and a right homology arm. As used herein, a transgene refers to any exogenous nucleic acid to be inserted into the genome of host cells.

In some instances, the transgene may comprise one or more open reading frames (ORFs) encoding polypeptides of interest. In some examples, the donor DNA template may carry one or more suitable promoters for driving expressing of the encoded polypeptides. Alternatively, the donor DNA template may not carry promoters associated with the coding sequence(s). In that case, the transgene may be inserted at a genomic site close to an endogenous promoter in host cells and the expression of the encoded polypeptides may be under the control of the endogenous promoter.

In other instances, the transgene may comprise a fragment of a gene, which, upon inserting into a genomic site of interest, forms a whole gene with either the upstream sequence or the downstream sequence at the insertion site to restore gene activity. For example, the transgene may comprise a fragment corresponding to a portion of a disease- associated gene where mutations occur. The transgene may contain variations relative to the disease-associated gene with the mutations fixed. Once inserted at a genomic site within the disease-associated gene, the transgene forms a functional whole gene with either the upstream sequence or downstream sequence at the insertion site to restore proper ftmction of the target gene, thereby benefiting treatment of the associated disease (e.g., a genetic disease).

The gene editing system provided herein can insert transgenes with a wide variety of sizes into a genomic site of interest, for example, ranging from 0.5 kb to 100 kb. In some instances, the transgene is at least 2-kb in size, e.g., at least 5-kb, at least 10-kb, at least 20- kb, at least 30-kb, at least 40-kb, at least 50-kb, at least 60-kb, at least 70-kb, at least 80-kb, at least 90-kb, or larger.

The transgene may be of a suitable size, taking into consideration the packaging capacity of the vectors that carry the transgene. In some instances, the transgene may be at least 1 kb, e.g., at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8kb, at least 9 kb, or larger. In case the donor DNA templated is carried by a vector, the size of the transgene would depend on the cloning capacity of the vector. For example, if an AAV vector is used, the transgene may be up to 4.5 kb in length (e.g., up to 4- kb or up to 3.5 -kb in length) given the capacity of AAV vectors.

In the donor DNA template, any of the transgenes disclosed herein is flanked by a left homology arm and a right homology arm, which comprise sequences homologous to sequences flanking the genomic site at which the transgene is to be inserted. For example, the left and right homology arms may be homologous to upstream and downstream sequences flanking the target sequence within the genomic site. The left and right homology arms may be of a suitable size, i.e., long enough to facilitate HMEJ-medicated DNA repair to allow for insertion of the transgene into the genomic site. In some instances, the homology arms may range from about 20-bp to about 1,000 bp. In some examples, the homology arms may range from about 150-bp to about 800 bp, e.g., about 300-bp to about 800-bp or about 600 bp to about 800-bp. In specific examples, the left and right homology arms are about 600-bp. In other examples, the homology arms are about 700-bp. In yet other examples, the homology arms are about 800-bp.

Any of the donor DNA template further comprises a first target sequence upstream to the left homology arm and a second target sequence downstream to the right homology arm. As used herein, the terms “upstream” and “downstream” refer to relative positions within a single nucleic acid (e.g., DNA) sequence. “Upstream” and “downstream” relate to the 5’ to 3’ direction, respectively, in which RNA transcription occurs (i.e., the 5’ to 3’ direction of the sense strand when the DNA molecule is double-stranded). A first sequence is upstream of a second sequence when the 3’ end of the first sequence occurs before the 5’ end of the second sequence. A first sequence is downstream of a second sequence when the 5’ end of the first sequence occurs after the 3’ end of the second sequence.

In some instances, the first and/or second target sequences may be located on the sense strand. In this case, the target sequence is at the 5’ end of the sense strand of the left homology arm and/or at the 3’ end of the sense strand of the right homology arm. Alternatively, the first and/or second target sequences may be located on the anti-sense strand. In that case, the target sequence is at the 3 ’ end of the anti-sense strand of the left homology arm and/or at the 5’ end of the anti-sense strand of the right homology arm. The donor DNA template also comprises a PAM motif at the 5’ end of the target sequence.

The first and second target sequences are either identical or substantially similar to the target sequence at the genomic site such as the same gRNA in the gene editing system can form base pairs with the complementary regions of the target sequences, thus recruiting the CRISPR nuclease to the target sequences. In some examples, the target sequences in the donor DNA template are identical to the target sequence at the genomic site.

In some instances, the target sequences in the donor DNA template may be adjacent to the left homology arm and/or the right homology arm. As used herein, the term “adjacent to” refers to a nucleotide sequence in close proximity to another nucleotide sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by up to 2 nucleotides, up to 5 nucleotides, up to 8 nucleotides, up to 10 nucleotides, up to 12 nucleotides, or up to 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by 2-5 nucleotides, 4-6 nucleotides, 4-8 nucleotides, 4-10 nucleotides, 6-8 nucleotides, 6-10 nucleotides, 6-12 nucleotides, 8-10 nucleotides, 8-12 nucleotides, 10-12 nucleotides, 10-15 nucleotides, or 12-15 nucleotides.

In some embodiments, the donor DNA template may be located on a vector, for example, a viral vector. Viral vector technology is well known in the art and described in a variety of virology and molecular biology manuals. Viruses useftil as vectors include, but are not limited to, phage viruses, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

In some examples, the vector can be an adeno-associated vector (AAV), which can deliver the donor DNA template to host cells. The serotype of the AAV vector shall depend on the host cells of interest, which is within the knowledge of those skilled in the art.

In some instances, the vector comprising the donor DNA template may farther comprise a coding sequence for the guide RNA. In some examples, the gRNA coding sequence may be in operable linkage to a suitable promoter. Examples include, but are not limited to, a U6 promoter, a Pol II promoter, or a Pol III promoter.

In some examples, the donor DNA template may be a double-stranded DNA molecule. In other examples, the donor DNA template may be a single-stranded DNA molecule.

D. Exemplary HMEJ-Mediated Gene Editing Systems

The exemplary HMEJ-mediated gene editing systems provided in this section are meant to be illustrative and by no means limits the scope of the present disclosure.

In some embodiments, the gene editing system provided herein comprises a CRISPR nuclease (e.g., the Type V nuclease such as the Casl2i2 nucleases disclosed herein) and a gRNA. In some instances, the CRISPR nuclease and the gRNA may form RNP complexes. Such a gene editing system may farther comprise the donor DNA template, which may be located on a viral vector, for example, an AAV vector.

In some embodiments, the gene editing system provided herein may comprise a CRISPR nuclease (e.g., the Type V nuclease disclosed herein) and a vector, which comprises a donor DNA template as disclosed herein and a nucleotide sequence encoding a gRNA. The gRNA-coding sequence may be in operable linkage to a suitable promoter, for example, a U6 promoter. In some instances, the vector can be a viral vector such as an AAV vector.

In some embodiments, the gene editing system provided herein may comprise a fast vector that comprises a nucleotide sequence encoding the CRISPR nuclease (e.g., the Type V nuclease disclosed herein), the coding sequence being in operable linkage to a suitable promoter. The gene editing system may farther comprise a second vector, which comprises a donor DNA template as disclosed herein and a nucleotide sequence encoding a gRNA. The gRNA-coding sequence may be in operable linkage to a suitable promoter, for example, a U6 promoter. In some instances, the first vector and/or the second vector can be a viral vector such as an AAV vector.

In some embodiments, the gene editing system provided herein may comprise a messenger RNA encoding the CRISPR nuclease (e.g., the Type V nuclease such as the Casl2i2 nucleases disclosed herein). The mRNA may be complexed with lipid excipients. In some instances, the mRNA may be attached to or encapsulated by lipid nanoparticles. The gene editing system may further comprise a vector, which comprises a donor DNA template as disclosed herein and a nucleotide sequence encoding a gRNA. The gRNA-coding sequence may be in operable linkage to a suitable promoter, for example, a U6 promoter. In some instances, the vector can be a viral vector such as an AAV vector.

In some embodiments, the gene editing system provided herein may comprise a messenger RNA encoding the CRISPR nuclease (e.g., the Type V nuclease such as the Casl2i2 nuclease disclosed herein) and a gRNA. The mRNA and optionally the gRNA may be complexed with lipid excipients. In some instances, the mRNA and optionally the gRNA may be attached to or encapsulated by lipid nanoparticles. The gene editing system may further comprise a vector, which comprises a donor DNA template as disclosed herein. In some instances, the vector can be a viral vector such as an AAV vector.

II. Gene Editing Methods

In some aspects, provided herein are gene editing methods for inserting transgenes, such as large transgenes at a genomic site of interest in host cells via, for example, homology- mediated end joining. Any of the gene editing systems provided herein can be used in the gene editing methods also provided herein.

A. Host Cells

Any of the gene editing systems disclosed herein can be delivered to a variety of cells to genetically edit a target gene, e.g., inserting a transgene into a genomic site of interest via, e.g., HMEJ.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell.

The gene editing system provided herein may be delivered to any type of host cells where gene editing is needed. In some embodiments, the host cells can be in vitro cultured cells. In other embodiments, the host cells may be located in the body of a subject such as a human subject.

In some instances, the host cells may be obtained from a subject (e.g., a human patient). In some instances, the host cells can be primary cells obtained from a subject such as a human subject. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. For example, in some embodiments, the differentiated cell is a liver cell (e.g., a hepatocyte), a biliary cell (e.g., a cholangiocyte), a stellate cell, a Kupffer cell, a liver sinusoidal endothelial cell, a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a fibroblast, or a sex cell. In some embodiments, the cell is a terminally differentiated cell. For example, in some embodiments, the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is a Tumor Infiltrating Lymphocyte (TIL). In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism.

In some instances, the host cells may be terminally differentiated cells, which may be obtained from a subject such as a human patient. In some examples, the host cells can be nondividing cells.

Alternatively, the host cells may be a cell line. In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell.

B. Delivery of Gene Editing System to Host Cells

In some embodiments, any of the gene editing systems or components thereof as disclosed herein may be delivered to host cells by methods known in the art. Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, microprojectile bombardment (“gene gun”), fagene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.

In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the Type V CRISPR nuclease, nucleic acids encoding the gRNA, and/or the donor DNA template), one or more transcripts thereof, and/or a pre-formed ribonucleoprotein to a cell. Exemplary intracellular delivery methods, include, but are not limited to: viruses or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnetofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, the present application farther provides cells produced by such methods, and organisms (such as animals, plants, or fangi) comprising or produced from such cells. In some embodiments, the gene editing system can be farther delivered with an agent (e.g., compound, molecule, or biomolecule) that affects DNA repair or DNA repair machinery. In some embodiments, a composition of the present invention is farther delivered with an agent (e.g., compound, molecule, or biomolecule) that affects the cell cycle.

In some embodiments, the gene editing system provided herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome or lipid nanoparticle, and delivered by known methods to a host cell.

In some embodiments, the components of the gene editing system, the CRISPR nuclease such as the Type V nuclease provided herein, the gRNA, and the donor DNA template, may be formulated into multiple compositions, each of which can be delivered to host cells by methods suitable for each composition. Alternatively, the components of the gene editing system may be formulated in one composition, which can be delivered to host cells via a suitable method as known to those skilled in the art.

In some embodiments, the host cells may be located in the body of a subject (e.g., a human subject). For example, the gene editing system may be administered to a subject in need of the designed gene editing via a suitable route.

In other examples, the gene editing system provided herein comprises a CRISPR nuclease (e.g., any of the Type V nucleases listed in Table 1 above), a gRNA and a vector (e.g., a viral vector such as an AAV vector) carrying a donor DNA template. The CRISPR nuclease and the gRNA may form an RNP complex, which can be delivered to host cells where gene editing is needed. The donor DNA template can be delivered to host cells via AAV virus infection.

In some examples, the gene editing system provided herein may comprise a messenger RNA (mRNA) encoding the CRISPR nuclease (e.g., any of the Type V nucleases listed in Table 1 above), a gRNA and a vector (e.g., a viral vector such as an AAV vector) carrying a donor DNA template. The mRNA and the gRNA may be formulated with lipid excipients, which may form LNPs. The mRNA and gRNA may be associated with or encapsulated by the LNPs. In this setting, the donor DNA template can be delivered to host cells (e.g., administer to a subject in need of the treatment via a suitable route) via AAV virus infection and the nuclease-encoding mRNA and gRNA can be delivered to host cells (e.g., administer to the subject in need of the treatment via a suitable route) via LNP -mediated delivery.

Any of the genetically modified cells produced using any of the gene editing system disclosed herein is also within the scope of the present disclosure.

C. Therapeutic Applications

Any of the gene editing systems or modified cells generated using such a gene editing system as disclosed herein may be used for treating a disease associated with the gene targeted by the gene editing system.

In some embodiments, provided herein is a composition comprising the gene editing system disclosed herein or components thereof. Such a composition can be a pharmaceutical composition. A pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation for a suitable delivery route, e.g., parenteral, intra-lesional, intra-organ or another route of administration. A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

A formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may fiirther comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

III. Kits for HMEJ-Medicated Gene Editing

The present disclosure also provides kits that can be used, for example, to carry out a gene editing method described herein for genetical editing of a target gene as disclosed herein. In some embodiments, the kits include an RNA guide or its encoding nucleic acid, a CRISPR nuclease (e.g., the Type V nuclease provided herein) or its encoding nucleic acid, and a donor DNA template as also disclosed herein. In some embodiments, the kits include components of the exemplary gene editing systems provided above.

The CRISPR nuclease (or polynucleotide encoding the CRISPR nuclease), the RNA guide or the encoding nucleic acid, and the donor DNA template can be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The components of the gene editing system provided herein may be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use.

The kits can additionally include, optionally, a buffer and/or instructions for use of the RNA guide, the CRISPR nuclease, and the donor DNA template.

General techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained hilly in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: apractical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (1RL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

Example 1: Plasmid Design for Testing Casl2i2-Mediated Knock-In in Human Cells

This Example describes the design of plasmid DNA donors used for testing different knock-in methods in human cells.

Five different types of donor constructs were designed: 1-cut HITI (homology independent target integration) donors, 1-cut MITI (microhomology dependent target integration) donors, 2-cut MITI donors, HMEJ (homology mediated end joining) donors, and HR (homologous recombination) donors. Designs of these donor constructs are illustrated in FIG. 1.

The HITI donor integrates into the target sequence by first generating a double stranded break, via a dsDNA nuclease or paired nickase, and ligating directly end-to-end to the two liberated blunt ends from the break. This HITI integration method frequently generates indels at the two ligation points at each end of the donor sequence due to the imprecise nature of NHEJ repair. To construct 1-cut HITI donors, either SpCas9 or Casl2i2 guide RNA sequences (DR+spacer) targeting the EMX1 gene were cloned into a guide expression plasmid following the U6 promoter. Upstream of the U6 promoter, a unique 890 bp sequence expressing the mCherry fluorescent protein and other exogenous sequence was inserted which resulted in a total plasmid size of 3.3 kb. One EMX1 target site was placed upstream of the mCherry sequence, including a compatible PAM and 20 bp spacer.

For Casl2i2 donors, which has a staggered PAM-distal cut outside the spacer sequence, 3 additional bp matching the corresponding genomic sequence were added after the target site. This design was intended to bias integration for the desired forward orientation — if insertion in the undesired reverse orientation occurs, the target site is reconstituted, and the insertion product is re-cut. The expected insertion transgene inserted by these constructs is the entire plasmid (3.3 kb). After nuclease cutting the full plasmid sequence is integrated into the genomic target site as illustrated in FIG. 1.

The MITI donors, unlike the HITI donors, contain nuclease or nickase target sites that result in double stranded breaks that have defined sequence overhangs that are designed to match to complementary overhangs generated at the genomic target site. The ‘sticky ends’ are able to anneal after ligation, sealing the breaks and leaving a precise integration. 1 -cut MITI donors were designed as similarly to HITI donors, except that the PAM-distal ends of the MITI donor target sites were designed to be reverse complements of the overhang predicted to be made by the genomic cut (FIG. 1). The intention of this design is to take advantage of the ability of Casl2i2 to generate staggered cuts, allowing for MITI — complementary “sticky ends” to facilitate the precise and efficient integration of the donor without reconstituting the cut site, leading to stable integration. Similar to 1-cut HITI donor, the full plasmid sequence is integrated into the genomic target site as illustrated in FIGs. 1A- 1C.

To construct 2-cut MITI donors, a 2.2 kb transgene sequence (in order 5’ to 3’: a kanamycin resistance gene (KanR), the UbC promoter, and a sequence encoding the mCherry protein) was flanked by 2 EMX1 MITI target sites and was cloned into the guide expression vector. The expected transgene insertion size for these donors is 2.2 kb (FIGs. 1A-1C).

Because blunt ligation via NHEJ competes with MITI “sticky end” repair, a mix of precise and imprecise integrations are typically observed. The HMEJ donors provide the most potential precision by including homology arms (sequence matching the left/right flanking sequence at the genomic target) to guide precisely how and where the transgene should be integrated. To construct HMEJ donors, the 2.2 kb transgene sequence described above for the 2-cut MITI donors was flanked by both left and right homology arms of various sizes (20, 150, 300, 600, or 800 bp) which matched the genomic sequence flanking the EMX1 target site. EMX1 target nuclease cut sites were inserted immediately flanking the homology arms. The expected insertion size for these constructs is 2.2 kb. The HR donors were identical to the HMEJ donors, except that the EMX1 target sites were replaced with a non-target control sequence not recognized by the EMX1 gRNA. Thus, the HMEJ and HR donors primarily differ in the presence of nuclease cut sites at the outer borders of the homology arms. HMEJ contains the cut sites, resulting in a linear DNA strand comprising a released linear DNA sequence of: homology arm - transgene - homology arm. The cut sites are absent in the HR donors and thus, rather than be excised out of the donor vector like with HMEJ, the homology arm - transgene - homology arm donor template is retained in the vector context, whether it be in an AAV, plasmid, dog bone, etc.

In order to express the nucleases that create the double stranded breaks, mammalian expression plasmids were created to express the Casl2i2 A and Nuclease C (see Table 1 above), as well as the SpCas9 nuclease, from the CMV promoter.

Table 3. Donor Template comparison for Knock-In in HEK293 cells

Example 2: Casl2i2-Mediated Knock-In in HEK293 Cells

This Example describes the experimental approach and outcomes of testing different knock-in methods in human cell culture. One donor plasmid and one nuclease expression plasmid (SpCas9 or Casl2i2vl.l) were co-transfected into HEK293 cells using Lipofectamine 2000. Experimental groups are listed in Table 3 above. After 3 days, cells were harvested, and genomic DNA was purified (Qiagen DNeasy 96 Blood and Tissue Kit). Knock-in efficiency was measured by ddPCR using primers and probes specific for the expected integration at the EMX target, normalized to a non-targeted EMX1 reference sequence.

Both the HMEJ and HR donor designs outperformed the HITI and MITI designs regardless of homology arm length and which nuclease was used to perform the double stranded break (FIG. 2). Increasing lengths of the homology arms led to increasing amounts of integration up to a homology arm length of 600 bp, maintaining high levels of integration beyond. Additionally, the addition of targets to liberate transgene & homology arms from the vector (HMEJ) increases the level of integration up to 2-fold for SpCas9.

Example 3: Casl2i2-Mediated Knock-In iPSC-Derived Hepatocytes

This Example describes measuring the efficiency of different knock-in methods in non-dividing iPSC-derived hepatocytes.

SpCas9-expressing mRNA was purchased from Trilink, and Casl2i2 A-expressing mRNA was generated through in vitro transcription, adding a 5’ cap and 3’ polyA. Synthetic guide RNAs targeting the EMX1 gene were purchased from IDT for both nucleases (see gRNA information in Table 4 below).

Wild-type iPSCs were differentiated in 96-well plates for 3 weeks into HLCs (hepatocyte-like cells) using a standardized protocol. Successful differentiation was confirmed by measuring the mRNA expression of the hepatocyte maturity markers albumin (ALB), alpha-1 antitrypsin (Al AT), and hepatocyte nuclear factor 4 alpha (HNF4A) by qPCR.

HLCs were then co-transfected with 500 ng donor plasmid, 250 ng nuclease mRNA, and 250 ng synthetic guide RNA using a lipid-based transfection method. 12 days posttransfection, HLCs were frozen for later processing and analysis. Genomic DNA was purified, and integration efficiency was measured by ddPCR, using primers and probes specific for the expected integration at the EMX target, normalized to a non-targeted EMX1 reference sequence.

Measurable integration was only observed for the HR or HMEJ donors (FIG. 3), with a significantly higher level of integration seen using the HMEJ donor over the HR donor when Casl2i2 A was used as the nuclease.

Example 4: Nuclease C-Mediated Knock-In in Mouse Liver

This Example describes testing the efficiency of Nuclease C-mediated corrective gene knock-in at the TTR locus in a humanized mouse model of ATTR amyloidosis (CARD1194, Kumamoto University, Zhao et al., Genes to Cells 2008) containing the pathogenic V30M mutation. See Table 1 above for structural information of Nuclease C. This approach uses an AAV8 virus to deliver the HMEJ donor and uses an LNP (lipid nanoparticle) to deliver Nuclease C-encoding mRNA and guide RNA. Knock-in at TTR using Nuclease C was compared to knock-in at the Actb locus using SpCas9 as a control (Yao et al., Cell Research 2017).

To generate TTR targeting AAV8 donors for this study (FIG 4A), an AAV plasmid backbone containing a constitutive EGFP expression cassette was used as the starting point. Upstream of the EGFP cassette, the 1188 bp integration transgene sequence was inserted. The transgene sequence includes the part of the human TTR coding sequence found immediately downstream of the TTR target site in the humanized mouse model. The TTR sequence differs from the mouse model TTR in that 1) it corrects the disease-causing V30M mutation back to the wildtype V30; 2) it introduces the stabilizing T119M mutation; 3) it is codon-optimized to prevent any homology-related readout or recombination complications; 4) it is followed by a P2A-mCherry sequence designed to be expressed when the transgene in integrated in frame at the humanized TTR locus. Left and right homology arms — each 600 bp in length and matching the humanized mouse model TTR coding sequence on either side of the target site — were inserted flanking the transgene sequence. The recoded TTR sequence and homology arm sequences are provided in Table 4 below:

Table 4. Nucleotide Sequences for TTR Transgene and Homology Arms

Two TTR target sites for Nuclease C, including compatible PAM sequences, were inserted flanking the homology arms (FIG. 4A). This resulted in a construct for generating AAV donors for HMEJ-mediated knock-in at the humanized TTR locus using Nuclease C (Used in groups 3-6, Table 6).

A control HMEJ donor using SpCas9-mediated knock-in to integrate an mCherry reporter into the mouse Actb locus was used as a comparison (Groups 1-2, Table 5, Yao et al., Cell Research 2017).

All AAV plasmid constructs were sent to Vector Biolabs for plasmid scale-up and AAV8 generation. AAV8 concentrations were confirmed using a ddPCR quantification method. To generate the LNPs used in this study, a gRNA and an mRNA expressing a CRISPR nuclease were loaded into lipid nanoparticles according to the general procedures of Schoenmaker, IJPharm, 2021.

Animals were dosed with AAV8 virus (l*10^A12 vg/mouse) by retro-orbital injection, followed approximately 24 hrs later by LNP dosing at 1 mg/kg by retro-orbital injection. Details of treatment groups are described in Table 5. Donors with the correct V30M TTR mutation (groups 3-6) were dosed in CARD1194 mice, which are in C57bl/6 background and are homozygous for the disease-causing hTTR V30M mutation. The HMEJ donors from group 1 and 2 (not TTR targeting) were dosed in wildtype C57bl/6 mice. Control groups 1, 3, and 5 received donor AAV, but no LNP delivery. Seven days after LNP dosing, animals were euthanized and perfused with PBS. The left liver lobe was collected and frozen on dry ice for tissue processing and ddPCR analysis, while the remaining liver lobes were fixed in 10% neutral buffered formalin and sent for immunostaining at HistoWiz, Inc.

Table 5. Dosing Details of In Vivo Study Testing Knock-In into Mouse Liver

Structural information for the gRNAs used in this study is provided in Table 6 below.

Liver samples were embedded, sectioned, and stained by HistoWiz using a Standard Operating Procedure and fully automated workflow. Tissues were stained with an anti-GFP polyclonal antibody (A-l 1122, Invitrogen) and an anti-RFP antibody (600-401-379, Rockland), followed by secondary antibody staining and DAPI DNA staining. Whole tissue sections were imaged using Akoya Vectra Polaris slide scanner.

Images were quantified using Fiji (ImageJ) image analysis software. Nuclei ROIs were defined based on DAPI staining, then the percent of nuclei that were mCherry positive above a determined background threshold were counted.

Approximately 10% of the cells receiving the HMEJ donor AAV showed mCherry expression at either the mouse Actb locus or the humanized TTR E1T3 locus, indicating precise knock-in to both sites that put the mCherry in frame for expression from the genomic promoter. Without the nuclease present to create double stranded breaks in the genome, only background mCherry expression was observed.

Table 6. Guide RNA (gRNA) Sequences

OTHER EMBODIMENTS All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

WHAT IS CLAIMED IS:

1. A gene editing system for inserting a nucleic acid into a genomic site, comprising:

(a) a CRISPR nucleases or a first nucleic acid encoding the CRISPR nuclease;

(b) a guide RNA (gRNA) or a second nucleic acid encoding the gRNA, wherein the gRNA comprises (i) a spacer sequence specific to a target sequence in the genomic site, and (ii) a direct repeat sequence recognizable by the CRISPR nuclease; and

(c) a donor DNA template comprising (i) a transgene; (ii) a left homology arm upstream to the transgene and a right homology arm downstream to the transgene; and (iii) two copies of the target sequence, one being upstream to the left homology arm and the other being downstream to the right homology arm; wherein the left homology arm and the right homology arm are homologous to the upstream and downstream sequences flanking the target sequence in the genomic site.

2. The gene editing system of claim 1, wherein the CRISPR nuclease is a Type V nuclease.

3. The gene editing system of claim 2, wherein the Type V nuclease comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1; optionally wherein the Type V nuclease comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

4. The gene editing system of claim 3, wherein the direct repeat sequence in the gRNA comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 5; optionally wherein the direct repeat sequence is SEQ ID NO: 5.

5. The gene editing system of claim 2, wherein the Type V nuclease comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3; optionally wherein the Type V nuclease comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

6. The gene editing system of claim 5, wherein the direct repeat sequence in the gRNA comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6; optionally wherein the direct repeat sequence is SEQ ID NO: 6.

7. The gene editing system of any one of claims 1 -6, wherein the left and right homology arms are 20-1,000-bp in length; optionally about 600-800-bp in length.

8. The gene editing system of any one of claims 1 -7, wherein the transgene is at least 2 kb in size, optionally 2.5-3 kb in size.

9. The gene editing system of any one of claims 1 -8, wherein the donor DNA template is located on a vector, which optionally is a viral vector.

10. The gene editing system of any one of claims 1 -9, wherein the gene editing system comprise the first nucleic acid encoding the CRISPR nuclease.

11. The gene editing system of claim 10, wherein the first nucleic acid is an expression vector.

12. The gene editing system of claim 11, wherein the expression vector further comprises the second nucleic acid encoding the gRNA.

13. The gene editing system of claim 10, wherein the first nucleic acid is a messenger RNA (mRNA).

14. The gene editing system of claim 13, wherein the gene editing system comprises lipid nanoparticles (LNPs), which are associated with the mRNA and optionally the gRNA.

15. The gene editing system of any one of claims 1 -9, wherein the gene editing system comprises the CRISPR nuclease.

16. The gene editing system of claim 15, wherein the CRISPR and the gRNA form a ribonucleoprotein (RNP) complex.

17. The gene editing system of any one of claims 1-16, wherein the genomic site is in a safe harbor locus.

18. The gene editing system of any one of claims 1-16, wherein the genomic site is in a gene associated with a disease.

19. A method for inserting a nucleic acid into a genomic site, the method comprising delivering into a mammalian host cell a gene editing system, which comprises:

(a) a CRISPR nucleases or a first nucleic acid encoding the CRISPR nuclease;

(b) a guide RNA (gRNA) or a second nucleic acid encoding the gRNA, wherein the gRNA comprises (i) a spacer sequence specific to a target sequence in the genomic site, and (ii) a direct repeat sequence recognizable by the CRISPR nuclease;

(c) a donor DNA template comprising (i) a transgene; (ii) a left homology arm upstream to the transgene and a right homology arm downstream to the transgene; and (iii) two copies of the target sequence, one being upstream to the left homology arm and the other being downstream to the right homology arm; wherein the left homology arm and the right homology arm are homologous to the upstream and downstream sequences flanking the target sequence in the genomic site; wherein the gene editing system cleaves at the target sequence in the genomic site and at the target sequences in the donor DNA template, and wherein the cleaved donor DNA template, which comprises the transgene, inserts into the genomic site via homology-mediated end joining.

20. The method of claim 19, wherein the gene editing system is set forth in any one of claims 2-18.

21. The method of claim 19 or claim 20, wherein the mammalian host cell is a human mammalian cell.

22. The method of any one of claims 19-21, wherein the mammalian cell is a nondividing mammalian cell.

23. The method of any one of claims 19-22, wherein the mammalian cell is in a cell culture.

24. The method of any one of claims 19-22, wherein the method comprises delivering the gene editing system to a subject in need thereof.

25. The method of claim 23, wherein the subject is a human patient