WO2024206620A1 - Messenger rna encoding casx - Google Patents

Abstract

Provided herein are messenger ribonucleic acid (mRNA) sequences encoding engineered CasX proteins for modifying a cell, in vitro, in vivo, or ex vivo. The mRNAs may be codon-optimized for expression in a human cell, and may be used with guide RNAs in systems for modifying target nucleic acids. Also provided are methods for delivery of the mRNA sequences and guide RNAs.

Description

MESSENGER RNA ENCODING CASX

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional applications 63/492,968 filed March 29, 2023, and 63/563,124 filed March 8, 2024, the contents of which are incorporated herein by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SCRB_056_01US_SeqList_ST26.xml; Size: 1,217,298 bytes; and Date of Creation: March 5, 2024) are herein incorporated by reference in its entirety.

BACKGROUND

[0003] The advent of CRISPR/Cas systems and the programmable nature of these minimal systems has facilitated their use as a versatile technology for genomic manipulation and engineering. However, current methods of delivery utilizing viral vectors such as AAV can result in long-lived expression and increased off-target editing, while delivery utilizing vectors comprising ribonucleoprotein (RNP) complexes can result in short-lived exposure and insufficient editing to achieve the desired therapeutic outcome.

[0004] The use of messenger RNA (mRNA) to encode CRISPR proteins is becoming an increasingly important approach for the delivery of such proteins for the treatment of a variety of diseases amenable to genetic editing. Lipid nanoparticles (LNP) are commonly used to encapsulate mRNA for efficient in vivo delivery of mRNA, as well as the guide ribonucleic acid (gRNA). Thus, there remains a need in the art for compositions of mRNAs encoding CRISPR proteins and methods of making such mRNAs, as well as systems of the mRNA and gRNA and formulations thereof.

SUMMARY

[0005] The present disclosure provides mRNA sequences encoding CasX proteins that have utility in the modification of a target nucleic acid of a gene in eukaryotic cells. The mRNAs can be formulated in lipid nanoparticles (LNP) for entry into target cells. The mRNAs are useful in a variety of methods for target nucleic acid modification, for which methods are also provided. Attorney Docket No. SCRB-056/02WO 333322-2431

INCORPORATION BY REFERENCE

[0006] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The contents of WO 2020/247882, WO 2020/247883, WO 2021/113772, WO 2022/120095, and WO 2023/235818, which disclose CasX variants and gRNA variants, and methods of delivering same, are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0008] FIG. 1 is a bar graph showing the quantification of percent editing measured as indel rate detected by next-generation sequencing (NGS) at the mouse PCSK9 locus in Hepal-6 cells transfected with the indicated engineered CasX mRNAs and targeting spacers and harvested at 20 hours post-transfection, as described in Example 1.

[0009] FIG. 2A is a plot illustrating the percent reduction of secreted PCSK9 level, relative to the non-targeting (NT) control, for primary human hepatocytes from lot #31 treated with the indicated doses of lipid nanoparticles (LNPs) formulated with CasX 515 or CasX 812 mRNA and a EGS' -targeting gRNA with spacer 6.1, as described in Example 2.

[0010] FIG. 2B is a plot illustrating the percent reduction of secreted PCSK9 level, relative to the non-targeting (NT) control, for primary human hepatocytes from lot #31 treated with the indicated doses of LNPs formulated with CasX 515 or CasX 812 mRNA and &PCSK9- targeting gRNA with spacer 6.8, as described in Example 2.

[0011] FIG. 2C is a plot illustrating the percent reduction of secreted PCSK9 level, relative to the non-targeting (NT) control, for primary human hepatocytes from lot #51 treated with the indicated doses of LNPs formulated with CasX 515 or CasX 812 mRNA and &PCSK9- targeting gRNA with spacer 6.1, as described in Example 2.

[0012] FIG. 2D is a plot illustrating the percent reduction of secreted PCSK9 level, relative to the non-targeting (NT) control, for primary human hepatocytes from lot #51 treated with Attorney Docket No. SCRB-056/02WO 333322-2431 the indicated doses of LNPs formulated with CasX 515 or CasX 812 mRNA and & PCSK9- targeting gRNA with spacer 6.8, as described in Example 2.

[0013] FIG. 3A is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the PCSK9 locus in primary human hepatocytes from lot #31 treated with the indicated doses of LNPs formulated with CasX 515 or CasX 812 mRNA and &PCSK9- targeting gRNA with spacer 6.8, as described in Example 2.

[0014] FIG. 3B is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the PCSK9 locus in primary human hepatocytes from lot #51 treated with the indicated doses of LNPs formulated with CasX 515 or CasX 812 mRNA and &PCSK9- targeting gRNA with spacer 6.1, as described in Example 2.

[0015] FIG. 3C is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the PCSK9 locus in primary human hepatocytes from lot #51 treated with the indicated doses of LNPs formulated with CasX 515 or CasX 812 mRNA and &PCSK9- targeting gRNA with spacer 6.8, as described in Example 2.

[0016] FIG. 4A is a schematic illustrating versions 1-3 of chemical modifications made to gRNA scaffold variant 235 (SEQ ID NO: 153), as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond. For the v2 profile, the addition of three 3’ uracils (3’UUU) is annotated with “U”s in the relevant circles.

[0017] FIG. 4B is a schematic illustrating versions 4-6 of chemical modifications made to gRNA scaffold variant 235 (SEQ ID NO: 153), as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0018] FIG. 5 is a plot illustrating the quantification of percent knockout of B2M in HepG2 cells co-transfected with 100 ng of CasX 491 mRNA and with the indicated doses of end- modified (vl) or unmodified (vO) 2 -targeting gRNAs with spacer 7.37, as described in Example 3. Editing level was determined by flow cytometry as the population of cells with loss of surface presentation of the HLA complex due to successful editing at the B2M locus.

[0019] FIG. 6 is a schematic illustrating versions 7-9 of chemical modifications made to gRNA scaffold variant 316 (SEQ ID NO: 154), as described in Example 3. Structural motifs Attorney Docket No. SCRB-056/02WO 333322-2431 are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0020] FIG. 7A is a schematic of gRNA scaffold variant 174 (SEQ ID NO: 152), as described in Example 3. Structural motifs are highlighted.

[0021] FIG. 7B is a schematic of gRNA scaffold variant 235 (SEQ ID NO: 153), as described in Example 3. Highlighted structural motifs are the same as in FIG. 6A. The differences between variant 174 and variant 235 lie in the extended stem motif and several single-nucleotide changes (indicated with asterisks). Scaffold variant 316 maintains the shorter extended stem from variant 174 but harbors the four substitutions found in scaffold 235.

[0022] FIG. 7C is a schematic of gRNA scaffold variant 316 (SEQ ID NO: 154), as described in Example 3. Highlighted structural motifs are the same as in FIG. 6A. Variant 316 maintains the shorter extended stem from variant 174 (FIG. 7 A) but harbors the four substitutions found in scaffold 235 (FIG. 7B).

[0023] FIG. 8 is a plot displaying a correlation between indel rate (depicted as edit fraction) at the PCSK9 locus as measured by NGS (x-axis) and secreted PCSK9 levels (ng/mL) detected by enzyme-linked immunosorbent assay (ELISA) (y-axis) in HepG2 cells lipofected with CasX 491 mRNA and ECS'AV-targeting gRNAs containing the indicated scaffold variant and spacer combination, as described in Example 3.

[0024] FIG. 9A is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the human 2 locus in HepG2 cells treated with the indicated doses of LNPs formulated with CasX 491 mRNA and the indicated 7>2A/-targeting gRNA, as described in Example 3.

[0025] FIG. 9B is a plot illustrating the quantification of percent knockout of B2M in HepG2 cells treated with the indicated doses of LNPs formulated with CasX 491 mRNA and the indicated 2A7-targeting gRNA, as described in Example 3. Editing level was determined by flow cytometry as population of cells that did not have surface presentation of the HLA complex due to successful editing at the B2M locus.

[0026] FIG. 10A is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the mouse ROSA26 locus in Hepal-6 cells treated with the indicated Attorney Docket No. SCRB-056/02WO 333322-2431 doses of LNPs formulated with CasX 676 mRNA #2 and the indicated R0SA26-targeting gRNA with either the vl or v5 modification profile, as described in Example 3.

[0027] FIG. 10B is a plot illustrating the quantification of percent editing measured as indel rate detected by NGS at the ROSA26 locus in mice treated with LNPs formulated with CasX 676 mRNA #2 and the indicated chemically-modified AOX426-targeting gRNA, as described in Example 3.

[0028] FIG. 11 is a bar graph showing the results of the editing assay measured as indel rate detected by NGS as the mouse PCSK9 locus in mice treated with LNPs formulated with CasX 676 mRNA #1 and the indicated chemically-modified PGS' -targeting gRNA, as described in Example 3. Untreated mice served as experimental control.

[0029] FIG. 12A is a schematic illustrating versions 1-3 of chemical modifications made to gRNA scaffold variant 316, as described in Example 3. Structural motifs are highlighted.

Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0030] FIG. 12B is a schematic illustrating versions 4-6 of chemical modifications made to gRNA scaffold variant 316, as described in Example 3. Structural motifs are highlighted.

Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

DETAILED DESCRIPTION

[0031] While exemplary embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosures claimed herein. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments of the disclosure. It is intended that the claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described Attorney Docket No. SCRB-056/02WO 333322-2431 herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

Definitions

[0033] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a host cell” includes two or more such host cells, reference to “an engineered CasX protein” includes one or more engineered CasX protein(s), reference to “a nucleic acid sequence” includes one or more nucleic acid sequences, and the like.

[0034] As used herein, the term “about” is understood by persons of ordinary skill in the art and may vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which the term “about” is used, “about” will mean up to plus or minus 10% of the particular term.

[0035] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also include any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 1-5 members refers to groups having 1, 2, 3, 4, or 5 members, and so forth.

[0036] The term “combinations thereof includes every possible combination of elements to which the term refers.

[0037] The term "exemplary" as used herein, refers to an example or illustration, and is not intended to imply any preference or value.

[0038] The term “CasX protein,” as used herein, refers to a family of proteins, including e.g., all naturally-occurring CasX proteins (“reference CasX”), as well as engineered CasX proteins with sequence modifications possessing one or more improved characteristics relative to a CasX protein from which it was derived, described more fully, herein below. [0039] The term “specificity” as used herein refers to the ratio of average level of on- targeting editing divided by the average level of off-target editing. Attorney Docket No. SCRB-056/02WO 333322-2431

[0040] The term “off-target effects” as used herein refers to unintended cleavage and mutations at untargeted genomic sites showing a similar but not an identical sequence compared to the target site. In some embodiments the off-target effects are determined in an in vitro cell-free assay. In some embodiments the off-target effects are determined in a cellbased assay.

[0041] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass singlestranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; doublestranded RNA; multi -stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0042] “Hybridizable” or “complementary” are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still hybridize to the target nucleic acid. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a 'bulge', ‘bubble’ and the like). Thus, the skilled artisan will understand that while individual bases within a sequence may not be complementary to another sequence, the sequence as a whole is still considered to be complementary.

[0043] A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (e.g., a protein, or an RNA), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene may include accessory element sequences including, but not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry Attorney Docket No. SCRB-056/02WO 333322-2431 sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. Coding sequences encode a gene product upon transcription or transcription and translation; the coding sequences of the disclosure may comprise fragments and need not contain a full-length open reading frame. A gene can include both the strand that is transcribed as well as the complementary strand containing the anticodons.

[0044] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0045] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0046] The term “adjacent to” with respect to polynucleotide or amino acid sequences refers to sequences that are next to, or adjoining each other in a polynucleotide or polypeptide. The skilled artisan will appreciate that two sequences can be considered to be adjacent to each other and still encompass a limited amount of intervening sequence, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or amino acids.

[0047] The term “regulatory element” is used interchangeably herein with the term “regulatory sequence,” and is intended to include promoters, enhancers, and other expression regulatory elements. It will be understood that the choice of the appropriate regulatory element will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0048] The term “accessory element” is used interchangeably herein with the term “accessory sequence,” and is intended to include non-coding sequences that enhance expression, trafficking of the nucleic acid, or the function of mRNA and include, inter alia, polyadenylation signals (poly(A) signal), enhancer elements, introns, posttranscriptional regulatory elements (PTREs), nuclear localization signals (NLS), deaminases, DNA glycosylase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g. in cis or in trans), self-cleaving sequences, and fusion Attorney Docket No. SCRB-056/02WO 333322-2431 domains, for example a fusion domain fused to a CRISPR protein. It will be understood that the choice of the appropriate accessory element or elements will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0049] The term "promoter" refers to a DNA sequence that contains a transcription start site and additional sequences to facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as a TATA box, and/or B recognition element (BRE) and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. A promoter can be proximal or distal to the gene to be transcribed. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties. A promoter of the present disclosure can include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter can be classified according to criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. A promoter can also be classified according to its strength. As used in the context of a promoter, “strength” refers to the rate of transcription of the gene controlled by the promoter. A “strong” promoter means the rate of transcription is high, while a “weak” promoter means the rate of transcription is relatively low.

[0050] A promoter of the disclosure can be a Polymerase II (Pol II) promoter. Polymerase II transcribes all protein coding and many non-coding genes. A representative Pol II promoter includes a core promoter, which is a sequence of about 100 base pairs surrounding the transcription start site, and serves as a binding platform for the Pol II polymerase and associated general transcription factors. The promoter may contain one or more core promoter elements such as the TATA box, BRE, Initiator (INR), motif ten element (MTE), downstream core promoter element (DPE), downstream core element (DCE), although core promoters lacking these elements are known in the art.

[0051] A promoter of the disclosure can be a Polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs such as the 5S rRNA, tRNAs, and Attorney Docket No. SCRB-056/02WO 333322-2431 other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed section of the gene) to support transcription, although upstream elements such as the TATA box are also sometimes used. All Pol III promoters are envisaged as within the scope of the instant disclosure.

[0052] The term “enhancer” refers to regulatory DNA sequences that, when bound by specific proteins called transcription factors, regulate the expression of an associated gene. Enhancers may be located in the intron of the gene, or 5’ or 3’ of the coding sequence of the gene. Enhancers may be proximal to the gene (z.e., within a few tens or hundreds of base pairs (bp) of the promoter), or may be located distal to the gene (z.e., thousands of bp, hundreds of thousands of bp, or even millions of bp away from the promoter). A single gene may be regulated by more than one enhancer, all of which are envisaged as within the scope of the instant disclosure.

[0053] As used herein, a “post-transcriptional regulatory element (PTRE),” such as a hepatitis PTRE, refers to a DNA sequence that, when transcribed creates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote expression of an associated gene operably linked thereto.

[0054] “Operably linked” means with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components; e.g., a promoter and an encoding sequence. The skilled artisan will appreciate that the two components need not be physically linked to be operably linked.

[0055] “Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA Attorney Docket No. SCRB-056/02WO 333322-2431 comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “enhancers” and “promoters”, above).

[0056] The term “recombinant polynucleotide” or “recombinant nucleic acid” refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0057] Similarly, the term “recombinant polypeptide” or “recombinant protein” refers to a polypeptide or protein which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a protein that comprises a heterologous amino acid sequence is recombinant.

[0058] The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Attorney Docket No. SCRB-056/02WO 333322-2431

[0059] “Dissociation constant”, or “Ka”, are used interchangeably and mean the affinity between a ligand “L” and a protein “P”; i.e., how tightly a ligand binds to a particular protein. It can be calculated using the formula Kd=[L] [P]/[LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively.

[0060] As used herein, "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, helper phospholipids, and PEG-modified lipids), as well as cholesterol. Specific components of LNP are described more fully, below. Lipid nanoparticles can be included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). The lipid nanoparticles of the disclosure can comprise a nucleic acid. Such lipid nanoparticles typically comprise neutral lipids, charged lipids, steroids and polymer conjugated lipids. The active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0061] As used herein, “lipid encapsulated” refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both. The nucleic acid (e.g., mRNA) can be fully encapsulated in the lipid nanoparticle.

[0062] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a target nucleic acid with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid are made to share a physical connection; e.g., can hybridize if the sequences share sequence similarity.

[0063] The disclosure provides systems and methods useful for editing a target nucleic acid sequence. As used herein “editing” is used interchangeably with “modifying” and "modification" and includes but is not limited to cleaving, nicking, deleting, knocking in, knocking out, and the like.

[0064] The disclosure provides compositions and methods useful for modifying a target nucleic acid. As used herein “modifying” includes but is not limited to cleaving, nicking, editing, deleting, knocking in, knocking out, and the like. Attorney Docket No. SCRB-056/02WO 333322-2431

[0065] By “cleavage” it is meant the breakage of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and doublestranded cleavage can occur as a result of two distinct single-stranded cleavage events.

[0066] The term "knock-out" refers to the elimination of a gene or the expression of a gene. For example, a gene can be knocked out by either a deletion or an addition of a nucleotide sequence that leads to a disruption of the reading frame. As another example, a gene may be knocked out by replacing a part of the gene with an irrelevant sequence. The term "knockdown" as used herein refers to reduction in the expression of a gene or its gene product(s). As a result of a gene knock-down, the protein activity or function may be attenuated or the protein levels may be reduced or eliminated.

[0067] As used herein, “homology-directed repair” (HDR) refers to the form of DNA repair that takes place during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, and uses a donor template to repair or knock-out a target DNA, leading to the transfer of genetic information from the donor to the target. Homology- directed repair can result in an alteration of the sequence of the target sequence by insertion, deletion, or mutation if the donor template differs from the target DNA sequence and part or all of the sequence of the donor template is incorporated into the target DNA.

[0068] As used herein, "non-homologous end joining" (NHEJ) refers to the repair of double-strand breaks in DNA by direct ligation of the break ends to one another without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). NHEJ often results in the loss (deletion) or insertion of nucleotide sequence near the site of the double- strand break.

[0069] As used herein "micro-homology mediated end joining" (MMEJ) refers to a mutagenic double-strand break repair mechanism, which always associates with deletions flanking the break sites without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). MMEJ often results in the loss (deletion) of nucleotide sequence near the site of the double- strand break.

[0070] A polynucleotide or polypeptide has a certain percent “sequence similarity” or “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, Attorney Docket No. SCRB-056/02WO 333322-2431 that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity (sometimes referred to as percent similarity, percent identity, or homology) can be determined in a number of different manners. To determine sequence similarity, sequences can be aligned using the methods and computer programs that are known in the art, including Basic Local Alignment Search Tool (BLAST), available over the world wide web at ncbi.nlm.nih.gov/BLAST. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). [0071] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues linked via peptide bonds and which may be composed of two or more polypeptide chains. Polypeptide chains can be of any length, and in some embodiments can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequences. Typically, the terms "polypeptide," "protein," and/or "peptide" refer to a polymer of at least two amino acid monomers joined together through peptide bonds. An amino acid may be the L-optical isomer or the D-optical isomer. More specifically, the terms "polypeptide," "protein," and "peptide" refer to a molecule composed of two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the protein. In some cases, a protein can be a portion of a larger protein, for example, a domain, a subdomain, a subunit, or a motif of the protein. In some cases, a protein can be a variant (or mutation) of a protein, wherein one or more amino acid residues are inserted into, deleted from, and/or substituted into the naturally occurring, or reference, amino acid sequence of a protein. A polypeptide can be a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Attorney Docket No. SCRB-056/02WO 333322-2431

Polypeptides can be modified, for example, by the addition of carbohydrate, phosphorylation, etc. Proteins can comprise one or more polypeptides.

[0072] A protein or a variant thereof can be naturally occurring or recombinant. Methods for detection and/or measurement of polypeptides in biological material are well known in the art and include, but are not limited to, Western-blotting, flow cytometry, ELIS As, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as e.g., an ELISA This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and/or measurement of polypeptides/proteins are described e.g., in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[0073] In the context of proteins, the term "fragment," or equivalent terms refers, as used herein refers to a portion of a protein that has less than the full length of the protein and optionally maintains one or more functions of the protein.

[0074] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e., an expression cassette, may be attached so as to bring about the replication or expression of the attached segment in a cell.

[0075] The term “naturally-occurring” or “unmodified” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.

[0076] As used herein, a “mutation” refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides as compared to a wildtype or reference amino acid sequence or to a wild-type or reference nucleotide sequence. [0077] As used herein the term “isolated” is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0078] A “host cell,” as used herein, denotes a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells are used as recipients for a nucleic acid (e.g., an AAV vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be Attorney Docket No. SCRB-056/02WO 333322-2431 completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an AAV vector.

[0079] As used herein, “administering” means a method of giving a dosage of a compound (e.g., a composition of the disclosure) or a composition (e.g., a pharmaceutical composition) to a subject.

[0080] A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats and other rodents.

[0081] As used herein, “treatment” or “treating,” are used interchangeably and refer to an approach for obtaining beneficial or desired results, including, but not limited to, a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can also be achieved with the eradication or amelioration of one or more of the symptoms or an improvement in one or more clinical parameters associated with the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

[0082] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a biologic, alone or as a part of a composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristic of a disease state or condition when administered in one or repeated doses to a subject such as a human or an experimental animal. Such effect need not be absolute to be beneficial.

[0083] A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats and other rodents.

[0084] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

I. General Methods

[0085] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, Attorney Docket No. SCRB-056/02WO 333322-2431 microbiology, cell biology, genomics and recombinant DNA, which can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4^th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0086] Where a range of values is provided, it is understood that endpoints are included and that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0087] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0088] It will be appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. In other cases, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is intended that all combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Attorney Docket No. SCRB-056/02WO 333322-2431

II. Messenger RNA (mRNA) encoding CasX Proteins

[0089] The present disclosure provides mRNA sequences encoding CasX nuclease proteins that have utility in the modification of target nucleic acids of genes in eukaryotic cells. The term “CasX protein”, as used herein, refers to a family of proteins, and encompasses all naturally-occurring CasX proteins (“reference CasX”), as well as CasX proteins engineered with sequence modifications (interchangeably referred to herein as “engineered CasX” or “engineered CasX proteins”) possessing one or more improved characteristics relative to a naturally occurring CasX protein, described more fully, below.

[0090] The engineered CasX proteins encoded by the mRNA sequences are Class 2, Type V nucleases. Although members of Class 2, Type V CRISPR-Cas systems have differences, they share characteristics that distinguish them from the Cas9 systems. Firstly, the Class 2, Type V nucleases possess a single RNA-guided RuvC domain-containing effector but no HNH domain, and they recognize TC motif PAM 5' upstream to the target region on the nontargeted strand, which is different from Cas9 systems which rely on G-rich PAM at 3' side of target sequences. Type V nucleases generate staggered double-stranded breaks distal to the PAM sequence, unlike Cas9, which generates a blunt end in the proximal site close to the PAM. In addition, Type V nucleases degrade ssDNA in trans when activated by target dsDNA or ssDNA binding in cis. In some embodiments, the engineered CasX proteins of the embodiments recognize a 5'-TC PAM motif and produce staggered ends cleaved solely by the RuvC domain.

[0091] CasX proteins of the disclosure comprise the following protein domains: (1) a nontarget strand binding (NTSB) domain, (2) a target strand loading (TSL) domain, (3) a helical I domain (which is further divided into helical I-I and I-II subdomains), (4) a helical II domain, (5) an oligonucleotide binding domain (OBD, which is further divided into OBD-I and OBD-II subdomains), and (6) a RuvC DNA cleavage domain (which is further divided into RuvC-I and II subdomains.

[0092] In some cases, a reference CasX protein is isolated or derived from Deltaproteobacter and comprises a sequence of SEQ ID NO: 1.

[0093] In some cases, a reference CasX protein is isolated or derived from Planctomycetes and comprises a sequence of SEQ ID NO: 2.

[0094] In some cases, a reference CasX protein is isolated or derived from Candidates Sungbacteria and comprises a sequence of SEQ ID NO: 3. Attorney Docket No. SCRB-056/02WO 333322-2431 a. Engineered CasX Proteins

[0095] The present disclosure provides mRNAs encoding engineered CasX proteins derived from one or more reference CasX proteins, wherein the engineered CasX comprise at least one modification in at least one domain of the reference CasX protein, including the sequences of SEQ ID NOS: 1-3. Any change in amino acid sequence of a reference CasX protein that leads to an improved characteristic of the CasX protein and that retains the ability to complex with the gRNA and modify the target nucleic acid is considered an engineered CasX protein of the disclosure. For example, engineered CasX proteins can comprise one or more amino acid substitutions, insertions, deletions, swapped domains from a second CasX, or any combinations thereof, relative to a reference CasX protein sequence. In some embodiment, the disclosure provides engineered CasX proteins wherein the CasX protein comprises a RuvC cleavage domain, wherein the RuvC cleavage domain comprises the sequence of amino acids 648-812 of SEQ ID NO: 2 with one or more amino acid modifications relative to the RuvC cleavage domain sequence of SEQ ID NO: 2. In some embodiments, the one or more amino acid modifications of the RuvC domain comprise a modification at a position selected from the group consisting of 1658, A708, and P793 relative to SEQ ID NO: 2. Mutations can be introduced in any one or more domains of the reference CasX protein or in a CasX variant to result in an engineered CasX protein, and may include, for example, deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain of the reference CasX protein or the CasX variant protein from which it was derived.

[0096] In some embodiments, an engineered CasX protein can bind and/or modify (e.g., nick, or catalyze a double strand break ) a target nucleic acid at a specific sequence targeted by an associated gRNA, which hybridizes to a sequence within the target nucleic acid sequence. In some embodiments, the engineered CasX comprises a nuclease domain having double-stranded cleavage activity that generates a double-stranded break within 18-26 nucleotides 5' of a PAM site on the target strand and 10-18 nucleotides 3' on the non-target strand, resulting in overhangs that can facilitate a higher degree of editing efficiency and modification by NHEJ or insertion of a donor template nucleic acid by HDR or HITI repair mechanisms of the host cell, compared to other CRISPR systems.

[0097] The engineered CasX protein encoded by the mRNAs of the disclosure have one or more improved characteristics compared to a reference CasX from which it was derived. Attorney Docket No. SCRB-056/02WO 333322-2431

Exemplary improved characteristics of the engineered CasX protein may include, but are not limited to, improved ability to utilize a greater spectrum of PAM sequences in the editing and/or binding of target nucleic acid, increased nuclease activity, improved editing efficiency, improved editing specificity for the target nucleic acid, decreased off-target editing or cleavage, increased percentage of a eukaryotic genome that can be efficiently edited, increased activity of the nuclease, and improved ribonucleoprotein (RNP) complex stability. In particular, the engineered CasX proteins of the disclosure have an enhanced ability to efficiently edit and/or bind target DNA, when complexed with a guide RNA scaffold as an RNP, utilizing a PAM TC motif, including PAM sequences selected from TTC, ATC, GTC, or CTC, compared to an RNP of a reference CasX protein and a reference gRNA. In the foregoing, the PAM sequence is located at least 1 nucleotide 5’ to the non-target strand of the protospacer having identity with the targeting sequence of the gRNA in an assay system compared to the editing efficiency and/or binding of an RNP comprising the reference CasX protein and reference gRNA in a comparable assay system.

[0098] In some embodiments, provided herein is an mRNA encoding an engineered CasX protein comprising a sequence of SEQ ID NOS: 4-7, as set forth in Table 1. In some embodiments, the mRNA encodes an engineered CasX protein consisting essentially of a sequence of SEQ ID NOS: 4-7 as set forth in Table 1. In other embodiments, the mRNA encodes an engineered CasX protein comprising a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least

81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least

85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least

88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least

92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least

96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least

99.5% identical to a sequence of SEQ ID NOS: 4-7 as set forth in Table 1, wherein the expressed engineered CasX protein retains the functional properties of the ability to form an RNP with a gRNA and retains nuclease activity. In some embodiments, the mRNA encodes an engineered CasX protein consisting essentially of a sequence of SEQ ID NO: 4 as set forth in Table 1. In some embodiments, the mRNA encodes an engineered CasX protein consisting essentially of a sequence of SEQ ID NO: 5 as set forth in Table 1. In some embodiments, the mRNA encodes an engineered CasX protein consisting essentially of a sequence of SEQ ID Attorney Docket No. SCRB-056/02WO 333322-2431

NO: 6 as set forth in Table 1. In some embodiments, the mRNA encodes an engineered CasX protein consisting essentially of a sequence of SEQ ID NO: 7 as set forth in Table 1. In some embodiments, the mRNA encodes an engineered CasX protein consisting essentially of a sequence of SEQ ID NO: 202 as set forth in Table 1. In some embodiments, the mRNA encodes an engineered CasX protein consisting essentially of a sequence of SEQ ID NO: 203 as set forth in Table l.In some embodiments, the engineered CasX protein retains the ability to form an RNP with a gRNA. In some embodiments, the engineered CasX protein retains nuclease activity.

Table 1: Engineered CasX Sequences

Attorney Docket No. SCRB-056/02WO 333322-2431

Attorney Docket No. SCRB-056/02WO 333322-2431

b. CasX Proteins with Domains from Multiple Source Proteins

[0099] Also contemplated within the scope of the disclosure are mRNAs encoding chimeric CasX proteins comprising protein domains from two or more different CasX proteins, such as two or more naturally occurring CasX proteins, or two or more CasX variants of reference CasX protein sequences as described herein. As used herein, a “chimeric CasX protein” refers to a CasX containing at least two domains isolated or derived from different sources, such as two naturally occurring proteins, which may, in some embodiments, be isolated or derived from different species. In some embodiments, the modification is a substitution of a part or all of a domain from a different CasX protein. In some embodiments, the engineered CasX comprising a sequence of SEQ ID NOS: 4-7 have a NTSB and a portion of the helical I-II domain derived from the reference CasX sequence of SEQ ID NO: 1, while the other domains are derived from the reference CasX sequence of SEQ ID NO: 2. The skilled artisan will understand that the chimeric CasX of the disclosure can have additional amino acid changes at select locations, and the resulting chimeric CasX proteins were determined to have improved characteristics relative to the reference CasX proteins.

[0100] In a particular embodiment, the chimeric helical I domain of the chimeric CasX proteins of SEQ ID NOS: 4-7 comprise amino acids 59-102 of SEQ ID NO: 2, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at Attorney Docket No. SCRB-056/02WO 333322-2431 least about 99% sequence identity thereto (helical I-I), and comprises amino acids 192-332 of SEQ ID NO: 1, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto (helical I-II). In one embodiment, CasX 515 (SEQ ID NO: 5) has an NTSB and the helical I-II domain derived from the reference CasX sequence of SEQ ID NO: 1, and an insertion of P793 relative to the sequence of CasX 491 (SEQ ID NO: 4), wherein the resulting CasX 515 exhibits enhanced specificity for the modification of a target nucleic acid relative to CasX 491. Sequences of the domains of CasX 515 (SEQ ID NO: 5) are provided in Table 2, below. The skilled artisan will understand that the domain boundaries indicated in Table 2 below are approximate, and that protein fragments whose boundaries differ from those given in the table below by 1, 2, or 3 amino acids may have the same activity as the domains described below.

Table 2: CasX 515 domain sequences

c. CasX Fusion Proteins

[0101] Also contemplated within the scope of the disclosure are mRNAs encoding engineered CasX proteins comprising a heterologous protein fused to the CasX. This includes engineered CasX comprising N-terminal or C-terminal fusions of the CasX to a heterologous Attorney Docket No. SCRB-056/02WO 333322-2431 protein or domain thereof. In some embodiments, the engineered CasX protein is fused to one or more proteins or domains thereof that has a different activity of interest, resulting in a fusion protein.

[0102] In some cases, a heterologous polypeptide (a fusion partner) for use with an engineered CasX in the systems of the disclosure provides for subcellular localization, z.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES) to escort the engineered CasX through the nuclear pore complex, a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like). In some embodiments, a subject RNA-guided polypeptide or a conditionally active RNA-guided polypeptide and/or subject engineered CasX fusion protein does not include a NLS so that the protein is not targeted to the nucleus (which can be advantageous, e.g., when the target nucleic acid sequence is an RNA that is present in the cytosol). In some embodiments, a fusion partner can provide a tag (z.e., the heterologous polypeptide is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).

[0103] The disclosure contemplates assembly of multiple NLS in various configurations for linkage to the engineered CasX protein utilized in the embodiments described herein. In some embodiments, a single NLS is linked at or near the N-terminus of the engineered CasX protein. In some embodiments, a single NLS is linked at or near the N-terminus and at or near the C-terminus of the engineered CasX protein. In some embodiments, the N-terminal NLS comprises one or more c-MYC NLS. In some embodiments, the C-terminal NLS comprises one or more c-MYC NLS. In some embodiments, 2, 3, 4 or more NLS are linked by linker peptides at or near the C-terminus and/or the N-terminus of the engineered CasX protein. The person of ordinary skill in the art will understand that an NLS at or near the N- or C-terminus of a protein can be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the N- or C-terminus. In some embodiments, the NLS linked to the N- terminus of the engineered CasX protein are identical to the NLS linked to the C-terminus. In Attorney Docket No. SCRB-056/02WO 333322-2431 other embodiments, the NLS linked to the N-terminus of the engineered CasX protein are different to the NLS linked to the C-terminus. In some cases, non-limiting examples of NLSs suitable for use with an engineered CasX in the systems of the disclosure include sequences having at least about 80%, at least about 90%, or at least about 95% identity or are identical to sequences derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 48); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 49); the c-MYC NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 50) or the sequence RQRRNELKRSP (SEQ ID NO: 51). In some embodiments, the NLS linked to the N-terminus of the engineered CasX protein is selected from the group consisting of the N- terminal sequences as set forth in Table 2. In some embodiments, the NLS linked to the C- terminus of the CasX protein is selected from the group consisting of the C-terminal sequences as set forth in Table 3. In some embodiments, NLSs suitable for use with an engineered CasX in the systems of the disclosure include sequences having at least about 80%, at least about 90%, or at least about 95% identity or are identical to one or more sequences of Table 2 or Table 3. The skilled artisan will understand that the disclosure of NLS sequences in Tables 2 and 3 as N or C terminal, respectively, is exemplary only. Any of the NLS sequences disclosed in Table 4 or 5 may be located at or near the N or C terminus, or both.

Table 2: N-terminal NLS sequences

Attorney Docket No. SCRB-056/02WO 333322-2431

*Residues in bold are NLS residues, while unbolded residues are linkers.

Table 3: C-terminal NLS sequences

Attorney Docket No. SCRB-056/02WO 333322-2431

* Residues in bold are NLS residues, while unbolded residues are linkers. Attorney Docket No. SCRB-056/02WO 333322-2431

[0104] In some embodiments, the one or more NLSs are linked to the encoded CasX protein or to adjacent NLS with a linker peptide wherein the linker peptide is selected from the group consisting of RS, SR, GGS, PPP, GGS, VGS, (G)n (SEQ ID NO: 116), (GS)n (SEQ ID NO: 117), (GSGGS)n (SEQ ID NO: 118), (GGSGGS)n (SEQ ID NO: 119), (GGGS)n (SEQ ID NO: 120), GGSG (SEQ ID NO: 121), GGSGG (SEQ ID NO: 122), GSGSG (SEQ ID NO: 123), GSGGG (SEQ ID NO: 124), GGGSG (SEQ ID NO: 125), GSSSG (SEQ ID NO: 126), GPGP (SEQ ID NO: 127), GGP, PPP, PPAPPA (SEQ ID NO: 128), PPPG (SEQ ID NO: 129), PPPGPPP (SEQ ID NO: 130), PPP(GGGS)n (SEQ ID NO: 131), (GGGS)nPPP (SEQ ID NO: 132), AEAAAKEAAAKEAAAKA (SEQ ID NO: 133), TPPKTKRKVEFE (SEQ ID NO: 134), GGSGGGS (SEQ ID NO: 135), GSGSGGG (SEQ ID NO: 136), and SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 137), where n is 1 to 5.

[0105] In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of an engineered CasX fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus of the engineered CasX protein enhanced by the addition of NLS may be performed by any suitable technique; e.g., a detectable marker may be fused to a reference or engineered CasX fusion protein such that location within a cell may be visualized by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly. Accumulation in the nucleus may also be determined indirectly. d. Sequences encoding engineered CasX

[0106] Provided herein are mRNA and DNA sequences encoding engineered CasX. In some embodiments, the sequences encoding the engineered CasX proteins were generated based using one or more parameters. Suitable methods of codon optimization are known in the art. Non-limiting examples of such parameters include the codon usage in human host cells (e.g., utilizing the codon adaptation index (CAI)) or codon-usage tables derived from biologies intended for use as therapeutics. In some embodiments, the sequences encoding the engineered CasX proteins are codon-optimized for expression in a human cell.

[0107] In some embodiments, the mRNA comprises one or more modifications. Modifications to an mRNA sequence can affect mRNA stability, protein translation and expression levels, and immunogenicity, and therefore impact on the efficacy of mRNA-based delivery. Attorney Docket No. SCRB-056/02WO 333322-2431

[0108] In some embodiments, the mRNA comprises optimized coding and non-coding sequences. Optimization of coding sequences and untranslated regions (UTRs) may promote protein expression when delivering an mRNA encoding a protein of interest, as opposed to a DNA template that would be transcribed into an mRNA. DNA templates are long-lived, can replicate, and can produce many RNA transcripts over their lifetimes. For DNA templates, efficiency of transcription and pre-mRNA processing are major determinants of protein expression levels. In contrast, mRNAs generally have a much shorter half-life, on the order of hours, as they are vulnerable to degradation in the cytoplasm, and cannot produce more copies of themselves. As such, mRNA stability and translation efficiency can determine protein expression levels for mRNA-based delivery, and the sequences of UTRs and coding sequences can influence mRNA stability and translation efficiency.

[0109] The disclosure provides an mRNA comprising a sequence of SEQ ID NO: 8, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity thereto. In some embodiments, the mRNA encodes an engineered CasX protein comprising a sequence of SEQ ID NO: 4.

[0110] The disclosure provides an mRNA comprising a sequence of SEQ ID NO: 9, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity thereto. In some embodiments, the mRNA encodes an engineered CasX protein comprising a sequence of SEQ ID NO: 5.

[OHl] The disclosure provides an mRNA comprising a sequence of SEQ ID NO: 10, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity thereto. In some embodiments, the mRNA encodes an engineered CasX protein comprising a sequence of SEQ ID NO: 6.

[0112] The disclosure provides an mRNA comprising a sequence of SEQ ID NO: 11, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity thereto. In some embodiments, the mRNA encodes an engineered CasX protein comprising a sequence of SEQ ID NO: 7. Attorney Docket No. SCRB-056/02WO 333322-2431

[0113] In some embodiments, the mRNA comprising a sequence encoding the engineered CasX protein comprises one or more Nl-methyl-pseudouri dine nucleotides. In some embodiments, all uridine nucleotides of the sequence encoding the CasX protein are replaced with Nl-methyl-pseudouri dine nucleotides. In some embodiments, 100% of the uridine nucleosides of the mRNA are replaced with N1 -methylpseudouridines. In some embodiments, all uridine nucleotides of the mRNA are replaced with N1 -methylpseudouridine nucleotides. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 12-15, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0114] In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 12, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA sequence comprises the sequence of SEQ ID NO: 12. In some embodiments, the mRNA comprises a sequence encoding a CasX protein that consists essentially of the sequence of SEQ ID NO: 12. In some embodiments, the mRNA encodes an engineered CasX protein comprising a sequence of SEQ ID NO: 4 (CasX 491).

[0115] In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 13, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA s comprises the sequence of SEQ ID NO: 13. In some embodiments, the mRNA comprises a sequence encoding an engineered CasX protein that consists essentially of the sequence of SEQ ID NO: 13. In some embodiments, the mRNA encodes an engineered CasX protein comprising a sequence of SEQ ID NO: 5 (CasX 515). [0116] In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 14, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 14. In some embodiments, the mRNA comprises a sequence encoding a CasX protein that consists essentially of the Attorney Docket No. SCRB-056/02WO 333322-2431 sequence of SEQ ID NO: 14. In some embodiments, the mRNA encodes an engineered CasX protein comprising a sequence of SEQ ID NO: 6 (CasX 676).

[0117] In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 15, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 15. In some embodiments, the mRNA comprises a sequence encoding a CasX protein that consists essentially of the sequence of SEQ ID NO: 15. In some embodiments, the mRNA encodes an engineered CasX protein comprising a sequence of SEQ ID NO: 7 (CasX 812).

[0118] Exemplary sequences encoding engineered CasX, including DNA sequences encoding engineered CasX, are provided as SEQ ID NOS: 16-19 of Table 4. The skilled artisan will appreciate that, for the DNA and RNA sequences such as those disclosed in Table 4, if the sequence is a DNA sequence, T can be substituted by U to generate the corresponding RNA. Similarly, for RNA sequences, U can be substituted with T to generate the corresponding DNA sequence.

Table 4: RNA and DNA sequences encoding Engineered CasX

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= Nl-methyl-pseudouridine e. 5’ cap

[0119] In some embodiments of the mRNA of the disclosure, the mRNA comprises a 5’ cap linked 5’ to the 5’ UTR of the mRNA sequence of any of the embodiments described herein. In some embodiments, the 5’ cap is a 7-methylguanylate cap. In some embodiments, the 5’ cap comprises m7G(5’)ppp(5’)mAG. In other embodiments, the 5' cap comprises m7G(5')ppp (5'(A,G(5')ppp(5')A or G(5')ppp(5')G. In some embodiments, an extra guanine nucleotide is incorporated following the terminal AG of the 5' cap to enhance transcription initiation, resulting in m7G(5’)ppp(5’)mAGG as the full 5’ cap structure. f. 5’ untranslated region (UTR)

[0120] The 5’ UTR of an mRNA molecule can be a key determinant of both the stability of the mRNA and how efficiently it is translated into protein. Specifically, the 5’ UTR, in conjunction with the 5’ cap structure, serves as a binding site and recruitment platform for the translation pre-initiation complex as well as additional regulatory proteins that may positively or negatively affect translation. Structures within the 5’ UTR can enhance translation by recruiting initiation factors or other protein or RNA factors, reduce translation by physically blocking ribosome binding and scanning, and contribute to the stability of the mRNA by affecting both hydrolysis and nuclease digestion.

[0121] An exemplary 5’ UTR sequence for use in the mRNA of the disclosure is provided in Table 5. Table 5 lists the RNA sequence, RNA sequence with N1 -methylpseudouridine substituted in place of uridine, and DNA sequence of the 5’ UTR.

Table 5: 5’ UTR sequences

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*‘r |/’ = Nl-methyl-pseudouridine

[0122] In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 20, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 20. In some embodiments, the 5’ UTR consists of the sequence of SEQ ID NO: 20. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 21, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, identity thereto. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 21. In some embodiments, the 5’ UTR consists of the sequence of SEQ ID NO: 21.

[0123] In some embodiments, the mRNA of the disclosure comprise a Kozak sequence. In some embodiments, the mRNA comprises the sequence GCCACC (SEQ ID NO: 26). In some embodiments, the mRNA comprises the sequence GCCACC (SEQ ID NO: 26) between the 5’ UTR and the sequence encoding the CasX. g. 3’ UTR

[0124] 3’ UTR sequences can have a significant impact on mRNA stability and translation efficiency and can determine both subcellular localization and tissue-specific expression. Factors influencing these properties include microRNA binding sites, AU-rich elements that recruit an array of RNA-binding proteins, Pumilio binding elements, and other binding sites for RNA-binding proteins. While many of these interactions with the 3’ UTR are known to negatively impact stability or expression, some can enhance translation. The effects of a 3’ UTR sequence can be highly cell-type specific due to differential expression of microRNAs and RNA binding proteins, which provides opportunities for engineering tissue-specific expression into a therapeutic mRNA.

[0125] An exemplary 3’ UTR sequence for use in the mRNA of the disclosure is provided in Table 6. Table 6 lists the RNA sequence, RNA sequence with N1 -methylpseudouridine substituted in place of uridine, and DNA sequence of the 3’ UTR.

Table 6: 3’ UTR sequences

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= Nl-methyl-pseudouridine

[0126] In some embodiments, the 3’ UTR comprises a mouse 3’ UTR. In some embodiments, the 3’ UTR comprises a mouse HBA gene 3’ UTR.

[0127] In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 23, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% identity thereto. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 23. In some embodiments, the 3’ UTR consists of the sequence of SEQ ID NO: 23. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 24, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% identity thereto. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 24. In some embodiments, the 3’ UTR consists of the sequence of SEQ ID NO: 24. h. Poly(A) sequence

[0128] The 3’ poly(A) tail can contribute to mRNA stability and translation efficiency. Generally, longer poly(A) tails are associated with increased mRNA stability, thereby allowing their translation and promoting high protein expression.

[0129] In some embodiments, the mRNAs of the disclosure comprise a poly(A) sequence having at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides. In some embodiments, the poly(A) sequence comprises 80 adenine nucleotides. In some embodiments, the poly(A) sequence for use in the mRNA of the disclosure comprises the nucleic acid sequence of Attorney Docket No. SCRB-056/02WO 333322-2431

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 156). In some embodiments, the poly(A) sequence for use in the mRNA of the disclosure comprises 79 adenine nucleotides. In some embodiments, the poly(A) sequence for use in the mRNA of the disclosure comprises the nucleic acid sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 155). i. mRNA sequences

[0130] In some embodiments, the mRNA comprises the following components in 5’ to 3’ orientation: a 5' UTR; a start codon; an NLS; a sequence encoding a CasX protein; an NLS; stop codon; and a 3' UTR. In some embodiments, the mRNA comprises the following components in 5’ to 3’ orientation: a 5’ cap; a 5' UTR; a start codon; an NLS; a sequence encoding a CasX protein; a stop codon; a 3' UTR; and a poly(A) sequence. In some embodiments, the mRNA comprises the following components in 5’ to 3’ orientation: a 5’ cap; a 5' UTR; a start codon; a sequence encoding a CasX protein; an NLS; a stop codon; a 3' UTR; and a poly(A) sequence. In some embodiments, the mRNA comprises the following components in 5’ to 3’ orientation: a 5’ cap; a 5' UTR; a start codon; a sequence encoding a CasX protein; a stop codon; a 3' UTR; and a poly(A) sequence.

[0131] Various naturally-occurring or modified nucleosides may be used to produce mRNA according to the present disclosure. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-l -methylpseudouridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine (“5 mC”), pseudouridine (“\|/U”), Attorney Docket No. SCRB-056/02WO 333322-2431 and/or 2-thio-uridine (“2sU”). In a particular embodiment, one or more of the uridine residues of the mRNA of the disclosure are replaced with 1-methyl-pseudouridine. See, e.g., U.S. Pat. No. 8,278,036 or WO2011012316, incorporated by reference herein, for a discussion of such residues and their incorporation into mRNA.

[0132] Exemplary mRNA sequences encoding the engineered CasX are provided in Table 7. Table 7 lists the RNA sequences, RNA sequences with N1 -methylpseudouridine substituted in place of uridine, and DNA sequences of the mRNAs. The sequences in Table 7 include, from 5’ to 3’, AGG nucleotides 5’ of the 5’ UTR, a 5’ UTR, GCCACC (SEQ ID NO: 26) nucleotides, a start codon, a sequence encoding a c-MYC NLS, a sequence encoding a linker, a sequence encoding CasX, a sequence encoding a linker, a sequence encoding a c- MYC NLS, a stop codon, a 3’ UTR, and a sequence corresponding to a partial Xbal restriction site (UCUAG, SEQ ID NO: 33; myCmyAG, SEQ ID NO: 34; or TCTAG, SEQ ID NO: 35, for RNA, N1 -methylpseudouridine substituted RNA, and DNA sequences, respectively).

[0133] In some embodiments, the disclosure provides an mRNA sequence encoding an engineered CasX protein comprising a sequence of SEQ ID NO: 4, the mRNA comprising the sequence of SEQ ID NO: 36 or SEQ ID NO: 40, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the disclosure provides an mRNA sequence encoding an engineered CasX protein comprising a sequence of SEQ ID NO: 5, the mRNA comprising the sequence of SEQ ID NO: 37, SEQ ID NO: 41, or SEQ ID NO: 200, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the disclosure provides an mRNA sequence encoding the engineered CasX protein comprising a sequence of SEQ ID NO: 6, the mRNA comprising the sequence of SEQ ID NO: 38 or SEQ ID NO: 42, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the disclosure provides an mRNA sequence encoding the engineered CasX protein comprising a sequence of SEQ ID NO: 7 , the mRNA comprising the sequence of SEQ ID NO: 39, SEQ ID NO: 43, or SEQ ID NO: 201, or a sequence having at least about Attorney Docket No. SCRB-056/02WO 333322-2431

70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

Table 7: Exemplary full-length mRNA sequences encoding CasX

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= Nl-methyl-pseudouridine

[0134] In some embodiments, the DNA sequence encoding the engineered CasX protein is selected from the group consisting of SEQ ID NOS: 44, 45, 46, and 47.

III. Systems for Genetic Editing of Target Nucleic Acid

[0135] The present disclosure provides systems comprising a messenger RNA (mRNA) encoding an engineered CasX protein and one or more guide ribonucleic acids (gRNA) for use in modifying target nucleic acids. In some embodiments, the modifying occurs in a cell. As used herein, the term “gRNA” covers naturally-occurring molecules and gRNA variants, Attorney Docket No. SCRB-056/02WO 333322-2431 including chimeric gRNA variants comprising domains from different gRNA. gRNAs of the disclosure comprise a scaffold and a targeting sequence complementary to a target nucleic acid of a gene in a cell to be modified. As used herein, a “system”, used interchangeably with “composition”, can comprise an mRNA encoding a CasX protein and one or more gRNAs of any of the embodiments disclosed herein, which can be utilized as gene editing pairs.

[0136] In some embodiments of the systems described herein, upon expression of the engineered CasX protein in a cell, the CasX protein forms a ribonucleoprotein (RNP) complex with the gRNA. The RNP targets and edits specific locations in a target nucleic acid sequence of the cell. The gRNA provides target specificity to the complex by including a targeting sequence (or “spacer”) having a nucleotide sequence that is complementary to a sequence of the target nucleic acid sequence to be modified, while the CasX protein provides the site-specific activity such as cleavage or nicking of the target sequence that is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence by virtue of its association with the gRNA. gRNAs and formulations of mRNAs and gRNAs for use in the editing of target nucleic acids are described herein, below.

[0137] In another aspect, the disclosure relates to gRNA scaffolds with linked targeting sequences complementary to (and are therefore able to hybridize with) a target nucleic acid sequence of a gene that have utility, when complexed with a CasX protein, in genome editing of a target nucleic acid in a cell. In some embodiments, the gRNA scaffolds of the disclosure are modified relative to gRNA variants by approaches including mutagenesis of individual nucleotides or domain swapping, as described herein. As used herein, “scaffold” refers to all parts to the guide with the exception of the targeting sequence.

[0138] It is envisioned that in some embodiments, multiple gRNAs are delivered in the systems for the modification of a target nucleic acid. For example, a pair of gRNAs with targeting sequences to different or overlapping regions of the target nucleic acid sequence can be used to bind and cleave at two different or overlapping sites within the gene, which is then edited by non-homologous end joining (NHEJ), homology-directed repair (HDR), homologyindependent targeted integration (HITI), micro-homology mediated end joining (MMEJ), single strand annealing (SSA) or base excision repair (BER). For example, when an editing event designed to delete one or more exons of the gene is desired, a pair of gRNAs can be used with the expressed CasX protein to bind and cleave at two different sites 5’ and 3’ of the targeted exon(s) within the gene to excise the intervening sequence. Both single-stranded Attorney Docket No. SCRB-056/02WO 333322-2431 cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events by the CasX protein. In some embodiments, indels are introduced in the target nucleic acid by the CasX:gRNA systems of the embodiments described herein and cellular repair systems that can disrupt the protein reading frame of the targeted gene. a. Reference gRNA and gRNA variants

[0139] As used herein, a “reference gRNA" refers to a CRISPR guide ribonucleic acid comprising a wild-type sequence of a naturally-occurring gRNA. Table 8 provides the sequences of reference gRNA tracr and scaffold sequences. In some embodiments, the disclosure provides gRNA sequences wherein the gRNA has a scaffold comprising a sequence having one or more nucleotide modifications relative to a reference gRNA sequence of any one of SEQ ID NOS: 139-151 of Table 8.

Table 8: Reference gRNA tracr and scaffold sequences

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[0140] The gRNAs of the systems of the disclosure comprise two segments: a targeting sequence and a protein-binding segment. The targeting segment of a gRNA includes a nucleotide sequence (referred to interchangeably as a guide sequence, a spacer, a targeter, or a targeting sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within the target nucleic acid sequence (e.g., a strand of a double stranded target DNA, a target ssRNA, a target ssDNA, etc.), described more fully below. The targeting sequence of a gRNA is capable of binding to a target nucleic acid sequence, including, in the context of the present disclosure, a coding sequence, a complement of a coding sequence, a non-coding sequence, and to accessory elements. The protein-binding segment (or “activator” or “protein-binding sequence”) interacts with (e.g., binds to) a CasX protein as a complex, forming an RNP (described more fully, below). The protein-binding segment is alternatively referred to herein as a “scaffold”, which is comprised of several regions, described more fully, below.

[0141] In the case of a reference gRNA, the gRNA occurs naturally as a dual guide RNA (dgRNA), wherein the targeter and the activator portions each have a duplex-forming segment that have complementarity with one another and hybridize to one another to form a double stranded duplex (dsRNA duplex for a gRNA). The term “targeter” or “targeter RNA” is used herein to refer to a crRNA-like molecule (crRNA: "CRISPR RNA") of a CasX dual guide RNA (and therefore of a CasX single guide RNA when the “activator” and the "targeter” are linked together, e.g., by intervening nucleotides). The crRNA has a 5' region that anneals with the tracrRNA followed by the nucleotides of the targeting sequence. In the case of the gRNA for use in the systems of the disclosure, the scaffolds are designed such that the activator and targeter portions are covalently linked to one another (rather than hybridizing to one another) and comprise a single molecule, and can be referred to as a “single-molecule gRNA,” “single guide RNA”, a “single-molecule guide RNA,” a “one- molecule guide RNA”, or a “sgRNA”. The gRNA of the disclosure are all single molecule versions.

[0142] Collectively, the assembled gRNAs of the disclosure comprise distinct structured regions, or domains: the RNA triplex, the scaffold stem loop, the extended stem loop, the pseudoknot, and the targeting sequence that, in the embodiments of the disclosure is specific for a target nucleic acid and is located on the 3’ end of the gRNA. The RNA triplex, the Attorney Docket No. SCRB-056/02WO 333322-2431 scaffold stem loop, the pseudoknot and the extended stem loop, together with the unstructured triplex loop that bridges portions of the triplex, together, are referred to as the “scaffold” of the gRNA. In some embodiments, the gRNA scaffolds of the disclosure comprise a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 138). In some embodiments, the gRNA scaffolds of the disclosure comprise a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 138) or a sequence having 1, 2, 3, 4, or 5 mismatches thereto.

[0143] Each of the structured domains contribute to the global RNA fold of the guide and retain functionality of the guide; particularly the ability to properly complex with the CasX protein. For example, the guide scaffold stem interacts with the helical I domain of CasX protein, while residues within the triplex, triplex loop, and pseudoknot stem interact with the OBD of the CasX protein. Together, these interactions confer the ability of the guide to bind and form an RNP with the CasX that retains stability, while the spacer (or targeting sequence) directs and defines the specificity of the RNP for binding a specific sequence of DNA.

[0144] Site-specific binding and/or cleavage of a target nucleic acid sequence (e.g., genomic DNA) by the CasX protein can occur at one or more locations (e.g., a sequence of a target nucleic acid) determined by base-pairing complementarity between the targeting sequence of the gRNA and the target nucleic acid sequence. Thus, for example, the gRNA of the disclosure have sequences complementarity to and therefore can hybridize with the target nucleic acid that is adjacent to a sequence complementary to a TC protospacer adjacent motif (PAM) motif or a PAM sequence, such as ATC, CTC, GTC, or TTC. Because the targeting sequence of a guide sequence hybridizes with a sequence of a target nucleic acid sequence, a targeting sequence can be modified by a user to hybridize with a specific target nucleic acid sequence, so long as the location of the PAM sequence is considered. By selection of the targeting sequences of the gRNA, defined regions of the target nucleic acid sequence or sequences bracketing a particular location within the target nucleic acid can be modified or edited using the CasX:gRNA systems described herein. In some embodiments, the targeting sequence of the gRNA has between 15 and 20 consecutive nucleotides. In some embodiments, the targeting sequence has 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 21 consecutive nucleotides. In some embodiments, the targeting sequence consists of 20 consecutive Attorney Docket No. SCRB-056/02WO 333322-2431 nucleotides. In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides.

[0145] In some embodiments, the CasX:gRNA system comprises a first gRNA and further comprises a second (and optionally a third, fourth, fifth, or more) gRNA, wherein the second gRNA or additional gRNA has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid sequence compared to the targeting sequence of the first gRNA such that multiple points in the target nucleic acid are targeted, and, for example, multiple breaks are introduced in the target nucleic acid by the CasX, which may result in the excision of the intervening sequence. It will be understood that in such cases, the second or additional gRNA is complexed with an additional copy of the CasX protein. c. gRNA modifications

[0146] In another aspect, the disclosure relates to gRNA for use in the gene-editing systems of the disclosure, which comprise one or more modifications relative to a gRNA scaffold from which it was derived. In some embodiments, a gRNA variant for use in the systems of the disclosure comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced domains relative to a gRNA sequence of the disclosure that improve a characteristic relative to the reference gRNA. Exemplary regions for modifications and swapped regions or domains include the RNA triplex, the pseudoknot, the scaffold stem loop, and the extended stem loop. In some embodiments, the gRNA variant of the disclosure comprises at least a first swapped region from a different gRNA, resulting in a chimeric gRNA. A representative example of such a chimeric gRNA is guide 316 (SEQ ID NO: 154), in which the extended step of gRNA scaffold 235 is replaced with the extended stem of gRNA scaffold 174, wherein the resulting 316 variant retains the ability to form an RNP with a CasX protein and exhibits an improved functional characteristic compared to the parent 235, when assessed in an in vitro or in vivo assay under comparable conditions.

[0147] All gRNAs that have one or more improved functions, characteristics, or add one or more new functions when the gRNA scaffold variant is compared to a gRNA scaffold from which it was derived, while retaining the functional properties of being able to complex with Attorney Docket No. SCRB-056/02WO 333322-2431 the CasX and guide the CasX ribonucleoprotein holocomplex to the target nucleic acid are envisaged as within the scope of the disclosure. In some embodiments, the gRNA has an improved characteristic selected from the group consisting of increased editing activity, increased pseudoknot stem stability, increased triplex region stability, increased scaffold stem stability, extended stem stability, reduced off-target folding intermediates, and increased binding affinity to a CasX protein, or any combination thereof. In some cases of the foregoing, the improved characteristic is assessed in an in vitro assay, including the assays of the Examples. In other cases of the foregoing, the improved characteristic is assessed in vivo. [0148] In other embodiments, the gRNA scaffold variant has improved manufacturability compared to the gRNA scaffold from which it was derived. In a particular embodiment, the 316 gRNA scaffold has a shorter sequence compared to the 235 scaffold from which it was derived, which confers the improvements of a higher fidelity in the ability to create the guide synthetically with the correct and complete sequence, as well as an enhanced ability to be successfully incorporated into an LNP.

[0149] Table 9 provides exemplary gRNA scaffold sequences for use in the systems of the disclosure.

Table 9: Exemplary gRNA Scaffold Sequences

[0150] Guide scaffolds can be made by several methods, including recombinantly or by solid-phase RNA synthesis. However, the length of the scaffold can affect the manufacturability when using solid-phase RNA synthesis, with longer lengths resulting in increased manufacturing costs, decreased purity and yield, and higher rates of synthesis failures. For use in lipid nanoparticle (LNP) formulations, solid-phase RNA synthesis of the scaffold is preferred to generate the quantities needed for commercial development. While previous experiments had identified gRNA scaffold 235 (SEDQ ID NO: 153) as having enhanced properties relative to gRNA scaffold 174 (SEQ ID NO: 152), its increased length Attorney Docket No. SCRB-056/02WO 333322-2431 potentially rendered its use for LNP formulations problematic. Accordingly, alternative sequences were sought. In some embodiments, the disclosure provides a gRNA wherein the gRNA scaffold and linked targeting sequence has a sequence less than about 120 nucleotides, less than about 110 nucleotides, or less than about 100 nucleotides.

[0151] In one embodiment, a scaffold was designed wherein the scaffold 235 sequence was modified by a domain swap in which the extended stem loop of scaffold 174 replaced the extended stem loop of the 235 scaffold, resulting in the chimeric gRNA scaffold 316, having the sequence ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUA GUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 154), having 89 nucleotides, compared with the 99 nucleotides of gRNA scaffold 235. The resulting 316 scaffold had the further advantage in that the extended stem loop does not contain CpG motifs. In some embodiments, the disclosure provides gRNA 316 variants that are chemically-modified, as described below. d. Chemically-modified gRNAs

[0152] In some embodiments, the gRNAs have one or more chemical modifications. In some embodiments, the chemical modification is the addition of a 2’O-methyl group to one or more nucleotides of the sequence. In some embodiments, the chemical modification is substitution of a phosphorothioate bond between two or more nucleotides of the sequence. In some embodiments, the first 1, 2, or 3 nucleotides of the 5’ end of the scaffold (i.e., A, C, and U in the case of gRNA 174, 235, and 316) are modified by the addition of a 2’O-methyl group and each of the modified nucleotides is linked to the adjoining nucleotide by a phosphorothioate bond. Similarly, the last 1, 2, or 3 nucleotides of the 3’ end of the targeting sequence linked to the 3’ end of the scaffold are similarly modified. Exemplary chemically modified gRNAs are described in the Examples, below. In some embodiments, the disclosure provides gRNA with chemical modifications comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 158-166, 168-176 and 178-186, as set forth in Table 17, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto. In some embodiments, the gRNA with chemical modifications comprises a scaffold of SEQ ID NOS: 158-166; 168-176; 178-186, i.e., a sequence of SEQ ID NOS: 158-166; 168-176; 178-186 with the 20 nucleotides of the 3' Attorney Docket No. SCRB-056/02WO 333322-2431 spacer represented in the foregoing sequences as undefined nucleotides substituted with a targeting sequence with chemical modifications that is complementary to a target nucleic to be modified. In some embodiments, the gRNA with chemical modifications exhibit improved stability compared to gRNA without chemical modifications. e. Complex Formation with CasX Protein

[0153] Upon expression of the components of the system, the gRNA is capable of complexing as an RNP with a CasX protein. In some embodiments, a gRNA has an improved ability to form an RNP complex with a CasX protein when compared to a reference gRNA or a gRNA variant from which it was derived. Improving ribonucleoprotein complex formation may, in some embodiments, improve the efficiency with which functional RNPs are assembled. In some embodiments, greater than 90%, greater than 93%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% of RNPs comprising a gRNA and its spacer are competent for gene editing or modification of a target nucleic acid.

IV. Lipid Nanoparticles (LNP)

[0154] In another aspect, the present disclosure provides lipid nanoparticles (LNP) for delivery of any one of the mRNAs encoding CasX described herein. In some embodiments, the LNP comprises any one of the mRNAs described herein and a gRNA. In some embodiments, the LNPs of the disclosure are tissue-specific, have excellent biocompatibility, and can deliver the mRNA with high efficiency, and thus can be used for the modification or repression of the target nucleic acid.

[0155] In their native forms, nucleic acid polymers are generally unstable in biological fluids and cannot penetrate into the cytoplasm of target cells, thus requiring delivery systems. Lipid nanoparticles (LNP) have proven useful for both the protection and delivery of nucleic acids to tissues and cells. Furthermore, the use of mRNA in LNPs to encode a CRISPR nuclease eliminates the possibility of undesirable genome integration compared to DNA vectors. Moreover, mRNA efficiently transfects both mitotic and non-mitotic cells, as it does not require to enter into the nucleus since it exerts its function in the cytoplasmic compartment.

[0156] Accordingly, in various embodiments, the disclosure encompasses lipid nanoparticles and compositions that may be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic Attorney Docket No. SCRB-056/02WO 333322-2431 acids to cells, both in vitro and in vivo. In certain embodiments, the disclosure encompasses methods of treating or preventing diseases or disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle that encapsulates or is associated with a suitable therapeutic agent.

[0157] In certain embodiments, the lipid nanoparticles are useful for the delivery of nucleic acids, including, e.g., the mRNA sequences of SEQ ID NOS: 36-43. Therefore, the lipid nanoparticles and compositions of certain embodiments of the disclosure may be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel ionizable lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA encoding the desired protein). In some embodiments, the lipid nanoparticles and compositions may be used to decrease the expression of target genes and proteins both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with one or more nucleic acids of the disclosure modifies the target nucleic acid.

[0158] In some embodiments, the mRNA of the disclosure encoding the CasX nuclease may be provided in a solution to be mixed with a lipid solution such that the mRNA may be encapsulated in the lipid nanoparticles. A suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations. For example, a suitable mRNA solution may contain an mRNA at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.25 mg/ml, 1.5 mg/ml, 1.75 mg/ml, or 2.0 mg/ml. In some embodiments, a suitable mRNA solution may contain an mRNA at a concentration ranging from about 0.01- 2.0 mg/ml, 0.01-1.5 mg/ml, 0.01-1.25 mg/ml, 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01-0.8 mg/ml, 0.01-0.7 mg/ml, 0.01-0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3 mg/ml, 0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9 mg/ml, 0.05-0.8 mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5 mg/ml, 0.05-0.4 mg/ml, 0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1 mg/ml, 0.1-1.0 mg/ml, 0.2-0.9 mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml, or 0.5-0.6 mg/ml. In some embodiments, a suitable mRNA solution may contain an mRNA at a concentration up to about 5.0 mg/ml, 4.0 mg/ml, 3.0 mg/ml, 2.0 mg/ml, 1.0 mg/ml, 0.9 Attorney Docket No. SCRB-056/02WO 333322-2431 mg/ml, 0.8 mg/ml, 0.7 mg/ml, 0.6 mg/ml, 0.5 mg/ml, 0.4 mg/ml, 0.3 mg/ml, 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml, 0.04 mg/ml, 0.03 mg/ml, 0.02 mg/ml, 0.01 mg/ml, or 0.05 mg/ml.

[0159] Early formulations of LNP utilizing permanently cationic lipids resulted in LNPs with positive surface charge that proved toxic in vivo, plus were rapidly cleared by phagocytic cells. By changing to ionizable cationic lipids bearing tertiary or quaternary amines, especially those with pKa < 7, resulting LNP achieve efficient encapsulation of nucleic acid polymers at low pH by interacting electrostatically with the negative charges of the phosphate backbone of mRNA, that also result in largely neutral systems at physiological pH values, thus alleviating problems associated with permanently-charged cationic lipids. Herein, "ionizable lipid" means an amine-containing lipid which can be easily protonated, and for example, it may be a lipid of which charge state changes depending on the surrounding pH. The ionizable lipid may be protonated (positively charged) at a pH below the pKa of a cationic lipid, and it may be substantially neutral at a pH over the pKa. In one example, the LNP may comprise a protonated ionizable lipid and/or an ionizable lipid showing neutrality. In some embodiments, the LNP has a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. The pKa of the LNP is important for in vivo stability and release of the nucleic acid payload of the LNP. In some embodiments, the LNP having the foregoing pKa ranges may be safely delivered to a target organ (for example, the liver, lung, heart, spleen, as well as to tumors) and/or target cell (hepatocyte, LSEC, cardiac cell, cancer cell, etc.) in vivo, and after endocytosis, exhibit a positive charge to release the encapsulated payload through electrostatic interaction with an anionic protein of the endosome membrane.

[0160] The ionizable lipid is an ionizable compound having characteristics similar to lipids generally, and through electrostatic interaction with a nucleic acid (for example, an mRNA of the disclosure), may play a role of encapsulating the nucleic acid within the LNP with high efficiency.

[0161] According to the type of the amine comprised in the ionizable lipid, (i) the nucleic acid encapsulation efficiency, (ii) PDI (poly dispersity index), and/or (iii) the nucleic acid delivery efficiency to tissue and/or cells constituting an organ (for example, hepatocytes or liver sinusoidal endothelial cells in the liver) of the LNP may be different. In certain embodiments, the ionizable cationic lipid comprises from about 46 mol % to about 66 mol % of the total lipid present in the particle. Attorney Docket No. SCRB-056/02WO 333322-2431

[0162] The LNP comprising an ionizable lipid comprising an amine may have one or more kinds of the following characteristics: (1) encapsulating a drug with high efficiency; (2) uniform size of prepared particles (or having a low PDI value); and/or (3) excellent nucleic acid delivery efficiency to organs such as liver, lung, heart, spleen, as well as to tumors, and/or cells constituting such organs (for example, hepatocytes, LSEC, cardiac cells, cancer cells, etc.).

[0163] The lipid composition usually consists of an ionizable amino lipid, a helper lipid (usually a phospholipid), cholesterol, and a polyethylene gly col-lipid conjugate (PEG-lipid) to improve the colloidal stability in biological environments by reducing aspecific absorption of plasma proteins and forming a hydration layer over the nanoparticles and are formulated at typical mole ratios of 50: 10:37-39: 1.5-2.5, with variations made to adjust individual properties. As the PEG-lipid forms the surface lipid, the size of the LNP can be readily varied by varying the proportion of surface (PEG) lipid to the core (ionizable cationic) lipids. In some embodiments, the PEG-lipid can be varied from ~1 to 5 mol% to modify particle properties such as size, stability, and circulation time. In particular, the cationic lipid form plays a crucial role both in nucleic acid encapsulation through electrostatic interactions and intracellular release by disrupting endosomal membranes. The mRNA are encapsulated within the LNP by the ionic interactions they form with the positively charged cationic (or ionizable) lipid. Non-limiting examples of ionizable cationic lipid components utilized in the LNP of the disclosure are selected from DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraen- 19-yl4-(dimethylamino)butanoate), DLin- KC2-DMA (2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane), and TNT (1, 3, 5-triazinane-2, 4, 6-trione) and TT (Nl,N3,N5-tris(2-aminoethyl)benzene-l,3,5-tricarboxamide). Non-limiting examples of helper lipids utilized in the LNP of the disclosure are selected from DSPC (1,2-distearoyl-sn- glycero-3 -phosphocholine), POPC (2-Oleoyl-l- palmitoyl-sn-glycero-3-phosphocholine) and DOPE (l,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine). Cholesterol and PEG-DMG ((R)- 2,3- bis(octadecyloxy)propyl-l -(methoxy polyethylene glycol 2000) carbamate) or PEG-DSG (l,2-Distearoyl-rac-glycero-3-methylpolyoxy ethylene glycol 2000) are components utilized for the stability, circulation, and size of the LNP.

[0164] In other embodiments, the ionizable cationic lipid in the LNPs of the disclosure may comprise, for example, one or more ionizable cationic lipids wherein the ionizable cationic lipid is a dialkyl lipid. In another embodiment, the ionizable cationic lipid is a trialkyl lipid. Attorney Docket No. SCRB-056/02WO 333322-2431

In one particular embodiment, the ionizable cationic lipid is selected from the group consisting of l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy- N,N-dimethylaminopropane (DLinDMA), 1,2-di-. gamma. -linolenyloxy-N,N- dimethylaminopropane (gamma. -DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- di oxolane (DLin-K-C2-DMA), 2, 2-dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), dilinoleylmethyl-3 -dimethylaminopropionate (DLin-M-C2-DMA), or salts thereof and mixtures thereof. In a particular embodiment, the ionizable cationic lipid is selected from the group consisting of l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-. gamma. - linolenyloxy-N,N-dimethylaminopropane (.gamma.-DLenDMA; a salt thereof, or a mixture thereof. In some embodiments, the N/P ratio (nitrogen from the cationic/ionizable lipid and phosphate from the nucleic acid) is in the range of is about 3:1 to 7:1, or about 4:1 to 6:1, or is 3:1, or is 4:1, or is 5: 1, or is 6:1, or is 7:1.

[0165] The phospholipid of the elements of the LNP according to one example plays a role of covering and protecting a core formed by interaction of the ionizable lipid and nucleic acid in the LNP, and may facilitate cell membrane permeation and endosomal escape during intracellular delivery of the nucleic acid by binding to the phospholipid bilayer of a target cell.

[0166] For the phospholipid, a phospholipid which can promote fusion of the LNP according to one example may be used without limitation, and for example, it may be one or more kinds selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-di oleoyl-sn- glycero-3 -phosphoethanolamine, l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine(POPE), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC), l,2-dioleoyl-sn-glycero-3-[phospho-L-serine](DOPS), 1,2-di oleoyl-sn-glycero-3-[phospho-L- serine] and the like. In one example, the LNP comprising DOPE may be effective in mRNA delivery (excellent drug delivery efficacy). Attorney Docket No. SCRB-056/02WO 333322-2431

[0167] The cholesterol of the elements of the LNP according to one example may provide morphological rigidity to lipid filling in the LNP and be dispersed in the core and surface of the nanoparticle to improve the stability of the nanoparticle.

[0168] Herein, "lipid-PEG (polyethyleneglycol) conjugate", "lipid-PEG", "PEG-lipid", "PEG-lipid", or "lipid-PEG" refers to a form in which lipid and PEG are conjugated and means a lipid in which a polyethylene glycol (PEG) polymer which is a hydrophilic polymer is bound to one end. The lipid-PEG conjugate contributes to the particle stability in serum of the nanoparticle within the LNP, and plays a role of preventing aggregation between nanoparticles. In addition, the lipid-PEG conjugate may protect nucleic acids from degrading enzyme during in vivo delivery of the nucleic acids and enhance the stability of nucleic acids in vivo and increase the half-life of the drug encapsulated in the nanoparticle. Examples of PEG-lipid conjugates include, but are not limited to, PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof. In certain embodiments, the PEG-lipid conjugate is selected from the group consisting of a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG- dialkyloxypropyl (PEG-DAA) conjugate, a PEG-phospholipid conjugate, a PEG-ceramide (PEG-Cer) conjugate, and a mixture thereof. In certain embodiments, the PEG-lipid conjugate is a PEG-DAA conjugate. In certain embodiments, the PEG-DAA conjugate in the lipid particle may comprise a PEG-didecyloxypropyl (Cio) conjugate, a PEG- dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG- dipalmityloxypropyl (Cie) conjugate, a PEG-distearyl oxy propyl (Cis) conjugate, or mixtures thereof. In certain embodiments, wherein the PEG-DAA conjugate is a PEG- dimyristyloxypropyl (C14) conjugate. In other embodiments, the lipid-PEG conjugate may be PEG bound to phospholipid such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide (PEG-CER, ceramide-PEG conjugate, ceramide-PEG, cholesterol or PEG conjugated to derivative thereof, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE(DSPE-PEG), and a mixture thereof, and for example, may be Cl 6- PEG2000 ceramide (N-palmitoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]}), DMG-PEG 2000, 14:0 PEG2000 PE.

[0169] In certain embodiments, the conjugated lipid that inhibits aggregation of particles comprises from about 0.5 mol % to about 3 mol % of the total lipid present in the particle. [0170] In one example, the average molecular weight of the lipid-PEG conjugate may be 100 daltons to 10,000 daltons, 200 daltons to 8,000 daltons, 500 daltons to 5,000 daltons, Attorney Docket No. SCRB-056/02WO 333322-2431

1,000 daltons to 3,000 daltons, 1,000 daltons to 2,600 daltons, 1,500 daltons to 2,600 daltons, 1,500 daltons to 2,500 daltons, 2,000 daltons to 2,600 daltons, 2,000 daltons to 2,500 daltons, or 2,000 daltons.

[0171] For the lipid in the lipid-PEG conjugate, any lipid capable of binding to polyethyleneglycol may be used without limitation, and the phospholipid and/or cholesterol which are other elements of the LNP may be also used. Specifically, the lipid in the lipid- PEG conjugate may be ceramide, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s- DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), or cholesterol, but not limited thereto.

[0172] In the lipid-PEG conjugate, the PEG may be directly conjugated to the lipid or linked to the lipid via a linker moiety. Any linker moiety suitable for binding PEG to the lipid may be used, and for example, includes an ester-free linker moiety and an ester-containing linker moiety. The ester-free linker moiety includes not only amido (-C(O)NH-), amino (- NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide but also combinations thereof (for example, a linker containing both a carbamate linker moiety and an amido linker moiety), but not limited thereto. The ester- containing linker moiety includes for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof, but not limited thereto.

[0173] In certain embodiments, the LNP comprising the nucleic acid(s) has a total lipid:mRNA mass ratio of from about 5: 1 to about 15: 1. In some embodiments, the weight ratio of the ionizable lipid and nucleic acid comprised in the LNP may be 1 to 20: 1, 1 to 15: 1, 1 to 10: 1, 5 to 20: 1, 5 to 15: 1, 5 to 10: 1, 7.5 to 20: 1, 7.5 to 15: 1, or 7.5 to 10: 1.

[0174] In one example, the LNP may comprise the ionizable lipid of 20 to 50 parts by weight, phospholipid of 10 to 30 parts by weight, cholesterol of 20 to 60 parts by weight (or 20 to 60 parts by weight), and lipid-PEG conjugate of 0.1 to 10 parts by weight (or 0.25 to 10 parts by weight, 0.5 to 5 parts by weight). The LNP may comprise the ionizable lipid of 20 to 50 % by weight, phospholipid of 10 to 30 % by weight, cholesterol of 20 to 60 % by weight (or 30 to 60 % by weight), and lipid-PEG conjugate of 0.1 to 10 % by weight (or 0.25 to 10 % by weight, 0.5 to 5 % by weight) based on the total nanoparticle weight. In other example, the LNP may comprise the ionizable lipid of 25 to 50 % by weight, phospholipid of 10 to 20 % by weight, cholesterol of 35 to 55 % by weight, and lipid-PEG conjugate of 0.1 to 10 % by Attorney Docket No. SCRB-056/02WO 333322-2431 weight (or 0.25 to 10 % by weight, 0.5 to 5 % by weight), based on the total nanoparticle weight.

[0175] In some embodiments, the approach to formulating the LNP of the disclosure (described more fully in the examples) is to dissolve lipids in an organic solvent such as ethanol, which is then mixed through a micromixer with the nucleic acid dissolved in an acidic buffer (usually pH 4). At this pH the ionizable cationic lipid is positively charged and interacts with the negatively-charged nucleic acid polymers. The resulting nanostructures containing the nucleic acids are then converted to neutral LNP when dialyzed against a neutral buffer during the ethanol removal step. The LNP formed by this have a distinct electron-dense nanostructured core where the ionizable cationic lipids are organized into inverted micelles around the encapsulated mRNA molecules, as opposed to the traditional bilayer liposomal structures.

[0176] In some embodiments, the LNP may have an average diameter of 20nm to 200nm, 20 to 180nm, 20nm to 170nm, 20nm to 150nm, 20nm to 120nm, 20nm to lOOnm, 20nm to 90nm, 30nm to 200nm, 30 to 180nm, 30nm to 170nm, 30nm to 150nm, 30nm to 120nm, 30nm to lOOnm, 30nm to 90nm, 40nm to 200nm, 40 to 180nm, 40nm to 170nm, 40nm to 150nm, 40nm to 120nm, 40nm to lOOnm, 40nm to 90nm, 40nm to 80nm, 40nm to 70nm, 50nm to 200nm, 50 to 180nm, 50nm to 170nm, 50nm to 150nm, 50nm to 120nm, 50nm to lOOnm, 50nm to 90nm, 60nm to 200nm, 60 to 180nm, 60nm to 170nm, 60nm to 150nm, 60nm to 120nm, 60nm to lOOnm, 60nm to 90nm, 70nm to 200nm, 70 to 180nm, 70nm to 170nm, 70nm to 150nm, 70nm to 120nm, 70nm to lOOnm, 70nm to 90nm, 80nm to 200nm, 80 to 180nm, 80nm to 170nm, 80nm to 150nm, 80nm to 120nm, 80nm to lOOnm, 80nm to 90nm, 90nm to 200nm, 90 to 180nm, 90nm to 170nm, 90nm to 150nm, 90nm to 120nm, or 90nm to lOOnm. The LNP may be sized for easy introduction into organs or tissues, including but not limited to liver, lung, heart, spleen, as well as to tumors. When the size of the LNP is smaller than the above range, it is difficult to maintain stability as the surface area of the LNP is excessively increased, and thus delivery to the target tissue and/or drug effect may be reduced. The LNP may specifically target liver tissue. The LNP may imitate metabolic behaviors of natural lipoproteins very similarly and may be usefully applied for the lipid metabolism process by the liver and therapeutic mechanism through this. During the delivery to hepatocytes or and/or LSEC (liver sinusoidal endothelial cells), the diameter of the fenestrae leading from the sinusoidal lumen to the hepatocytes and LSEC is about 140 nm in Attorney Docket No. SCRB-056/02WO 333322-2431 mammals and about 100 nm in humans, so the composition for delivery having a diameter in the above ranges may have excellent delivery efficiency to hepatocytes and LSEC than the LNP having the diameter outside the above range.

[0177] According to one example, the LNP comprised in the composition for nucleic acid delivery into target cells may comprise the ionizable lipid : phospholipid : cholesterol : lipid- PEG conjugate in the range described above or at a molar ratio of 20 to 50: 10 to 30:30 to 60:0.5 to 5, at a molar ratio of 25 to 45: 10 to 25:40 to 50:0.5 to 3, at a molar ratio of 25 to 45: 10 to 20:40 to 55:0.5 to 3, or at a molar ratio of 25 to 45: 10 to 20:40 to 55: 1.0 to 1.5. The LNP comprising components at a molar ratio in the above range may have excellent delivery efficiency specific to cells of target organs.

[0178] The LNP according to one example exhibits a positive charge under the acidic pH condition by showing a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7, and may encapsulate a nucleic acid with high efficiency by easily forming a complex with a nucleic acid through electrostatic interaction with a therapeutic agent such as a nucleic acid showing a negative charge, and it may be usefully used as a composition for intracellular or in vivo drug delivery of a drug (for example, nucleic acid). Herein, "encapsulation" refers to encapsulating a delivery substance for surrounding and embedding it in vivo efficiently, and the drug encapsulation efficiency (encapsulation efficiency) mean the content of the drug encapsulated in the LNP for the total drug content used for preparation.

[0179] The encapsulation efficiency of the nucleic acids of the composition in the LNP may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 94% or more, or 95% or more. In other embodiments, the encapsulation efficiency of the nucleic acids of the composition in the LNP is over 80% to 99% or less, over 80% to 97% or less, over 80% to 95% or less, 85% or more to 95% or less, 87% or more to 95% or less, 90% or more to 95% or less, 91% or more to 95% or less, 91% or more to 94% or less, over 91% to 95% or less, 92% or more to 99% or less, 92% or more to 97% or less, or 92% or more to 95% or less. As used herein, "encapsulation efficiency" means the percentage of LNP particles containing the nucleic acids to be incorporated within the LNP. In some embodiments, the mRNA encoding the CasX of the disclosure are fully encapsulated in the LNP.

[0180] The target organs to which a nucleic acid is delivered by the LNP include, but are not limited to the liver, lung, heart, spleen, as well as to tumors. The LNP according to one Attorney Docket No. SCRB-056/02WO 333322-2431 example is liver tissue-specific and has excellent biocompatibility and can deliver the nucleic acids of a composition with high efficiency, and thus it can be usefully used in related technical fields such as lipid nanoparticle-mediated gene therapy. In a particular embodiment, the target cell to which the nucleic acids are delivered by the LNP according to one example may be a hepatocyte and/or LSEC in vivo. In other embodiments, the disclosure provides LNP formulated for delivery of the nucleic acids of the embodiments to cells ex vivo.

[0181] The disclosure also provides a pharmaceutical composition comprising an mRNA encoding a CasX described herein, and a pharmaceutically acceptable carrier.

[0182] In certain embodiments, the LNP comprising the nucleic acid(s) has an electron dense core.

[0183] In some embodiments, the disclosure provides LNPs comprising: (a) an mRNA encoding the CasX described herein; (b) one or more ionizable cationic lipids or salts thereof comprising from about 50 mol % to about 85 mol % of the total lipid present in the particle; (c) one or more non-cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and (d) one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle. In some embodiments, the LNP comprises the mRNA encoding the CasX and a gRNA.

[0184] In some embodiments, the disclosure provides LNPs comprising: (a) an mRNA encoding the CasX described herein; (b) a ionizable cationic lipid or a salt thereof comprising from about 52 mol % to about 62 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 36 mol % to about 47 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle. In one particular embodiment, the formulation is a four-component system comprising about 1.4 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 57.1 mol % ionizable cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3 mol % cholesterol (or derivative thereof). In some embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA.

[0185] In another embodiment, the LNP comprises: (a) an mRNA encoding the CasX described herein; (b) a ionizable cationic lipid or a salt thereof comprising from about 46.5 mol % to about 66.5 mol % of the total lipid present in the particle; (c) cholesterol or a Attorney Docket No. SCRB-056/02WO 333322-2431 derivative thereof comprising from about 31.5 mol % to about 42.5 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle. In one particular embodiment, the formulation is a three-component system which is phospholipid-free and comprises about 1.5 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 61.5 mol % ionizable cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, and about 36.9 mol % cholesterol (or derivative thereof). In some embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA.

[0186] Additional formulations are described in PCT Publication No. WO 09/127060 and published US patent application publication numbers US 2011/0071208 Al and US 2011/0076335 Al, the disclosures of which are herein incorporated by reference in their entirety.

[0187] In another embodiment, the LNP comprises: (a) an mRNA encoding the CasX described herein; (b) one or more ionizable lipids or salts thereof comprising from about 2 mol % to about 50 mol % of the total lipid present in the particle; (c) one or more noncationic lipids comprising from about 5 mol % to about 90 mol % of the total lipid present in the particle; and (d) one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol % to about 20 mol % of the total lipid present in the particle. In some embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA.

[0188] In another embodiment, the LNP comprises: (a) an mRNA encoding the CasX described herein; (b) a ionizable cationic lipid or a salt thereof comprising from about 30 mol % to about 50 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 47 mol % to about 69 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 3 mol % of the total lipid present in the particle. In one particular embodiment, the formulation is a four-component system which comprises about 2 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 40 mol % ionizable cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 10 mol % DPPC (or DSPC), and about 48 mol % cholesterol (or derivative thereof). In some embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA. Attorney Docket No. SCRB-056/02WO 333322-2431

[0189] In another embodiment, the LNP comprises: (a) an mRNA encoding the CasX described herein; (b) one or more ionizable cationic lipids or salts thereof comprising from about 50 mol % to about 65 mol % of the total lipid present in the particle; (c) one or more non-cationic lipids comprising from about 25 mol % to about 45 mol % of the total lipid present in the particle; and (d) one or more conjugated lipids that inhibit aggregation of particles comprising from about 5 mol % to about 10 mol % of the total lipid present in the particle. In some embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA.

[0190] In another embodiment, the LNP comprises: (a) an mRNA encoding the CasX described herein; (b) a ionizable cationic lipid or a salt thereof comprising from about 50 mol % to about 60 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 35 mol % to about 45 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 5 mol % to about 10 mol % of the total lipid present in the particle. In some embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA.

[0191] In certain embodiments, the non-cationic lipid mixture in the formulation comprises: (i) a phospholipid of from about 5 mol % to about 10 mol % of the total lipid present in the particle; and (ii) cholesterol or a derivative thereof of from about 25 mol % to about 35 mol % of the total lipid present in the particle. In one particular embodiment, the formulation is a four-component system which comprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about 54 mol % ionizable cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7 mol % DPPC (or DSPC), and about 32 mol % cholesterol (or derivative thereof).

[0192] In another embodiment, the LNP comprises: (a) an mRNA encoding the CasX described herein; (b) a ionizable cationic lipid or a salt thereof comprising from about 55 mol % to about 65 mol % of the total lipid present in the particle; (c) cholesterol or a derivative thereof comprising from about 30 mol % to about 40 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 5 mol % to about 10 mol % of the total lipid present in the particle. In one particular embodiment, the formulation is a three- component system which is phospholipid-free and comprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about 58 mol % ionizable cationic lipid (e.g., DLin-K-C2- DMA) or a salt thereof, and about 35 mol % cholesterol (or derivative thereof). In some Attorney Docket No. SCRB-056/02WO 333322-2431 embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA.

[0193] In another embodiment, the LNP comprises: (a) an mRNA encoding the CasX described herein; (b) a ionizable cationic lipid or a salt thereof comprising from about 48 mol % to about 62 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid comprises about 7 mol % to about 17 mol % of the total lipid present in the particle, and wherein the cholesterol or derivative thereof comprises about 25 mol % to about 40 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 0.5 mol % to about 3.0 mol % of the total lipid present in the particle. In some embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA.

[0194] The disclosure provides pharmaceutical compositions comprising the LNP described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0195] In some embodiments, the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intraportal vein injection, intraperitoneal, intramuscular, subcutaneous, intraocular, and oral routes.

[0196] Excipients can include a salt, an isotonic agent, a serum protein, a buffer or other pH-controlling agent, an anti-oxidant, a thickener, an uncharged polymer, a preservative or a cryoprotectant. Excipients used in compositions of the disclosure may further include an isotonic agent and a buffer or other pH-controlling agent. These excipients may be added for the attainment of preferred ranges of pH (about 6.0-8.0) and osmolarity (about 50-400 mmol/L). Examples of suitable buffers are acetate, borate, carbonate, citrate, phosphate and sulfonated organic molecule buffer. Such buffers may be present in a composition in concentrations from 0.01 to 1.0% (w/v). An isotonic agent may be selected from any of those known in the art, e.g. mannitol, dextrose, glucose and sodium chloride, or other electrolytes. In some embodiments the isotonic agent may be glucose or sodium chloride. The isotonic agents may be used in amounts that impart to the composition the same or a similar osmotic pressure as that of the biological environment into which it is introduced. The concentration of isotonic agent in the composition will depend upon the nature of the particular isotonic agent used and may range from about 0.1 to 10%. When glucose is used, it is preferably used in a concentration of from 1 to 5% w/v, more particularly 5% w/v. When the isotonic agent is sodium chloride, it is preferably employed in amounts of up to 1% w/v, in particular 0.9% Attorney Docket No. SCRB-056/02WO 333322-2431 vi/v. The compositions of the invention may further contain a preservative. Examples of preservatives include polyhexamethylene-biguanidine, benzalkonium chloride, stabilized oxychloro complexes (such as those known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, and thimerosal. Typically, such preservatives are present at concentrations from about 0.001 to 1.0%. Furthermore, the compositions of the invention may also contain a cryopreservative agent. Preferred cryopreservatives are glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, dextran of molecular weight preferable below 100,000 g/mol, glycerol, and polyethylene glycols of molecular weights below 100,000 g/mol or mixtures thereof. Most preferred are glucose, trehalose and polyethylene glycol. Typically, such cryopreservatives are present at concentrations from about 0.01 to 10%.

[0197] Additional pharmaceutical formulations appropriate for administration are applicable in the methods and compositions disclosed herein (see, e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (2023) 23rd ed., Elsevier Publishing; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993).

V. Vectors

[0198] The disclosure provides vectors comprising DNA that serve as a template for transcription of the mRNAs and gRNAs described herein. In some cases, the vectors are utilized for the expression and recovery of the mRNA and gRNA components of the gene editing pair. In other cases, the vectors are utilized for the delivery of the encoding polynucleotides to target cells for the editing of the target nucleic acid, as described more fully, below. In some embodiments, the vectors comprising the DNA include bacterial plasmids, viral vectors, and the like. In some embodiments, an mRNA and a gRNA are templated on the same vector. In some embodiments, an mRNA and a gRNA are templated on different vectors. Suitable vectors are described, for example, in W02022120095A1, WO2020247882A1 and WO2023240162A1, incorporated by reference herein.

[0199] As described in W02022120095A1, WO2020247882A1, and WO2023240162A1, depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector. Attorney Docket No. SCRB-056/02WO 333322-2431

[0200] In some embodiments, the vector is a recombinant expression vector. A recombinant expression vector sequence can be packaged into a virus or virus-like particle (also referred to herein as a "particle" or "virion") for subsequent infection and transformation of a cell, ex vivo, in vitro or in vivo. Such particles or virions will typically include proteins that encapsidate or package the vector genome. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant retroviral vector. Such vectors are described in detail in W02022120095A1, WO2020247882A1, and WO/2022/125843, incorporated by reference herein.

VI. Systems and Methods for Modification of Target Nucleic Acids

[0201] The mRNA encoding CasX proteins of the disclosure have utility in systems designed to modify or edit a target nucleic acid of a gene in a population of cells, when used with a guide ribonucleic acid having a targeting sequence complementary to the target nucleic acid to be modified or edited. Such systems are useful for various applications, including as therapeutics, diagnostics, and for research. The programmable nature of the systems provided herein allows for the precise targeting to achieve the desired effect (nicking, cleaving, modifying, etc.) at one or more regions of predetermined interest in the gene target nucleic acid of a gene in a eukaryotic cell. In some embodiments, it may be desirable to knock-down or knock-out expression of the gene in the subject comprising mutations. In other embodiments, it may be desirable to correct or compensate for the mutation such that a wild-type or a functional gene product is expressed.

[0202] A variety of strategies and methods can be employed to modify the target nucleic acid sequence in a cell using the systems provided herein. Depending on the system components utilized, the modifying or editing event may be a cleavage event followed by introducing random insertions or deletions (indels) or other mutations (e.g., a substitution, duplication, or inversion of one or more nucleotides), for example by utilizing the imprecise non-homologous DNA end joining (NHEJ) repair pathway, which may generate, for example, a frame shift mutation. Alternatively, the editing event may be a cleavage event, or with systems employing two gRNAs targeted to different regions of the target nucleic acid, a dual-cut that results in excision of the intervening sequence. In some embodiments of the Attorney Docket No. SCRB-056/02WO 333322-2431 method, the modification comprises introducing an in-frame mutation in the target nucleic acid. In some embodiments of the method, the modification comprises introducing a frameshifting mutation in the target nucleic acid. In some embodiments of the method, the modification comprises introducing a premature stop codon in the coding sequence in the target nucleic acid. In some embodiments of the method, the modification results in expression of a non-functional protein in the modified cells of the population. As a result of a gene knock-down, the protein activity or function may be attenuated or the protein levels may be reduced or eliminated. In some embodiments of the method, the modification results in at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% reduced expression of the protein in the modified cells of the population in comparison to cells in which the gene has not been modified. In other embodiments, the disclosure provides systems and methods for correcting mutations in the gene wherein a corrective sequence is knocked-in by introducing insertions or deletions at select locations. The systems can be specifically designed for use in the methods to modify the target nucleic acid of a gene in eukaryotic cells; either in vitro, ex vivo, or in vivo in a subject. In some embodiments of the method, the modifying of the cell occurs in vitro. In some embodiments of the method, the modifying of the cell occurs ex vivo, wherein the modified cells can be administered to a subject. In some embodiments of the method, the modifying of the cell occurs in vivo.

[0203] In some embodiments of the method, the cell is a eukaryotic cell. In some embodiments of the method, the eukaryotic cell is selected from the group consisting of a rodent cell, a mouse cell, a rat cell, a primate cell, and a non-human primate cell. In some embodiments of the method, the eukaryotic cell is a human cell. In some embodiments of the method, the cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell, a germ cell, a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B-cell, an NK cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplanted expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, a fibroblast cell, an osteoblast cell, a chondrocyte cell, an exogenous cell, an endogenous cell, a stem cell, a hematopoietic stem Attorney Docket No. SCRB-056/02WO 333322-2431 cell, a bone-marrow derived progenitor cell, a myocardial cell, a skeletal cell, a fetal cell, an undifferentiated cell, a multi-potent progenitor cell, a unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, an autologous cell, and a post-natal stem cell.

[0204] In some embodiments, the systems provided herein for modification of the target nucleic acid comprise an mRNA encoded by a sequence selected from the group consisting of SEQ ID NOS: 36-43, or a sequence at least 60% identical, at least 70% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto. In some embodiments, the systems provided herein for modification of the target nucleic acid comprise an mRNA encoded by a sequence selected from the group consisting of SEQ ID NOS: 36-43. In a particular embodiment, the systems are formulated in LNP that encapsulate the mRNA sequence encoded by a sequence selected from the group consisting of SEQ ID NOS: 36-43. In other embodiments, the mRNAs are encoded by DNA that are incorporated into vectors, such as an adenoviral vector, a recombinant Adeno- Associated Viral (AAV) vector, a herpes simplex virus (HSV) vector, or a retroviral vector, e.g., a lentiviral vector, described in WO2020247882A1 and WO 2022120095, incorporated by reference herein, for delivery of the CasX of the disclosure. In other embodiments, the mRNAs of the disclosure are used to produce CasX protein for incorporation into a virus-like particle (VLP).

[0205] In one embodiment of the method, the system is introduced into the cells using LNP encompassing mRNA encoding the engineered CasX of any of the embodiments disclosed herein. In some embodiments, the LNP encompasses an mRNA of the sequence of SEQ ID NO: 37 or SEQ ID NO: 41, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity thereto, encoding the engineered CasX 515 (SEQ ID NO: 5). In another embodiment, the LNP encompasses an mRNA of the sequence of SEQ ID NO: 39 or SEQ ID NO: 43, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at Attorney Docket No. SCRB-056/02WO 333322-2431 least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity thereto, encoding the engineered CasX 812 (SEQ ID NO: 7). In another embodiment, the LNP encompasses an mRNA of the sequence of SEQ ID NO: 36 or SEQ ID NO: 40, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity thereto, encoding the engineered CasX 491 (SEQ ID NO: 4). In another embodiment, the LNP encompasses an mRNA of the sequence of SEQ ID NO: 38 or SEQ ID NO: 42, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity thereto, encoding the engineered CasX 676 (SEQ ID NO: 6). In some embodiments of the foregoing, the LNP further encompass a gRNA of the disclosure. In some embodiments of the method, the cells to be modified are selected from the group consisting of rodent cells, mouse cells, rat cells, and non-human primate cells. In other embodiments of the method, the cells to be modified are human cells. In some embodiments of the method, the modification of the population of cells occurs in vivo in a subject, wherein the subject is selected from the group consisting of a rodent, a mouse, a rat, a non-human primate, and a human. In some embodiments of the methods, the modified cell is a hepatocyte, or a cell of the intestine, the kidney, the central nervous system, a smooth muscle cell, macrophage or a cell of arterial walls such as the endothelium.

[0206] The LNP can be administered by a route of administration selected from the group consisting of intravenous, intraarterial, intraportal vein injection, intraperitoneal, intramuscular, intracerebroventricular, intracisternal, intrathecal, intracranial, intralumbar, intraocular, subcutaneous, and oral routes.

[0207] The systems and methods described herein can be used to engineer a variety of cells in which mutations in are associated with disease, e.g., cells of the liver, the intestine, the kidney, the central nervous system, smooth muscle cells, macrophages or cells of arterial walls such as the endothelium, to produce a cell or cells in which the comprising mutations is corrected or knocked-out.

[0208] In some embodiments, the disclosure provides compositions for use in the manufacture of a medicament for the treatment a subject having a disease. In some embodiments, the composition comprises an mRNA encoding a CasX protein of any of the Attorney Docket No. SCRB-056/02WO 333322-2431 embodiments disclosed herein. In some embodiments, the composition comprises a gRNA with a targeting sequence complementary to a gene with a mutation associated with a disease and an mRNA encoding a CasX protein of any of the embodiments disclosed herein. In some embodiments, the composition comprises an LNP of any of the embodiments disclosed herein. In some embodiments, the composition comprises a combination of the foregoing.

VII. Kits

[0209] The disclosure provides kits comprising any of the mRNA, gRNA, vectors, systems, LNP or compositions described herein and a suitable container. In some embodiments, the kit comprises instructions for use. In some embodiments, the kit comprises a buffer, an excipient, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[0210] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.

299911683

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EXAMPLES

Example 1: Demonstration that altering the UTR sequences of the engineered CasX mRNA can affect CasX-mediated editing

[0211] 5’ and 3 ’ UTRs can be essential and required for efficient translation of mRNA. Here, experiments were performed to demonstrate that altering the 5’ and 3’ UTR sequences of the engineered CasX mRNA affects CasX-mediated editing at a target locus when CasX mRNA and targeting gRNAs were delivered in vitro via transfection.

Materials and Methods:

In vitro transcription (IVT) of CasX mRNA:

[0212] CasX 676 mRNA was generated by IVT. Briefly, constructs encoding for a 5 ’UTR region, a codon-optimized CasX 676 with flanking c-MYC NLS, and a 3 ’UTR region were cloned into a plasmid containing a T7 promoter and 80-nucleotide poly(A) tail. The resulting plasmid was linearized prior to use for IVT reactions, which were carried out with CleanCap® AG and Nl-methyl-pseudouridine. For the 5’ cap, the CleanCap® AG contains a m7G(5')ppp(5')mAG structure, where “m7G” denotes N⁷-methylguanosine, “mA” denotes 2’0-methyladenosine, and (5’)ppp(5’) denotes a 5’ to 5’ triphosphate bridge. An extra guanine nucleotide was incorporated following the CleanCap® AG to enhance transcription initiation, resulting in the incorporation of m7G(5’)ppp(5’)mAGG as the full 5’ cap structure. Meanwhile, the substitution of the uridine ribonucleoside to Nl-methyl-pseudouridine improves mRNA performance and reduces mRNA immunogenicity.

[0213] IVT reactions were subsequently subjected to DNase digestion to remove template DNA and purification using an oligo-dT column. In this example, two mRNAs encoding CasX 676 with different pairs of 5 ’ and 3 ’ UTRs were generated for assessment in vitro. The encoding sequences of the two CasX mRNA configurations are detailed in Table 10. Full-length RNA sequences encoding the CasX mRNA with the chemical modifications are listed in Table 11.

Table 10: Encoding sequences of the two CasX mRNA molecules assessed in this example*

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Components are listed in a 5’ to 3’ order within the constructs

Table 11: Full-length RNA sequences of CasX mRNA molecules assessed in this example. The 5’ cap (m7G(5’)ppp(5’)mAGG), discussed in the example herein, is not shown in the table. Modification ‘mq/’ = Nl-methyl-pseudouridine

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Synthesis of gRNAs:

[0214] In this example, gRNAs targeting the mouse PCSK9 locus were designed using gRNA scaffold 174 with a vl modification profile (see Example 3) and chemically synthesized. The sequences of the EGS' -targeting spacers are listed in Table 12.

Table 12: Sequences of spacers targeting the mouse PCSK9 locus assayed in this example

Transfection of CasX mRNA and gRNA into mouse Hepal-6 cells in vitro'.

[0215] Editing at the mouse PCSK9 locus was assessed by delivering in vitro transcribed CasX mRNA (CasX mRNA #1 or CasX mRNA #2; see Table 10) and synthesized gRNAs targeting PCSK9 into Hepal-6 cells via transfection. Briefly, each well of 20, 000 Hepal-6 cells were lipofected with in vitro transcribed mRNA coding for CasX 676 and a ECS' -targeting gRNA. After a media change, transfected cells were harvested at 20 hours post-transfection for Attorney Docket No.: SCRB-056/02WO 33322-2431 editing assessment at the CGS' locus by next-generation sequencing (NGS). As experimental controls, individual transfections of CasX mRNA #1 and CasX mRNA #2 without gRNAs were performed.

NGS processing and analysis:

[0216] Genomic DNA (gDNA) from harvested cells were extracted using the Zymo Quick- DNA Miniprep Plus kit following the manufacturer’s instructions. Target amplicons were formed by amplifying regions of interest from 50-100 ng of extracted gDNA with a set of primers targeting the human PCSK9 locus. These gene-specific primers contained an additional sequence at the 5' ends to introduce Illumina reads 1 and 2 sequences. Further, they contained a 16-nucleotide random sequence that functioned as a unique molecular identifier (UMI). The quality and quantification of the amplicon was assessed using a Fragment Analyzer DNA analyzer kit (Agilent, dsDNA 35-1500 bp). Amplicons were sequenced on the Illumina MiSeq™ according to the manufacturer’s instructions. Raw fastq sequencing files were processed by trimming for quality and adapter sequences and merging read 1 and read 2 into a single insert sequence; insert sequences were then analyzed by the CRISPResso2 (v 2.0.29) program. The percentage of reads modified in a window around the 3' end of the spacer was determined. The activity of the CasX molecule was quantified as the total percent of reads that contain insertions, substitutions, and/or deletions anywhere within this window for each.

Results:

[0217] CasX-mediated editing at the mouse PCSK9 locus was used to evaluate the effects of incorporating different 5’ and 3’ UTRs into the engineered CasX mRNA. The plot in FIG. 1 shows the quantification of percent editing measured as indel rate at the PCSK9 locus in mouse Hepal-6 cells transfected with CasX 676 mRNA #1 or CasX 676 mRNA #2 with the indicated CCS' -targeting gRNAs. The data demonstrate that for all targeting spacers tested in this experiment, CasX mRNA #2, the mRNA with a synthetic 5’ UTR and a mouse HBA 3’ UTR, consistently exhibited higher editing levels at the mouse PCSK9 locus compared to editing levels achieved by CasX mRNA #1, the mRNA with human HBA 5’ and 3’ UTRs. Specifically, the highest level of editing rate achieved was with spacer 27.116, where use of CasX mRNA #2 resulted in -35% editing efficiency compared to -20% editing level by CasX mRNA #1 (FIG. 1).

[0218] The results demonstrate that altering the 5 ’UTR and 3 ’UTR sequences of the CasX mRNA can affect the editing activity of CasX at a target locus in a cell-based assay, and that Attorney Docket No.: SCRB-056/02WO 33322-2431 the combination of a synthetic 5’ UTR and mouse HBA 3’ UTR was particularly effective for generating an mRNA encoding CasX that produced a high level of editing.

Example 2: CasX mRNA and /Y.SA -targeting gRNA can be delivered via LNPs to achieve editing at the human PCSK9 locus in vitro

[0219] Experiments were performed to demonstrate that delivery of LNPs encapsulating CasX mRNA and a PGS'AV-targeting gRNA can induce editing at the endogenous human PCSK9 locus in primary human hepatocytes. Here, CasX 515 was selected for assessment given its improvement in specificity while maintaining activity compared to the earlier prototype CasX 491, and CasX 812 was selected given its increased specificity.

Materials and Methods:

[0220] Generation of CasX mRNAs encoding for CasX 515 and CasX 812 was performed by IVT, following similar methods described earlier in Example 1. Briefly, constructs encoding for a synthetic 5 ’UTR, a codon-optimized CasX 515 or CasX 812 with flanking c-MYC NLS, and a 3 ’UTR derived from the mouse hemoglobin alpha (mHBA) were cloned into a plasmid containing a T7 promoter and 79-nucleotide poly(A) tail. The resulting plasmid was linearized prior to use for IVT reactions, which were carried out with CleanCap® AG and N1 -methylpseudouridine (as described in Example 1). The DNA sequences encoding the CasX 515 or CasX 812 mRNA molecules are listed in Table 13, with the corresponding mRNA sequences with the chemical modifications listed in Table 14. The protein sequences for CasX 515 and CasX 812 resulting from expression of the IVT mRNA molecules are listed in Table 15.

Table 13: Encoding sequences of the two CasX mRNA molecules assessed in this example*.

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*Components are listed in a 5’ to 3’ order within the constructs

Table 14: Full-length RNA sequences of CasX mRNA molecules assessed in this example. The CleanCap® AGG 5’ cap is not shown in the table. Modification ‘my’ =

N 1-methyl-pseudouridine

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Table 15: Full-length protein sequences of CasX molecules assessed in this example.

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Synthesis of gRNAs:

[0221] In this example, gRNAs targeting the human PCSK9 locus were designed using gRNA scaffold 316 (SEQ ID NO: 154) and chemically synthesized. The sequences of the ECS' -targeting gRNAs with the vl modification profile (as described in Example 3, below) are listed in Table 16. A schematic of the sites of chemical modifications for a ‘vl’ profile of the gRNA scaffold variant 316 is shown in FIG. 12A.

Table 16: Sequences of chemically modified gRNAs targeting the human PCSK9 locus assayed in this example

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Formulation of lipid nanoparticles (LNPs):

[0222] CasX mRNA and gRNA were encapsulated into LNPs using GenVoy-ILM™ lipids on the Precision NanoSystems Inc. (PNI) Ignite™ Benchtop System and following the manufacturer’s guidelines. GenVoy-ILM™ lipids are manufactured by PNI, with a proprietary composition of ionizable lipid:DSPC:cholesterol:stabilizer at 50: 10:37.5:2.5 mol%.

[0223] Briefly, to formulate LNPs, equal mass ratios of CasX mRNA and gRNA were diluted in PNI Formulation Buffer, pH 4.0. GenVoy-ILM™ was diluted 1 : 1 in anhydrous ethanol. mRNA/gRNA co-formulations were performed using a 6: 1 N/P ratio (N/P: nitrogen from the cationic/ionizable lipid and phosphate from the nucleic acid). The RNA and lipids were run through a PNI laminar flow cartridge at a predetermined flow rate ratio (RNA: Genvoy-ILM™) on the PNI Ignite™ Benchtop System. After formulation, the LNPs were diluted in PBS, pH 7.4, to decrease the ethanol concentration and increase the pH, which increases the stability of the particles. Buffer exchange of the mRNA/sgRNA-LNPs was achieved by overnight dialysis into PBS, pH 7.4, at 4°C using 10k Slide-A-Lyzer™ Dialysis Cassettes (Thermo Scientific™). Following dialysis, the mRNA/gRNA-LNPs were concentrated to > 0.5 mg/mL using 100 kDa Amicon®-Ultra Centrifugal Filters (Millipore) and then filter-sterilized. Formulated LNPs were analyzed on a Stunner (Unchained Labs) to determine their diameter and poly dispersity index (PDI). Encapsulation efficiency and RNA concentration was determined by RiboGreen™ assay using Invitrogen's Quant-iT™ Ribogreen™ RNA assay kit. LNPs were used in various experiments as described herein to deliver CasX mRNA and gRNA to target cells and tissue.

Delivery of LNPs encapsulating CasX mRNA and targeting gRNA into primary human hepatocytes:

[0224] Two lots (lot #31 and lot #51) of primary human hepatocytes derived from two different donors (Lonza Biologies), were used in these experiments to assess CasX:gRNA- mediated editing at the human PCSK9 locus when delivered by LNPs. For each lot, -50,000 cells, cultured in Williams’ E media supplemented with FBS, PenStrep, L-glutamine, ITS (insulin, transferrin, sodium selenite), dexamethasone, and Z-VAD-FMK, were seeded per well in a 96-well plate. The next day, seeded cells were treated with varying concentrations of LNPs, Attorney Docket No.: SCRB-056/02WO 33322-2431 which were prepared in five 3-fold serial dilutions starting at 1,200 ng. These LNPs were formulated to encapsulate CasX 515 or CasX 812 mRNA and a EGS' -targeting gRNA incorporating scaffold variant 316 with either spacer 6.1 or 6.8 (vl; see Table 16). Media was changed two days after LNP treatment, and cells were cultured for three additional days prior to harvesting 1) the media supernatant to measure PCSK9 secretion levels and 2) treated cells for gDNA extraction for editing assessment at the PCSK9 locus by NGS. Briefly, for editing assessment, amplicons were amplified from 200 ng of extracted gDNA with primers targeting the human PCSK9 locus and processed as described in Example 1. PCSK9 secretion levels were measured by ELISA using the BioLegend® ELISA MAX™ kit following the manufacturer’s instructions. Treatment with LNPs co-encapsulating a non-targeting gRNA with CasX 515 mRNA served as an experimental control.

Results:

[0225] Two lots of primary human hepatocytes were treated with LNPs, which coencapsulated either CasX 515 or CasX 812 mRNA and a CS P-targeting gRNA, at various doses and harvested five days post-treatment to assess effects on PCSK9 secretion (FIGS. 2A- 2D) and editing at the PCSK9 locus (FIGS. 3 A-3C). The results in FIGS. 2A-2D demonstrate that the effects from treatment with LNPs to deliver either CasX 515 or CasX 812 mRNA were comparable, such that similar levels of reduced PCSK9 secretion were observed in a dosedependent manner. Furthermore, the data in FIGS. 3A-3C show that use of either CasX 515 or CasX 812 mRNA resulted in similar levels of editing at the PCSK9 locus in primary human hepatocytes in a dose-dependent manner, corroborating findings observed in FIGS. 2A-2D.

[0226] Altogether, the results from these experiments demonstrate that delivery of LNPs encapsulating an mRNA encoding CasX and a EGS' -targeting gRNA was able to induce efficacious editing at the endogenous human PCSK9 locus in primary human hepatocytes, which resulted in substantial reduction in secreted PCSK9 levels.

Example 3: Design and assessment of modified gRNAs in improving editing when delivered together with CasX mRNA in vitro and in vivo

[0227] Experiments were performed to identify new gRNA variant sequences, and demonstrate that chemical modifications of these gRNA variants enhance the editing efficiency of the CasX:gRNA system when delivered in vitro in conjunction with CasX mRNA. Attorney Docket No.: SCRB-056/02WO 33322-2431

Materials and Methods:

Synthesis of gRNAs:

[0228] All gRNAs tested in this example were chemically synthesized and were derived from gRNA scaffolds 174, 235, and 316. The sequences of gRNA scaffolds 174, 235, and 316 and their chemical modification profiles are listed in Table 17. The sequences of the resulting gRNAs, including spacers targeting PCSK9, B2M, or RO SA26, and their chemical modification profiles assayed in this example are listed in Table 18. A schematic of the structure of gRNA scaffold variants 174, 235, and 316 are shown in FIGS. 7A-C, respectively, and the sites of chemical modifications of the gRNA variants are shown schematically in FIGS. 4 A, 4B, 6, 12 A, and 12B.

Table 17: Sequences of gRNA scaffolds with their different chemical modification profiles (denoted by version number), where “NNNNNNNNNNNNNNNNNNNN” is a spacer placeholder. Chemical modifications: * = phosphorothioate bond; m = 2’OMe modification

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Table 18: Sequences of gRNAs with their different chemical modification profiles

(denoted by version number) assayed in this example. Chemical modifications: * = phosphorothioate bond; m = 2’OMe modification

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Note that gRNAs annotated with a vl’ design contain one less phosphorothioate bond on the 3’ end of the gRNA. gRNAs annotated with vl* contain one extra phosphorothioate bond on Attorney Docket No.: SCRB-056/02WO 33322-2431 the 3 ’end of the gRNA. gRNAs annotated with a v9* contain an extra phosphorothioate bond on the 3 ’ end of the gRNA.

Biochemical characterization of gRNA activity:

[0229] Target DNA oligonucleotides with fluorescent moi eties on the 5’ ends were purchased commercially (sequences listed in Table 19). Double-stranded DNA (dsDNA) targets were formed by mixing the oligos in a 1 : 1 ratio in lx cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCh), following by heating to 95°C for 10 minutes, and then allowing the solution to cool to room temperature. CasX ribonucleoproteins (RNPs) were reconstituted with CasX 491 and the indicated gRNAs at a final concentration of 1 pM with 1.2-fold excess of the indicated gRNA in lx cleavage buffer. RNPs were allowed to form at 37°C for 10 minutes.

[0230] The effects of various structural and chemical modifications to the gRNA scaffold on the cleavage rate of CasX 491 RNPs were determined. Cleavage reactions were prepared with final RNP concentrations of 200 nM and final target concentrations of 10 nM, and reactions were carried out at 16°C and initiated by the addition of the labeled target DNA substrate (Table 19). Aliquots of reactions were taken at 0.25, 0.5, 1, 2, 5, and 10 minutes and quenched by adding an equal volume of 95% formamide with 20 mM EDTA. Samples were denatured at 95°C for 10 minutes and resolved on a 10% urea-PAGE gel. Gels were imaged on a Typhoon™ laser-scanner platform and quantified using ImageQuant™ TL 8.2 image analysis software (Cytiva™). The apparent first-order rate constant of non-target strand cleavage (kcieave-) was determined for each CasX:gRNA combination.

[0231] To determine the competent fraction formed by each gRNA, cleavage reactions were prepared with final RNP concentrations of 100 nM and final target concentrations of 100 nM. Reactions were carried out at 37°C and initiated by the addition of the labeled target substrate (Table 19). Aliquots were taken at 0.5, 1, 2, 5, 10, and 30 minutes and quenched by adding an equal volume of 95% formamide with 25 mM EDTA. Samples were denatured by heating at 95°C for 10 minutes and resolved on a 10% urea-PAGE gel. Gels were imaged and quantified as above. CasX was assumed to act as a single-turnover enzyme under the assayed conditions, as indicated by the observation that sub-stoichiometric amounts of enzyme would fail to cleave a greater-than-stoichiometric amount of target substrate even under extended time-scales, and instead would approach a plateau that scaled with the amount of enzyme present. Thus, the Attorney Docket No.: SCRB-056/02WO 33322-2431 fraction of target substrate cleaved over long time-scales by an equimolar amount of RNP would be indicative of the fraction of RNP that was properly formed and active for cleavage. The cleavage traces were fitted with a biphasic rate model, as the cleavage reaction clearly deviated from monophasic under this concentration regime. The plateau of each fit was determined and reported as the active fraction for each RNP in Table 22.

Table 19: Sequences of target DNA substrate oligonucleotides with fluorescent moieties on the 5’ ends used for biochemical characterization of gRNA activity. /700/ = IRDye700; /800/ = IRDye800

In vitro transcription of CasX mRNA:

[0232] DNA templates encoding for CasX 491 (see Table 20 for encoding sequences) used for in vitro transcription were generated by PCR using forward primers containing a T7 promoter, followed by agarose gel extraction of the appropriately sized DNA. 25 ng/pL final concentration of template DNA was used in each in vitro transcription reaction that was carried out following the manufacturer's recommended protocol with slight modifications. Following in vitro transcription reaction incubation for 2-3 hours at 37°C, which were carried out with CleanCap® AG and Nl-methyl-pseudouridine, DNAse digestion of template DNA and column-based purification using the Zymo RNA miniprep kit were performed. The poly(A) tail was added using E. coli PolyA Polymerase following the manufacturer's protocol, followed by column-based purification as stated above. Poly(A) tailed in vitro transcribed RNA was eluted in RNAse free water, analyzed on an Agilent TapeStation for integrity, and flash frozen prior to storage at -80°C.

Table 20: Encoding sequences of the CasX mRNA molecules assessed in this example*

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Components are listed in a 5’ to 3’ order within the constructs

In vitro delivery of gRNA and CasX mRNA via transfection:

[0233] Editing at the PCSK9 locus and consequential effects on secreted PCSK9 levels were assessed for conditions using CasX 491 mRNA co-delivered with a EGS' -targeting gRNA with scaffold variant 174 compared to conditions where a ECS' -targeting gRNA with scaffold variant 316 was used. 100 ng of in vitro transcribed mRNA coding for CasX 491 with a P2A and mScarlet fluorescent protein was transfected into HepG2 cells with version 1 (vl) of gRNAs 174-6.7, 174-6.8, 316-6.7, and 316-6.8 (see Table 18) using lipofectamine. After a media change, the following were harvested at 28 hours post-transfection: 1) transfected cells were harvested for editing assessment at the PCSK9 locus by NGS; 2) media supernatant was harvested to measure secreted PCSK9 protein levels by ELISA. For editing analysis by NGS, amplicons were amplified from 200 ng of extracted gDNA with a set of primers targeting the PCSK9 locus and processed as described earlier in Example 1. Secreted PCSK9 levels in the media supernatant were also analyzed using a fluorescence resonance energy transfer-based immunoassay from CISBio following the manufacturer’s instructions. Here, a gRNA using scaffold 174 with spacer 7.37 (vO; see Table 18), which targeted the endogenous B2M (beta-2- microglobulin) locus, served as the non-targeting (NT) control. These results are shown in FIG. 8.

[0234] To compare the editing potency of version 0 (vO) and version 1 (vl) of 2A7-targeting gRNAs, ~6E4 HepG2 hepatocytes were seeded per well of a 96-well plate. 24 hours later, Attorney Docket No.: SCRB-056/02WO 33322-2431 seeded cells were co-transfected using lipofectamine with 100 ng of in vitro transcribed mRNA coding for CasX 491 and different doses (1, 5, or 50 ng) of either vO or vl of the 7>2A/-targeting gRNA containing scaffold variant 174 and spacer 7.37 (see Table 18). Six days posttransfection, cells were harvested for B2M protein expression analysis via immunostaining of the B2M-dependent HLA protein, followed by flow cytometry using the Attune™ NxT flow cytometer. These results are shown in FIG. 5.

[0235] VI through v6 variants of chemically-modified CS V-targeting gRNAs (Table 18) were assessed fortheir effects on editing potency and consequential effects on secreted PCSK9 levels in vitro. Briefly, 100 ng of in vitro transcribed mRNA coding for CasX variant 491 and a P2A and mScarlet fluorescent protein was transfected into HepG2 cells with 50 ng of the indicated chemically-modified gRNA using lipofectamine. After a media change, the following were harvested at 28 hours post-transfection: 1) transfected cells for editing assessment at the PCSK9 locus by NGS as described above; 2) media supernatant to measure secreted PCSK9 protein levels by ELISA, as described above. Here, a 7>2A/-targeting gRNA was used as a nontargeting control. These results are shown in Table 23.

[0236] LNP co-formulations were generated as described in Example 2.

Delivery of LNPs encapsulating CasX mRNA and targeting gRNAs in vitro'.

[0237] -50,000 HepG2 cells, cultured in DMEM/F-12 media containing 10% FBS and 1% PenStrep, were seeded per well in a 96-well plate. The next day, seeded cells were treated with varying concentrations of LNPs, which were prepared in six 2-fold serial dilutions starting at 250 ng. These LNPs were formulated to encapsulate CasX 491 mRNA and a 7>2A/-targeting gRNA incorporating either scaffold variant 174 or 316 with spacer 7.9 (vl; see Table 18). Media was changed 24 hours after LNP treatment, and cells were cultured for six additional days prior to harvesting for gDNA extraction for editing assessment at the B2M locus by NGS and B2M protein expression analysis via HLA immunostaining, followed by flow cytometry using the Attune NxT flow cytometer. Briefly, for editing assessment, amplicons were amplified from 200 ng of extracted gDNA with primers targeting the human B2M locus and processed as described in Example 1. The results of these assays are shown in FIGS. 9A and 9B.

[0238] -20,000 mouse Hepal-6 hepatocytes were seeded per well in a 96-well plate. The following day, seeded cells were treated with varying concentrations of LNPs, which were prepared in eight 2-fold serial dilutions starting at 1000 ng. These LNPs were formulated to Attorney Docket No.: SCRB-056/02WO 33322-2431 encapsulate CasX 676 mRNA #2 (see Table 20) and a RO SA 26-targeting gRNA incorporating scaffold variant 316 with spacer 35.2 (vl or 5; see Table 18). Media was changed 24 hours post-treatment with LNPs, and cells were cultured for seven additional days prior to harvesting for gDNA extraction for editing assessment at the ROSA26 locus by NGS. Briefly, amplicons were amplified from extracted gDNA with primers targeting the mouse ROSA26 locus and processed as described in Example 1. The results of this experiment are shown in FIG. 10A. Delivery of LNPs encapsulating CasX mRNA and targeting gRNA in vivo:

[0239] To assess the effects of using vl and v5 of scaffold 316 in vivo, CasX 676 mRNA #2 (see Table 20) and a RO SA 26-targeting gRNA using scaffold 316 with spacer 35.2 (vl or v5; see Table 18) were encapsulated within the same LNP using a 1 : 1 mass ratio for mRNA:gRNA. LNP co-formulations were performed as described in Example 2. Formulated LNPs were buffer-exchanged to PBS for in vivo injection. LNPs were administered intravenously through the retro-orbital sinus into 4-week old C57BL/6 mice. Mice were observed for five minutes after injection to ensure recovery from anesthesia before being placed into their home cage. Naive, uninjected animals served as experimental controls. Six days post-administration, mice were euthanized, and the liver tissue was harvested for gDNA extraction using the Zymo Research Quick DNA/RNA Miniprep kit following the manufacturer’s instructions. Target amplicons were then amplified from the extracted gDNA with a set of primers targeting the mouse ROSA26 locus and processed for editing assessment by NGS as described earlier in Example 1. The results of this experiment are shown in FIG. 10B.

[0240] To compare the effects of using v7, v8, and v9 of scaffold 316 on editing at the PCSK9 locus in vivo, CasX 676 mRNA #1 (see Table 21 for sequences) and a PC SK9 -targeting gRNA using scaffold 316 with spacer 27.107 (vl, v7, v8, or v9; see Table 18), were encapsulated within the same LNP using a 1 : 1 mass ratio for mRNA:gRNA for each gRNA. LNPs were administered retro-orbitally into 6-week old C57BL/6 mice, as described above, and mice were euthanized seven days post-injection to harvest liver tissue for gDNA extraction for editing assessment by NGS at the PCSK9 locus. The results of this experiment are shown in FIG. 11.

Table 21: Encoding sequences of CasX 676 mRNA #1 molecule

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Components are listed in a 5’ to 3’ order within the constructs

Results:

Assessing the effects of various chemical modifications on gRNA activity:

[0241] Several studies involving Cas9 have demonstrated that chemical modifications of the gRNA resulted in significantly improved editing activity when delivered with Cas9 mRNA. Following delivery of Cas9 mRNA and gRNA into target cells, unprotected gRNA is susceptible to degradation during the mRNA translation process. Addition of chemical modifications such as 2’0-methyl (2’0Me) groups and phosphorothioate bonds can reduce the susceptibility of the gRNA to cellular RNases, but also have the potential to disrupt folding of the gRNA and its interactions with the CRISPR-Cas protein. Given the lack of structural similarity between CasX and Cas9, as well as their respective gRNAs, appropriate chemical modification profiles must be designed and validated de novo. Using published structures of wild-type CasX from Deltaproteobacteria (PDB codes 6NY1, 6NY2, and 6NY3) as a reference, residues that appeared potentially amenable to modification were selected. However, the published structures were of a wild type CasX ortholog and gRNA distinct from the species used as the basis for the engineered variants presented here, and they also lacked the resolution to confidently determine interactions between protein side-chains and the RNA backbone. These limitations introduced a significant amount of ambiguity into determining which nucleotides might be safely modified. As a result, six profiles of chemical modifications (denoted as versions) were designed for initial testing, and these six profiles are illustrated in Attorney Docket No.: SCRB-056/02WO 33322-2431

FIGS. 4A and 4B. The vl profile was designed as a simple end-protected structure, where the first and last three nucleotides were modified with 2’0Me and phosphorothioate bonds. In the v2 profile, a 3’ UUU tail was added to mimic the termination sequence used in cellular transcription systems and to move the modified nucleotides outside of the region of the spacer involved in target recognition. The v3 profile included the end protection as in vl, as well as the addition of 2’0Me modifications at all nucleotides identified to be potentially modifiable based on structural analysis. The v4 profile was modeled based on v3, but with all the modifications in the triplex region removed, as this structure was predicted to be more sensitive to any perturbation of the RNA helical structure and backbone flexibility. The v5 profile maintained chemical modifications in the scaffold stem and extended stem regions, while the v6 profile harbored modifications only in the extended stem. The extended stem is a region that would become fully exposed to solvent in the RNP and is amenable to replacement by other hairpin structures, and therefore presumably relatively insensitive to chemical modifications. [0242] The minimally modified vl gRNA was initially assessed compared to an unmodified gRNA (vO) to determine the potential benefit of such chemical modifications on editing when the gRNA was co-delivered with CasX mRNA to target cells. Modified (vl) and unmodified (vO) 2A/-targeting gRNAs with spacer 7.37 were co-transfected with CasX mRNA into HepG2 cells, and editing at the B2M locus was measured by loss of surface presentation of the B2M-dependent HLA complex, as detected by flow cytometry (FIG. 5). The data demonstrate that use of the vl gRNA resulted in substantially greater loss of B2M expression compared to the levels seen with vO gRNA across the various doses, thereby confirming that end modifications of the gRNA increased CasX-mediated editing activity upon delivery of the CasX mRNA and gRNA.

[0243] The broader set of gRNA chemical modification profiles were assessed using PCSK9- targeting gRNAs using scaffold variant 235 and spacers 6.7 and 6.8 to determine whether the additional chemical modifications would be able to support the formation of active RNPs. In vitro cleavage assays described above were performed to determine kcieave and fraction competence for these engineered gRNAs harboring the various chemical modification profiles. The results from these in vitro cleavage assays are shown in Table 22. The data demonstrate that gRNAs with the v3 profiles exhibited no activity, an indication that the addition of some chemical modifications significantly interfered with RNP formation or activity. Adding v4 chemical modifications resulted in a reasonable cleavage rate in the excess RNP condition, but Attorney Docket No.: SCRB-056/02WO 33322-2431 exhibited very low fraction competence. The difference between v3 and v4 modifications confirmed that modifications in the triplex region prevented the formation of any active RNP, either due to the inability of the gRNA to fold properly or a disruption in the gRNA-protein interactions. The reduced fraction competence resulting from appending v4 modifications suggest that while the gRNA was able to successfully assemble with the CasX protein to form a cleavage-competent RNP, a large majority of the gRNA was misfolded, or that the appended chemical modifications reduced the affinity of the gRNA for the CasX protein and impeded the efficiency of RNP formation. Application of the v5 or v6 profiles resulted in competent fractions that were comparable to, but slightly lower than, those obtained for reactions using the vl and v2 modifications. While the kcieave values were relatively consistent between v5 and v6 gRNAs, both v5 and v6 gRNAs achieved nearly half of the kcieave values for vl and v2 gRNAs. The reduced kcieave value for v6 gRNA was particularly surprising, given the lack of expected interaction between the gRNA and CasX protein in the modified extended stem. However, for both v5 and v6 gRNAs, it is possible that the reduced flexibility of the gRNA, resulting from the 2’0Me modifications, inhibited structural changes in the RNP required for efficient cleavage, or that the modified initial base-pairs of the hairpin involved in CasX protein interaction had been negatively impacted by the inclusion of the 2’0Me groups.

Table 22: Parameters of cleavage activity assessed for CasX RNPs with the various PC.SA -targeting gRNAs using scaffold 235 and harboring the indicated chemical modification profile, denoted by version number.

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[0244] The chemically-modified EGS' -targeting gRNAs based on scaffold 235 were subsequently assessed for editing in a cell-based assay. CasX mRNA and chemically modified ECS' -targeting gRNAs were co-transfected into HepG2 cells using lipofectamine. Editing levels were measured by indel rate at the PCSK9 locus by NGS and secreted PCSK9 levels by ELISA, and the data are displayed in Table 23. The data demonstrate that use of v3 and v4 gRNAs resulted in minimal editing activity at the PCSK9 locus, consistent with findings from the biochemical in vitro cleavage assays shown in Table 22. Meanwhile, use of v5 and v6 gRNAs resulted in editing levels, measured by indel rate and PCSK9 secretion, that were slightly lower than the levels attained with use of vl and v2 gRNAs (Table 23). Specifically, the results show that use of vl and v2 gRNAs, which harbored end modifications, resulted in -80-85% editing at the PCSK9 locus, indicating that adding chemical modifications to the gRNA ends was sufficient to achieve efficient editing with CasX. While the data demonstrate that use of v5 and v6 gRNAs resulted in efficient editing in vitro, near-saturating levels of editing were observed with use of the vl gRNA in this experiment where a single dose of the gRNA was transfected. As a result, the use of a single dose rendered it challenging to assess clearly the effects of the chemical modifications on editing under guide-limiting conditions. Therefore, profiles vl and v5 were chosen for further testing, as vl contains the simplest modification profile, and v5 is the most heavily modified profile whose application demonstrated robust activity in vitro (Tables 22 and 23).

Table 23: Editing levels measured by indel rate at PCSK9 locus by NGS and secreted PCSK9 levels by ELISA in HepG2 cells co-transfected with CasX 491 mRNA and various chemically-modified /X.SA -targeting gRNAs using scaffold 235 and either spacer 6.7 or 6.8. Attorney Docket No.: SCRB-056/02WO 33322-2431

[0245] The vl and v5 profiles were further tested in another cell-based assay to assess their effects on editing efficiency. LNPs were formulated to co-encapsulate CasX mRNA #2 and vl and v5 chemically-modified ROSA26-targeting gRNAs using the newly-designed gRNA scaffold 316 (described further in the following sub-section). The “v5” profile was modified slightly for application to the 316 scaffold. Three 2’ OMe modifications in the non-base-paired region immediately 5’ of the extended stem were removed to restrict modifications to the two stemloop regions. Hepal-6 hepatocytes were treated with the resulting LNPs at various doses and harvested eight days post-treatment to assess editing at the ROSA26 locus, measured as indel rate detected by NGS (FIG. 10A). The data demonstrate that treatment with LNPs delivering the v5 ROSA26-targeting gRNA resulted in markedly lower editing levels across the range of doses compared to the levels achieved with the vl counterpart (FIG. 10A). There are several possible explanations for the differences in relative activity observed with use of v5 Attorney Docket No.: SCRB-056/02WO 33322-2431 gRNA in FIG. 10A relative to that observed in Table 23. The first and most likely possible explanation is that the single dose used to achieve editing shown in Table 23 was too high to measure differences in activity accurately between use of v5 gRNA and vl gRNA. It is also possible that the removal of the modifications outside the stemloop motifs in the 316 version of v5 negatively impacted guide activity. While it is possible that these modifications provide stability benefits that outweigh an activity cost imparted by the stemloop modifications, this seems unlikely given that increasing levels of modification have so far resulted in decreased activity. A final possible explanation is that the modifications in the v5 profile might negatively impact LNP formulation or behavior through differential interactions between the modified nucleotide backbone and the ionizable lipid of the LNP, potentially resulting in less efficient gRNA encapsulation or in less efficient gRNA release following internalization.

[0246] LNPs co-encapsulating the CasX mRNA #2 and vl and v5 chemically-modified ROSA26-targeting gRNAs based on scaffold 316 were further tested in vivo. FIG. 10B shows the results of the editing assay as percent editing measured as indel rate at the ROSA26 locus. The data demonstrate that use of the v5 gRNA resulted in ~5-fold lower editing compared to that achieved with use of the vl gRNA, under more relevant testing conditions of in vivo LNP delivery. These findings support the reduced cleavage rate observed biochemically for the v5 gRNA in Table 22, an indication that the v5 modifications have interfered with some aspect of CasX activity. Given the consistent decrease in activity detected in v5 and v6 profiles (Table 22), the reduced editing may be attributed to modifications in the extended stem region. Although the extended stem of the gRNA has minimal interactions with the CasX protein, it is possible that addition of 2’0Me groups at the first base-pair disrupted either the CasX protein- gRNA interactions or the complex RNA fold where the extended stem meets the pseudoknot and triplex regions. More specifically, inclusion of the 2’0Me groups might have adversely affected the basal base-pairs of the gRNA extended stem and residues R49, K50, and K51 of the CasX protein. Finally, structural studies of CasX have suggested that flexibility of the gRNA is required for efficient DNA cleavage (Liu J, et al, CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566:218-223 (2019); Tsuchida CA, et al, Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity. Mol Cell 82(6): 1199-1209 (2022)). Thus, the addition of the 2’0Me groups throughout the extended stem might have enforced a more rigid A-form helical structure and prevented the needed flexibility for the gRNA for efficient cleavage. Furthermore, it is possible that the Attorney Docket No.: SCRB-056/02WO 33322-2431 additional modifications in the scaffold stem in the v5 and v6 profiles might be detrimental to activity, though this is currently unclear given the limited comparisons between the v5 and v6 profiles.

[0247] Additional modification profiles were designed with the goal of enhancing gRNA stability while mitigating the adverse effects on RNP cleavage activity. Using recently published structures of wild-type CasX from Planctomycetes (PDB codes 7WAY, 7WAZ, 7WB0, 7WB1), which has a higher homology to the engineered CasX variants being assessed, additional chemical modification profiles for gRNAs were designed and are illustrated in FIG. 6. These profiles illustrate the addition of 2’0Me groups and phosphorothioate bonds to a newly-designed gRNA scaffold variant, which is described in the ensuing sub-section. These new gRNA chemical modification profiles were designed based on the initial data demonstrating sufficient editing activity observed in Table 23 with use of the v5 gRNA that suggested that modifications to the extended stem and scaffold stem regions would not negatively impact activity. The v7 profile was designed to include 2’0Me at residues likely to be modifiable throughout the gRNA structure, which excluded the triplex region, given the dramatic negative effects of adding such modifications observed earlier with the v3 profile. More conservative profiles, v8 and v9, were also designed, as illustrated in FIG. 6. For the v8 construct, modifications were removed in the pseudoknot and triplex loop region, but were retained in the scaffold stem, extended stem, and their flanking single-stranded regions, in addition to the 5’ and 3’ termini. For the v9 profile, modifications were removed in the singlestranded regions flanking the stemloops, but were retained in the stemloops themselves, in addition to the pseudoknot, triplex loop, and 5’ and 3’ termini. The additional chemical modification profiles v7, v8, and v9 of the newly designed gRNA scaffold variant 316 (discussed further below) were assessed in vivo at the PCSK9 locus. The results of the editing assay in vivo quantified as percent editing at the PCSK9 locus measured as indel rate as detected NGS are illustrated in FIG. 11. Despite the fact that low editing efficiency was detected overall, the data demonstrate that use of v7, v8, and v9 gRNAs resulted in lower editing levels at the PCSK9 locus compared to the indel rate achieved with use of the vl gRNA (FIG. 11). Given the findings in FIGS. 10A-10B showing inferior editing activity attained with the v5 gRNA, it is unsurprising that v7, v8, and v9 profiles similarly demonstrated comparatively lower editing activity. As illustrated in FIG. 6, the v7, v8, and v9 profiles include Attorney Docket No.: SCRB-056/02WO 33322-2431 modifications throughout the extended stem region, which might have interfered with RNP activity.

Comparison of gRNA scaffold variant 174 and 316 using an in vitro cleavage assay:

[0248] Previous work had established gRNA scaffold variant 235 as the top-performing scaffold variant across multiple delivery conditions. However, the longer length of scaffold 235 (119 bp, when using a 20 bp spacer) relative to gRNAs including scaffold 174 (109 bp, when using a 20 bp spacer) increased the difficulty of solid-phase RNA synthesis, which would result in increased manufacturing costs, decreased purity and yield, and higher rates of synthesis failures. To address these issues but retain the improved activity of using scaffold variant 235, a chimeric gRNA scaffold was designed primarily on the basis of the scaffold 235 sequence, but the extended stemloop of scaffold 235 was replaced with the shorter extended stemloop of scaffold variant 174 (FIGS. 7A-7C). The resulting chimeric scaffold, named scaffold 316, was synthesized in parallel with scaffold 174 and CGS' -targeting spacers 6.7 and 6.8, and 2Af-targeting spacer 7.9 harboring the vl chemical modification profile, with 2’0Me and phosphorothioate bonds on the first and last three nucleotides of all gRNAs (see Table 18). Scaffold variant 174 was chosen as the comparator rather than variant 235 because variant 174 was the best previously characterized scaffold with the same length as variant 316. [0249] In vitro cleavage activity was assessed for gRNAs with scaffold 174 and 316 and spacers 6.7 and 6.8. Cleavage assays were carried out with 20-fold excess RNP over a matching dsDNA target. Cleavage rates were quantified for all four guides, and the results are shown in Table 24. The data demonstrate that in the context of spacer 6.7, use of either scaffold 174 or 316 resulted in similar cleavage rates, with scaffold 316 resulting in marginally faster cleavage than that achieved with scaffold 174. In the context of spacer 6.8, the difference in cleavage activity was more pronounced: CasX RNPs using scaffold 316 were able to cleave DNA nearly twice as quickly as CasX RNPs using scaffold 174 (Table 24).

[0250] Assays were also performed with equimolar amounts of RNP and DNA target over a longer time course to assess the fraction of expected RNP active for cleavage. As the CasX RNP is essentially single-turnover over the tested timescale, and the concentrations used are expected to be substantially higher than the KD of the DNA-binding reaction, the amount of cleaved DNA should approximate the amount of active RNP. For either spacer 6.7 or 6.8, the active fraction of CasX RNPs incorporating scaffold 316 was 25-30% higher than for CasX RNPs using scaffold 174 (Table 24). These data suggest that a higher fraction of gRNA using Attorney Docket No.: SCRB-056/02WO 33322-2431 scaffold 316 was properly folded for association with the CasX protein, or that the gRNA using scaffold 316 was able to associate more strongly with the CasX protein. Compared to scaffold 174, scaffold 316 harbors mutations expected to stabilize the pseudoknot and triplex structures required for proper gRNA folding. The increased stability of these motifs in particular, which were more likely to misfold than the simple hairpins found elsewhere in the gRNA structure, might result in a slightly higher fraction of the gRNAs folding into an active conformation.

Table 24: Parameters of cleavage activity assessed for CasX RNPs with gRNAs containing scaffold variant 174 or 316 with the version 1 (vl) chemical modification profile.

Comparison of gRNA scaffold variant 174 and 316 in a cell-based assay:

[0251] An editing assessment using gRNA scaffold variant 174 compared to variant 316 was performed in a cell-based assay. CasX 491 mRNA and the version 1 (vl) of PCSK9-targeting gRNAs using spacers 6.7 and 6.8 were lipofected into HepG2 cells. Treated cells were harvested 28 hours post-transfection for analysis of editing levels at the PCSK9 locus by NGS and secreted PCSK9 levels by ELISA, and the data are presented in FIG. 8. The data demonstrate that use of any of the ECS' -targeting gRNA tested resulted in efficient editing at the PCSK9 locus and substantial reduction in PCSK9 secretion compared to the nontargeting control using the 2A -targeting gRNA. The results also show that use of scaffold 316 resulted in more effective editing at the PCSK9 locus than that observed with use of scaffold 174 (~10 percentage point increase in editing rate achieved with scaffold 316 over scaffold 174). This finding is further supported by the ELISA results, such that use of scaffold 316 resulted in more effective reduction of PCSK9 secretion compared to that achieved with use of scaffold 174. Attorney Docket No.: SCRB-056/02WO 33322-2431

[0252] Scaffold variants 174 and 316 were also assessed in an editing assay where LNPs were formulated to co-encapsulate CasX 491 mRNA and 7>2A7-targeting gRNA harboring either scaffold variant. HepG2 cells were treated with the resulting LNPs at various doses and harvested seven days post-treatment to assess editing at the B2M locus, measured as indel rate detected by NGS (FIG. 9A) and loss of surface presentation of the B2M-dependent HLA complex, as detected by flow cytometry (FIG. 9B). The results from both assays demonstrate that treatment with LNPs to deliver the 2A7-targeting gRNA using scaffold 316 resulted in higher editing potency at the 7>2A7 locus compared to LNPs delivering the gRNA using scaffold 174 at each dose (FIGS. 9A and 9B). Specifically, at the highest dose of 250 ng, use of scaffold 316 resulted in an editing level that was nearly two-fold higher than the level attained with using scaffold 174. This substantial increase in editing efficacy when using scaffold 316 versus scaffold 174, compared to the comparatively modest difference in activity observed from the in vitro cleavage assays, might be attributed to the destabilization of gRNA structure and folding during LNP formulation. The low pH conditions and association of cationic lipids during LNP formulation could adversely affect parts of the gRNA structure and result in unfolding. Consequently, it would be necessary for the gRNA to refold quickly in the cytoplasm upon delivery, both to bind the CasX protein to form the RNP and to evade RNase degradation. The stability -increasing mutations in scaffold 316 compared to scaffold 174 might provide a substantial benefit in supporting proper gRNA refolding in the cytoplasm after LNP delivery, while the deliberate folding protocol carried out for the gRNA prior to biochemical experiments likely reduced the impact of these mutations.

299869213

Claims

CLAIMS What is claimed is:

1. A messenger ribonucleic acid (mRNA) comprising a sequence encoding an engineered CasX protein comprising a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 8, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

2. The mRNA of claim 1, wherein the sequence encoding the engineered CasX protein comprises a sequence selected from the group consisting of SEQ ID NOS: 8-11.

3. The mRNA of claim 1, wherein the sequence encoding the engineered CasX protein comprises the sequence of SEQ ID NO: 9.

4. The mRNA of any one of claims 1-3, wherein the engineered CasX protein comprises a sequence selected from the group consisting of SEQ ID NOS: 4-7, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

5. The mRNA of claim 4, wherein the engineered CasX protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 4-7.

6. The mRNA of any one of claims 1-5, comprising one or more of: a. a 5’ cap; b. a 5’ untranslated region (UTR) sequence; c. a sequence encoding a nuclear localization sequence (NLS); d. a sequence encoding a peptide linker; e. a 3 ’ UTR sequence; and f. a polyadenylation (poly(A)) sequence.

7. The mRNA of claim 6, wherein the 5’ UTR comprises the nucleic acid sequence of SEQ ID NO: 20, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

8. The mRNA of claim 7, wherein the 5’ UTR comprises the nucleic acid sequence of SEQ ID NO: 20.

9. The mRNA of any one of claims 6-8, wherein the 3’ UTR comprises a mouse hemoglobin alpha (HBA) 3’ UTR.

10. The mRNA of any one of claims 6-9, wherein the 3’ UTR comprises the nucleic acid sequence of SEQ ID NO: 23, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

11. The mRNA of claim 10, wherein the 3’ UTR comprises the nucleic acid sequence of SEQ ID NO: 23.

12. The mRNA of any one of claims 6-11, wherein the NLS is a c-MYC NLS.

13. The mRNA of claim 12, wherein the c-MYC NLS comprises the sequence of PAAKRVKLD (SEQ ID NO: 50).

14. The mRNA of any one of claims 6-13, wherein the peptide linker comprises the amino acid sequence of GS or SR.

15. The mRNA of any one of claims 1-14, wherein the mRNA comprises, from 5’ to 3’ : a. a sequence encoding a first c-MYC NLS; b. a sequence encoding a first peptide linker; c. the codon-optimized sequence encoding the engineered CasX protein; d. a sequence encoding a second peptide linker; and e. a sequence encoding a second c-MYC NLS.

16. The mRNA of claim 15, wherein: a. the first c-MYC NLS and the first linker are encoded by the sequence of SEQ ID NO: 27; and/or b. the second c-MYC NLS and the second linker are encoded by the sequence of SEQ ID NO: 30.

17. The mRNA of any one of claims 6-16, wherein the poly(A) sequence comprises at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides.

18. The mRNA of claim 17, wherein the poly(A) sequence comprises at least about 70, or at least about 80 adenine nucleotides.

19. The mRNA of claim 17 or claim 18, wherein the poly(A) sequence comprises the sequence of SEQ ID NO: 155 or SEQ ID NO: 156.

20. The mRNA of any one of claims 1-19, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 36-39, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

21. The mRNA of any one of claims 1-19, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 36-39.

22. The mRNA of any one of claims 1-19, wherein the mRNA comprises the sequence of SEQ ID NO: 37.

23. The mRNA of any one of claims 6-22, wherein the 5’ cap is linked to the 5’ UTR in a 5 ’-5’ linkage.

24. The mRNA of any one of claims 6-23, the 5’ cap is a 7-methylguanylate cap, optionally wherein the 5’ cap comprises m7G(5’)ppp(5’)mAG, m7G(5')ppp (5'(A,G(5')ppp(5')A, or G(5')ppp(5')G.

25. The mRNA of any one of claims 1-24, wherein the mRNA comprises one or more nucleoside analogs, chemically modified bases, biologically modified bases, intercalated bases, modified sugars, modified phosphate groups, and/or nonstandard nucleotide residues.

26. The mRNA of any one of claims 1-25, wherein the mRNA comprises one or more nonstandard nucleotides.

27. The mRNA of claim 26, wherein at least one of the one or more nonstandard nucleotides is selected from 5-methyl-cytidine (“5 mC”), pseudouridine (“\|/U”), and/or 2- thio-uridine (“2sU”).

28. The mRNA of claim 27, wherein the pseudouridine is N1 -methylpseudouridine.

29. The mRNA of claim 28, wherein 100% of uridine nucleosides of the mRNA sequence are replaced with N1 -methylpseudouridine.

30. The mRNA of claim 29, comprising a sequence selected from the group consisting of SEQ ID NOS: 40-43, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

31. The mRNA of claim 29, comprising a sequence selected from the group consisting of SEQ ID NOS: 40-43.

32. The mRNA of claim 29, comprising a sequence of SEQ ID NO: 41, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

33. The mRNA of claim 29, comprising a sequence of SEQ ID NO: 41.

34. An mRNA comprising a sequence encoding an engineered CasX protein, wherein the mRNA comprises: a. a 5’ untranslated region (UTR) comprising the nucleic acid sequence of SEQ ID NO: 20, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto; b. the sequence encoding the engineered CasX protein; and c. a 3’ UTR comprising the nucleic acid sequence of SEQ ID NO: 23, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

35. The mRNA of claim 34, wherein the 5’ UTR comprises the nucleic acid sequence of SEQ ID NO: 20.

36. The mRNA of claim 34 or claim 35, wherein the 3’ UTR comprises the nucleic acid sequence of SEQ ID NO: 23.

37. The mRNA of any one of claims 34-36, wherein the engineered CasX protein comprises a sequence selected from the group consisting of SEQ ID NOS: 4-7, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

38. The mRNA of claim 37, wherein the engineered CasX protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 4-7.

39. The mRNA of any one of claims 34-38, wherein the sequence encoding the engineered CasX protein is codon-optimized.

40. The mRNA of any one of claims 34-39, wherein the sequence encoding the engineered CasX protein is codon-optimized for expression in a human cell.

41. The mRNA of any one of claims 34-40, wherein the sequence encoding the engineered CasX protein comprises a sequence selected from the group consisting of SEQ ID NOS: 8-11, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

42. The mRNA of claim 41, wherein the sequence encoding the engineered CasX protein comprises a sequence selected from the group consisting of SEQ ID NOS: 8-11.

43. The mRNA of claim 41, wherein the codon-optimized sequence encoding the engineered CasX protein comprises the sequence of SEQ ID NO: 9.

44. The mRNA of any one of claims 34-43, comprising one or more of: a. a 5’ cap; b. a sequence encoding a nuclear localization signal (NLS); c. a sequence encoding a peptide linker; and d. a polyadenylation (poly(A)) sequence.

45. The mRNA of claim 44, wherein the NLS is a c-MYC NLS.

46. The mRNA of claim 45, wherein the c-MYC NLS comprises the sequence of PAAKRVKLD (SEQ ID NO: 50).

47. The mRNA of any one of claims 44-46, wherein the peptide linker comprises the amino acid sequence of GS or SR.

48. The mRNA of any one of claims 34-47, wherein the mRNA comprises, from 5’ to 3’ : a. a 5' UTR; b. a sequence encoding a first c-MYC NLS; c. a sequence encoding a first peptide linker; d. the sequence encoding the engineered CasX protein; e. a sequence encoding a second peptide linker; f. a sequence encoding a second c-MYC NLS; and g. a 3' UTR.

49. The mRNA of claim 48, wherein: a. the first c-MYC NLS and the first linker are encoded by the sequence of SEQ ID NO: 27; and/or b. the second c-MYC NLS and the second linker are encoded by the sequence of SEQ ID NO: 30.

50. The mRNA of any one of claims 34-49, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 36-39, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

51. The mRNA of claim 50, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 36-39.

52. The mRNA of claim 50, wherein the mRNA comprises a sequence of SEQ ID NO: 37.

53. The mRNA of any one of claims 44-52, wherein the poly(A) sequence comprises at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides.

54. The mRNA of claim 53, wherein the poly(A) sequence comprises at least about 70, or at least about 80 adenine nucleotides.

55. The mRNA of claim 53 or claim 54, wherein the poly(A) sequence comprises the sequence of SEQ ID NO: 155 or SEQ ID NO: 156.

56. The mRNA of any one of claims 44-55, wherein the 5’ cap is linked to the 5’ UTR in a 5 ’-5’ linkage.

57. The mRNA of any one of claims 44-56, wherein the 5’ cap is a 7-methylguanylate cap, optionally wherein the cap comprises m7G(5’)ppp(5’)mAG, m7G(5')ppp (5'(A,G(5')ppp(5')A, or G(5')ppp(5').

58. The mRNA of any one of claims 44-57, wherein the mRNA comprises one or more nucleoside analogs, chemically modified bases, biologically modified bases, intercalated bases, modified sugars, modified phosphate groups, and/or nonstandard nucleotide residues.

59. The mRNA of any one of claims 44-58, wherein the mRNA comprises one or more nonstandard nucleotides.

60. The mRNA of claim 59, wherein at least one of the one or more nonstandard nucleotides is selected from 5-methyl-cytidine (“5 mC”), pseudouridine (“\|/U”), and/or 2- thio-uridine (“2sU”).

61. The mRNA of claim 60, wherein the pseudouridine is N1 -methylpseudouridine.

62. The mRNA of claim 61, wherein 100% of uridine nucleosides of the mRNA sequence are replaced with N1 -methylpseudouridine.

63. The mRNA of claim 62, comprising a sequence selected from the group consisting of SEQ ID NOS: 40-43, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

64. The mRNA of claim 62, comprising a sequence selected from the group consisting of SEQ ID NOS: 40-43.

65. The mRNA of claim 62, comprising a sequence of SEQ ID NO: 41, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

66. The mRNA of claim 62, comprising a sequence of SEQ ID NO: 41.

67. A system comprising the mRNA of any one of claims 1-62, and a guide ribonucleic acid (gRNA) comprising a scaffold and a targeting sequence complementary to a target nucleic acid in a cell linked 3' to the scaffold.

68. The system of claim 67, wherein the scaffold comprises a scaffold stem loop comprising the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 138), or a sequence having 1, 2, 3, 4, or 5 mismatches thereto.

69. The system of claim 67 or claim 68, wherein the scaffold comprises a sequence selected from the group consisting of SEQ ID NOS: 152-154.

70. The system of any one of claims 67-69, wherein the targeting sequence of the gRNA has 15, 16, 17, 18, 19, or 20 consecutive nucleotides.

71. The system of any one of claims 67-70, wherein the gRNA is chemically modified.

72. The system of claim 71, wherein the chemical modification is addition of a 2’0- methyl group to one or more nucleotides of the sequence.

73. The system of claim 71 or claim 72, wherein the chemical modification is substitution of a phosphorothioate bond between two or more nucleotides of the sequence.

74. The system of claim 73, wherein the chemical modification is an addition of a 2’0- methyl group to the 3 nucleotides of the 5' and 3’ ends of the gRNA and wherein each of the modified nucleotides is linked to the adjoining nucleotide by a phosphorothioate bond.

75. The system of any one of claims 71-74, wherein the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 158-166; 168-176; and 178-186, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

76. The system of claim 75, wherein the chemically modified gRNA comprises the sequence of SEQ ID NO: 178, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

77. The system of claim 75 or claim 76, wherein the 20 nucleotides of the 3' targeting sequence are substituted with a targeting sequence that is complementary to a target nucleic to be modified.

78. The system of any one of claims 71-77, wherein the chemical modification results in reduced susceptibility of the gRNA to degradation by a cellular ribonuclease compared to an unmodified gRNA.

79. The system of any one of claims 63-78, wherein the mRNA and the gRNA are encapsulated within a lipid nanoparticle (LNP).

80. The system of claim 79, wherein the LNP comprises one or more components selected from the group consisting of one or more ionizable lipids, one or more helper phospholipids, one or more PEG-modified lipids, and/or cholesterol or a derivative thereof.

81. The system of any one of claims 63-80, for use in modifying a target nucleic acid of a cell.

82. A vector comprising a nucleic acid sequence encoding the mRNA of any one of claims 1-62 and/or the gRNA of any one of claim 67-81.

83. The vector of claim 82, wherein the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) vector, a virus-like particle (VLP), a plasmid, a minicircle, a nanoplasmid, and a DNA vector.

84. A method of modifying a target nucleic acid in a cell, comprising introducing into the cell the system of any one of claims 67-81 or the vector of claim 82 or claim 83, resulting in the modification of the target nucleic acid.

85. The method of claim 84, wherein the engineered CasX protein is expressed within the cell and is capable of forming a ribonucleoprotein (RNP) complex with the gRNA.

86. The method of claim 84, wherein the engineered CasX protein is expressed within the cell and forms an RNP complex with the gRNA.

87. The method of any one of claims 84-86, wherein the cell is a eukaryotic cell.

88. The method of claim 87, wherein the eukaryotic cell is selected from the group consisting of a rodent cell, a mouse cell, a rat cell, and a non-human primate cell.

89. The method of claim 87, wherein the eukaryotic cell is a human cell.

90. The method of any one of claims 84-89, wherein the cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell, a germ cell, a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B-cell, an NK cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplanted expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, a fibroblast cell, an osteoblast cell, a chondrocyte cell, an exogenous cell, an endogenous cell, a stem cell, a hematopoietic stem cell, a bone-marrow derived progenitor cell, a myocardial cell, a skeletal cell, a fetal cell, an undifferentiated cell, a multi-potent progenitor cell, a unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, an autologous cell, and a post-natal stem cell.

91. The method of any one of claims 85-90, wherein the modifying comprises binding of the RNP to the target nucleic acid and introducing a double-stranded break in the target nucleic acid.

92. The method of claim 91, wherein the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the target nucleic acid.

93. The method of any one of claims 84-92, wherein the modifying of the cell occurs in vitro or ex vivo.

94. The method of any one of claims 84-92, wherein modifying of the cell occurs in vivo in a subject.

95. The method of claim 94, wherein the subject is selected from the group consisting of a rodent, a mouse, a rat, and a non-human primate.

96. The method of claim 94, wherein the subject is a human.

97. The method of claim 95 or claim 96, wherein the modifying occurs in the cells of the subject having a mutation in an allele of a gene, wherein the mutation causes a disease or disorder in the subject.

98. The method of claim 97, wherein the modifying changes the mutation to a wild-type sequence of the gene, results in the expression of a functional gene product, or reduces expression of a toxic gene product.

99. The method of claim 97, wherein the modifying knocks down or knocks out the gene causing the disease or disorder in the subject.

100. A pharmaceutical composition comprising, the system of any one of claims 67-81, or the vector of claim 82 or claim 83, and a pharmaceutically acceptable excipient.

101. The pharmaceutical composition of claim 100, wherein the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intraportal vein injection, intraperitoneal, intramuscular, subcutaneous, intraarterial, intracerebroventricular, intracistemal, intrathecal, intracranial, intralumbar, intraperitoneal, intraocular, and oral routes.

102. The pharmaceutical composition of claim 100 or claim 101, wherein the pharmaceutical composition is in a liquid, lyophilized, or frozen form.

103. The pharmaceutical composition of any one of claims 100-102, wherein the pharmaceutical composition is in a pre-filled syringe for a single injection.

104. The mRNA of any one of claims 1-66, the system of any one of claims 67-81, the vector of claim 82 or claim 83, or the pharmaceutical composition of any one of claims 100- 103 for the manufacture of a medicament for the treatment of a subject having a disease.

105. A kit comprising the mRNA of any one of claims 1-66, the system of any one of claims 67-80, or the vector of claim 82 or claim 83 and a suitable container.

106. The kit of claim 105, comprising a buffer, an excipient, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.