Engineered class 2 type V CRISPR system

Abstract

Engineered Class 2 Type V nucleases and guide ribonucleic acid scaffolds useful for editing target nucleic acids are provided herein. Methods of making and using such systems to modify nucleic acids are also provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 348,413, filed on June 2, 2022, U.S. Provisional Patent Application No. 63 / 350,400, filed on June 8, 2022, and U.S. Provisional Patent Application No. 63 / 350,770, filed on June 9, 2022, the contents of each of which are hereby incorporated by reference in their entirety.

[0002] Incorporation by Reference of Sequence Listing The contents of the electronic sequence listing (SCRB_041_03WO_SeqList_ST26.xml, size: 90,175,867 bytes, created on May 23, 2023) are hereby incorporated by reference in their entirety.

Background Art

[0003] Bacterial and archaeal CRISPR - Cas systems confer a form of acquired immunity against phages and viruses. Intensive research over the past decade has revealed the biochemistry of these systems. The CRISPR - Cas system consists of Cas proteins involved in the acquisition, targeting, and cleavage of foreign DNA or RNA, and a CRISPR array containing direct repeat sequences adjacent to short spacer sequences that guide the Cas proteins to the target. Class 2 CRISPR - Cas is a streamlined version in which a single Cas protein bound to RNA is involved in binding to and cleavage of the target sequence. The programmable nature of these minimal systems facilitates their use as versatile technologies that are revolutionizing the field of genome engineering.

[0004] To date, only a few widely used Class 2 CRISPR / Cas systems have been discovered. Among them, Type V is unique in that it recognizes a 5’ PAM sequence different from the 3’ PAM sequence recognized by Cas9 and utilizes a single integrated RuvC-like endonuclease (RuvC) domain to form staggered cuts in target nucleic acids with 5, 7, or 10 nt 5’ overhangs (Yang et al., PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease. Cell 167:1814 (2016)). However, wild-type Type V Cas nucleases and guide sequences have low editing efficiency. Thus, there is a need in the art for additional Class 2 Type V CRISPR / Cas systems (e.g., combinations of Cas proteins and guide RNAs) that are optimized and provide improvements compared to earlier generation systems for use in various therapeutic, diagnostic, and research applications. SUMMARY OF THE INVENTION

[0005] The present disclosure relates to a system of an engineered CasX protein and an engineered guide ribonucleic acid scaffold (ERS) having a linked targeting sequence used to modify a target nucleic acid of a gene in a eukaryotic cell. In some embodiments, the present disclosure provides an engineered CasX protein comprising one or more modifications compared to one or more domains of the CasX protein from which it is derived. These engineered CasXs exhibit one or more improved characteristics compared to a reference CasX or a CasX variant from which it is derived, and the engineered CasX retains the ability to form a ribonucleoprotein (RNP) complex with the ERS and retains nuclease activity.

[0006] In another aspect, the present disclosure provides an engineered guide ribonucleic acid scaffold (ERS) that can bind to a class 2 type V protein comprising engineered CasX of the present disclosure, wherein the ERS comprises a single-guide composition that can bind to the engineered CasX. The ERS provides an engineered guide ribonucleic acid scaffold (ERS) that contains one or more modifications in one or more regions as compared to a parental gRNA, such as a reference gRNA or a gRNA variant. In some embodiments, the modified region of the gRNA scaffold comprises one or more of (a) the 5' end of the scaffold, (b) the extension stem, (c) the scaffold stem, (d) the triple helix, (e) the triple helix loop, and (f) the pseudoknot stem.

[0007] In some embodiments, the present disclosure provides a gene editing pair system comprising an engineered CasX protein and an ERS of any of the embodiments described herein, wherein the gene editing pair exhibits at least one improved characteristic as compared to a gene editing pair of CasX and gRNA from which the engineered CasX protein and the ERS are derived.

[0008] In some embodiments, the present disclosure provides polynucleotides and vectors encoding the engineered CasX proteins, ERSs, and gene editing pairs described herein. In some embodiments, the vector is a viral vector such as an adeno-associated virus (AAV) vector. In other embodiments, the vector is a CasX delivery particle (XDP) comprising the RNP of the gene editing pair.

[0009] In some embodiments, the present disclosure provides methods for making engineered CasX proteins. In other embodiments, the present disclosure provides methods for making ERSs.

[0010] In some embodiments, the present disclosure provides a kit comprising the polynucleotides, vectors, engineered CasX proteins, ERSs, and gene editing pairs described herein, and an LNP composition.

[0011] In some embodiments, the present disclosure provides a method of editing a target nucleic acid, the method comprising contacting the target nucleic acid with an engineered CasX protein and an ERS embodiment described herein, wherein the contacting results in editing or modification of the target nucleic acid.

[0012] In some embodiments, the present disclosure provides a method of editing a target nucleic acid in a cell population, the method comprising contacting the cells with one or more of the gene editing pairs described herein, wherein the contacting results in editing or modification of the target nucleic acid in the cell population.

[0013] In another aspect, a gene editing pair for use in a method of treatment, a composition comprising the gene editing pair, or a vector comprising or encoding the gene editing pair is provided herein, the method comprising editing or modifying a target nucleic acid, optionally wherein the editing is a mutation in an allele of a gene, the mutation causing a disease or disorder in a subject, and preferably wherein the editing changes the mutation to the wild-type allele of the gene or knocks down or out the allele of the gene that causes the disease or disorder in the subject.

[0014] In another aspect, the present disclosure provides a composition comprising an engineered CasX, an ERS, and a gene editing pair for use in the manufacture of a medicament for use in the treatment of a subject having a disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The novel features of the invention are set forth with particularity in the appended claims. A further understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings.

[0016]

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Mode for Carrying Out the Invention

[0017] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0018] 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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this embodiment, 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. Those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the present invention. Definitions

[0019] The terms "polynucleotide" and "nucleic acid" as used interchangeably herein refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms "polynucleotide" and "nucleic acid" include single-stranded DNA, double-stranded DNA, multi-stranded DNA, single-stranded RNA, double-stranded RNA, multi-stranded RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers that include purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0020] "Hybridizable" or "complementary" are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) is capable of non-covalently binding to another nucleic acid (i.e., a nucleic acid that specifically binds to a complementary nucleic acid) in a sequence-specific, antiparallel-like manner under appropriate temperature and solution ionic strength conditions in vitro and / or in vivo, i.e., forming Watson-Crick base pairs and / or G / U base pairs, "annealing" to it, or "hybridizing" to it. The sequence of a polynucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable and is understood to have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still be able to hybridize to the target nucleic acid sequence. Further, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structures or hairpin structures, "bulges", etc.).

[0021] For the purposes of the present disclosure, a "gene" includes a DNA region encoding a gene product (e.g., a protein, RNA), as well as all DNA regions that regulate the production of the gene product (regardless of whether such regulatory sequences are adjacent to the coding sequence and / or the transcriptional sequence). Thus, a gene can include regulatory element sequences including, but not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions. The coding sequence encodes a gene product upon transcription or transcription and translation, and the coding sequences of the present disclosure can include fragments and need not include a full-length open reading frame. A gene can include not only the transcribed strand but also the complementary strand containing the anticodon.

[0022] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence is related to the sequence following the transcription start point. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0023] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence is related to the sequence located 5' to the coding region or the transcription start point. For example, most promoters are located upstream of the transcription start site.

[0024] The term "adjacent to" with respect to a polynucleotide or amino acid sequence refers to sequences that are next to each other in a polynucleotide or polypeptide, or sequences that are adjacent to each other. One of ordinary skill in the art will understand that two sequences are considered adjacent to each other and can still include a limited amount of intervening sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or amino acids.

[0025] 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 selection of appropriate regulatory elements depends on the coding component to be expressed (e.g., a protein or RNA), or on whether the nucleic acid includes multiple components that require different polymerases or multiple components that are not intended to be expressed as a fusion protein.

[0026] The term "accessory element" is used interchangeably herein with the term "accessory array" and is intended to include, among other things, a polyadenylation signal (poly(A) signal), enhancer element, intron, post-transcriptional regulatory element (PTRE), nuclear localization signal (NLS), deaminase, DNA glycosylase inhibitor, additional promoter, factor that stimulates CRISPR-mediated homology-directed repair (e.g., cis or trans), transcriptional activator or repressor, self-cleaving sequence, and fusion domain, e.g., a fusion domain fused to an engineered CasX protein. The selection of appropriate accessory element(s) is understood to depend on the expressed encoded component (e.g., protein or RNA) or on whether the nucleic acid includes multiple components that require different polymerases or multiple components that are not intended to be expressed as a fusion protein.

[0027] The term "promoter" refers to a DNA sequence that includes a transcription start site and additional sequences to facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as the TATA box and / or the B recognition element (BRE), and assist or promote the transcription and expression of related transcribable polynucleotide sequences and / or genes (or transgenes). Promoters can be synthetically generated or can be derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can also include chimeric promoters that contain combinations of two or more heterologous sequences to confer certain properties. The promoters of the present disclosure can include variants of promoter sequences that are similar but not identical in composition to known or other promoters provided herein. Promoters can be classified according to criteria related to the expression patterns of related coding sequences or genes or transcribable sequences or genes operably linked to promoters such as constitutive, developmental, tissue-specific, inducible, etc. Promoters can also be classified according to their strength. When used in the context of a promoter, "strength" refers to the transcription rate of a gene controlled by the promoter. A "strong" promoter means a high transcription rate, and a "weak" promoter means a relatively low transcription rate.

[0028] The promoters of the present disclosure can be polymerase II (Pol II) promoters. Polymerase II transcribes all protein-coding genes and many non-coding genes. A typical Pol II promoter is a sequence of about 100 base pairs surrounding the transcription start site and includes a core promoter that serves as a binding platform for Pol II polymerase and related general transcription factors. Promoters can include one or more core promoter elements such as the TATA box, BRE, initiator (INR), motif 10 element (MTE), downstream core promoter element (DPE), downstream core element (DCE), etc., but core promoters lacking these elements are known in the art.

[0029] The promoter of the present disclosure can be a polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs such as 5S rRNA, tRNA, and other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed portion of the gene) to assist transcription, although upstream elements such as the TATA box may also be used. All Pol III promoters are assumed to be within the scope of the present disclosure.

[0030] The term "enhancer" refers to a regulatory DNA sequence that, when bound by specific proteins called transcription factors, regulates the expression of the associated gene. Enhancers can be located in the intron of a gene, or 5' or 3' of the coding sequence of the gene. Enhancers can be located proximal to the gene (i.e., within dozens or hundreds of base pairs (bp) of the promoter) or distal to the gene (i.e., thousands, tens of thousands, or even millions of bp away from the promoter). A single gene may be regulated by two or more enhancers, all of which are assumed to be within the scope of the present disclosure.

[0031] As used herein, a "post-transcriptional regulatory element (PTRE or TRE)", such as a hepatitis PTRE, refers to a DNA sequence that, when transcribed, can assume a tertiary structure that exhibits post-transcriptional activity to enhance or promote the expression of the associated gene operably linked thereto.

[0032] As used herein, "recombinant" means the product of various combinations of cloning, restriction, and / or ligation steps that result in a construct in which a particular nucleic acid (DNA or RNA) has a structural coding or non-coding sequence distinguishable from the endogenous nucleic acids found in nature. Generally, a DNA sequence encoding a 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 that can be expressed from a recombinant transcription unit contained in a cell or cell-free transcription and translation system. Such sequences can typically be provided in the form of an open reading frame that is not interrupted by internal non-translated sequences or introns typically present in eukaryotic genes. Genomic DNA containing related sequences can also be used in the formation of recombinant genes or transcription units. The sequences of non-translated DNA may be present 5' or 3' to the open reading frame, and such sequences do not interfere with the manipulation or expression of the coding region and, in fact, can act to regulate the production of the desired product by various mechanisms (see "enhancers" and "promoters" above).

[0033] The term "recombinant polynucleotide" or "recombinant nucleic acid" refers to something that does not occur naturally, for example, something made by the artificial combination of two otherwise separated segments of a sequence by human intervention. This artificial combination is often achieved by either chemical synthesis means or the artificial manipulation of isolated segments of nucleic acids, such as genetic engineering techniques. Such an artificial combination is usually done to replace codons with redundant codons encoding the same or a conserved amino acid, often while introducing or removing sequence recognition sites. Alternatively, this is done by joining together nucleic acid segments having desired functions to generate a combination of desired functions. This artificial combination is often achieved by either chemical synthesis means or the artificial manipulation of isolated segments of nucleic acids, such as genetic engineering techniques.

[0034] Similarly, the terms "recombinant polypeptide" or "recombinant protein" refer to polypeptides or proteins that do not occur naturally and are made, for example, by the artificial combination of two separately isolated segments of amino acid sequence by human intervention. Thus, for example, a protein containing a heterologous amino acid sequence is recombinant.

[0035] 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 sharing a physical connection between the target nucleic acid and the guide nucleic acid, for example, if these sequences share sequence similarity, they can hybridize.

[0036] "Dissociation constant" or "K d " are used interchangeably and refer to the affinity between a ligand "L" and a protein "P", i.e., how strongly the ligand binds to a particular protein. This can be calculated using the equation K d = [L][P] / [LP], where [P], [L], and [LP] represent the molar concentrations of the protein, ligand, and complex, respectively.

[0037] The present disclosure provides systems and methods useful for editing target nucleic acid sequences. As used herein, "editing" is used interchangeably with "modifying" and "modification" and includes, but is not limited to, cleavage, nicking, editing, deletion, knock-in, knockout, etc.

[0038] "Cleavage" means cleavage 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 phosphodiester bonds. Both single-strand cleavage and double-strand cleavage are possible, and double-strand cleavage can result from two separate single-strand cleavage events.

[0039] The term "knockout" refers to the removal 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 can be knocked out by replacing a part of the gene with an unrelated sequence. The term "knockdown" as used herein refers to a decrease in the expression of a gene or its gene product. As a result of gene knockdown, protein activity or function may be attenuated, or the protein level may be decreased or eliminated.

[0040] As used herein, "homology-directed repair" (HDR) refers to a form of DNA repair that occurs during the repair of double-strand breaks in a cell. This process requires nucleotide sequence homology and uses a donor template to repair or knockout the target DNA, resulting in the transfer of genetic information from the donor to the target. If the donor template is different from the target DNA sequence and part or all of the donor template sequence is incorporated into the target DNA, homology-directed repair can result in the modification of the target sequence by insertion, deletion, or mutation.

[0041] As used herein, "non-homologous end joining" (NHEJ) refers to the repair of double-strand breaks in DNA by the direct ligation of the cut ends to each other (in contrast to homology-directed repair, which requires a homologous sequence to guide the repair) without the need for a homologous template. NHEJ often results in the loss (deletion) of nucleotide sequences near the site of the double-strand break.

[0042] As used herein, "microhomology-mediated end joining" (MMEJ) refers to a mutagenic DSB repair mechanism that is always associated with deletions adjacent to the cut site and does not require a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide the repair). MMEJ often results in the loss (deletion) of nucleotide sequences near the site of the double-strand break.

[0043] A polynucleotide or polypeptide has a certain "sequence similarity" or "sequence identity" percentage with another polynucleotide or polypeptide, which means that when aligned, the percentage of bases or amino acids is the same and they are in the same relative positions when these two sequences are compared. Sequence similarity (sometimes called similarity percentage, identity percentage, or homology) can be determined in several different ways. To determine sequence similarity, the sequences can be aligned using methods and computer programs known in the art, including BLAST, which is available at the World Wide Web site ncbi.nlm.nih.gov / BLAST. The percentage of complementarity between specific stretches of nucleic acid sequences within a nucleic acid can be determined using any convenient method. Exemplary methods include the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403 - 410, Zhang and Madden, Genome Res., 1997, 7, 649 - 656), or the method by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), for example, the method by using the default settings that use Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 - 489).

[0044] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length that can include encoded and non - encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. This term includes, but is not limited to, fusion proteins having heterologous amino acid sequences.

[0045] As used herein, the term "vector" or "expression vector" refers to a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment, i.e., an expression cassette, is attached and which can thereby cause the replication or expression of the attached segment in a cell.

[0046] As used herein, the terms "naturally occurring", "unmodified", or "wild-type" when applied to a nucleic acid, polypeptide, cell, or organism refer to a nucleic acid, polypeptide, cell, or organism as found in nature.

[0047] As used herein, "mutation" refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides as compared to a wild-type or reference amino acid sequence or wild-type or reference nucleotide sequence.

[0048] As used herein, the term "isolated" is intended to describe a polynucleotide, polypeptide, or cell that is in an environment different from that in which it naturally occurs. An isolated recombinant host cell can be present in a mixed population of recombinant host cells.

[0049] As used herein, "host cell" means a cell derived from a eukaryotic cell, prokaryotic cell, or multicellular organism (e.g., cell line) cultured as a single cell entity, and these eukaryotic or prokaryotic cells are used as recipients of nucleic acids (e.g., AAV vectors) and include the progeny of the original cells that have been genetically recombined by the nucleic acids. It is understood that the progeny of a single cell may not necessarily be identical in form or genomic or total DNA complement to the original parent due to natural mutations, accidental mutations, or intentional mutations. A "recombinant host cell" (also referred to as a "genetically recombinant host cell") is a host cell into which a heterologous nucleic acid, e.g., an AAV vector, has been introduced.

[0050] As used herein, the term "tropism" optionally and preferably refers to the preferential entry of CasX delivery particles (referred to as XDPs) into a particular cell type or tissue type, and / or preferential interaction with the cell surface that facilitates entry into a particular cell type or tissue type, followed by expression (e.g., transcription and optionally translation) of the sequence carried by the cell by XDPs.

[0051] As used herein, the term "pseudotype" or "pseudotyping" refers to a viral envelope protein replaced with that of another virus having desirable characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein (especially those described hereinafter in this specification), which enables HIV to infect a broader range of cells because the HIV envelope protein targets the virus mainly to CD4+ presenting cells.

[0052] As used herein, the term "tropism factor" refers to a component incorporated into the surface of an XDP that provides tropism for a particular cell type or tissue type. Non-limiting examples of tropism factors include glycoproteins, antibody fragments (e.g., scFv, nanobodies, linear antibodies, etc.), receptors, and ligands for target cell markers.

[0053] "Target cell marker" refers to a molecule expressed by a target cell that can serve as a ligand for an antibody fragment or tropism factor and may be present on the surface of the target tissue or cell, including but not limited to cell surface receptors, cytokine receptors, antigens, tumor-associated antigens, glycoproteins, oligonucleotides, enzyme substrates, antigenic determinants, or binding sites.

[0054] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in a protein having similar side chains. For example, the group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; the group of amino acids having amide-containing side chains consists of asparagine and glutamine; the group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains consists of lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0055] As used herein, the term "antibody" encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, single domain antibodies such as VHH antibodies, and antibody fragments, so long as they exhibit the desired antigen binding activity or immunological activity. Antibodies represent a large family of molecules including several types of molecules such as IgD, IgG, IgA, IgM, and IgE.

[0056] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies, single-chain diabodies, linear antibodies, single domain antibodies, single domain camelid antibodies, single-chain variable fragment (scFv) antibody molecules, and multispecific antibodies formed from antibody fragments.

[0057] As used herein, "treatment" or "treating" are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic and / or prophylactic benefits. Therapeutic benefit means eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can be achieved by eradication or amelioration of one or more of the symptoms, or improvement of one or more clinical parameters associated with the underlying disease, such that improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder.

[0058] As used herein, the terms "therapeutically effective amount" and "therapeutically effective dose" refer to the amount of a drug or biologic agent, alone or as part of a composition, which when administered to a subject, such as a human or experimental animal, in a single dose or in multiple doses, can provide some detectable and beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition. Such an effect need not be absolute in order to be beneficial.

[0059] As used herein, "administering" means a method of providing a dosage of a compound (e.g., a composition of the disclosure) or composition (e.g., a pharmaceutical composition) to a subject.

[0060] "Subject" is a mammal. Mammals include, but are not limited to, domestic animals, non-human primates, humans, dogs, rabbits, mice, rats, and other rodents.

[0061] As used herein, "treatment" or "treating" are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic and / or prophylactic benefits. Therapeutic benefit means eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can be achieved by eradication or amelioration of one or more of the symptoms, or improvement of one or more clinical parameters associated with the underlying disease, such that improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder.

[0062] As used herein, the terms "therapeutically effective amount" and "therapeutically effective dose" refer to the amount of a drug or biologic that, when administered to a subject, such as a human or experimental animal, in a single dose or multiple doses, can provide some detectable and beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition, either alone or as part of a composition. Such an effect need not be absolute in order to be beneficial.

[0063] As used herein, "administering" means a method of providing a dosage amount of a compound (e.g., a composition of the present disclosure) or a composition (e.g., a pharmaceutical composition) to a subject.

[0064] A "subject" is a mammal. Mammals include, but are not limited to, domestic animals, non-human primates, humans, rabbits, mice, rats, and other rodents.

[0065] All publications, patents, and patent applications mentioned herein are incorporated herein 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.

[0066] I. General Methods The practice of the present invention, unless otherwise indicated, uses conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are found in standard textbooks such as A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th 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.

[0067] When ranges of values are provided, it is understood that the endpoints are included and that each intervening value between the upper and lower limits of the range and any other stated value or intervening value within the stated range is included to the tenth of the unit of the lower limit, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and also are included in accordance with any specifically excluded limit values within the stated range. When the stated range includes one or both of the limit values, ranges excluding either or both of those included limit values are also included.

[0068] 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 invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials related to which the publications are cited.

[0069] It should be noted that, 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.

[0070] It will be understood that certain features of the present disclosure, which are described in connection with separate embodiments for clarity, may be provided in combination in a single embodiment in other cases. In other instances, the various features of the present disclosure that are described in connection with a single embodiment for brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments of the present disclosure are specifically encompassed by the present disclosure and are intended to be disclosed herein in the same manner as if each and every combination were individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and their elements are also specifically encompassed by the present disclosure and are intended to be disclosed herein in the same manner as if each and every such sub-combination were individually and explicitly disclosed herein.

[0071] II. Systems for Gene Editing and Gene Editing Targets In a first aspect, the present disclosure provides a system (eCasX:ERS system) comprising an engineered CasX nuclease protein and an engineered guide ribonucleic acid scaffold (ERS) for use in modifying or editing a target nucleic acid of a gene comprising a coding region and a non-coding region. Generally, any part of a gene can be targeted using the programmable systems and methods provided herein.

[0072] As used herein, the term "system", used interchangeably with "composition", can include the engineered CasX nuclease protein of the present disclosure and one or more ERSs (having a linked targeting sequence) as a gene editing pair, a nucleic acid encoding the engineered CasX nuclease protein and ERS, and a vector or particle delivery formulation comprising the nucleic acid or the engineered CasX protein and ERS of the present disclosure.

[0073] In some embodiments, the present disclosure provides a system specifically designed to modify the target nucleic acid of a gene in a eukaryotic cell either in vitro, ex vivo, or in vivo in a subject. The engineered CasX of the present disclosure is a class 2 type V CRISPR nuclease. Although members of the class 2 type V CRISPR-Cas nucleases differ, they share some common features that distinguish them from the Cas9 system. First, type V nucleases have an RNA-guided single effector that contains an RuvC domain but not an HNH domain, and they recognize a TC motif PAM upstream of the 5' of the target region on the non-target strand, which is different from the Cas9 system that depends on a G-rich PAM on the 3' side of the target sequence. Type V nucleases generate staggered double-strand breaks distal to the PAM sequence, unlike Cas9, which generates blunt ends at the proximal site near the PAM. In addition, type V nucleases degrade ssDNA in trans when activated by cis-targeted dsDNA or ssDNA binding. In some embodiments, the present disclosure provides an engineered CasX protein that is designed with multiple mutations compared to the CasX from which it is derived, and the engineered CasX retains the ability to form a complex with the guide ribonucleic acid and has improved properties while retaining nuclease activity.

[0074] A system comprising an engineered CasX protein and an engineered guide ribonucleic acid scaffold (ERS), wherein the ERS, together with a targeting sequence linked to the 3' end of the scaffold, is called a gene editing pair, is provided herein. The ERS and the engineered CasX protein can bind together by non-covalent interactions to form a gene editing pair complex, herein called a ribonucleoprotein (RNP) complex (in all cases, it is understood that the ERS will have a linked targeting sequence for use in editing a target nucleic acid). In some embodiments, the use of pre-complexed RNPs of engineered CasX and ERS provides an advantage in delivering system components to cells or a target nucleic acid for editing of the target nucleic acid. In the RNP, the ERS provides target specificity to the RNP complex by including a targeting sequence (i.e., a "spacer") having a nucleotide sequence that is complementary to and can bind to the sequence of the target nucleic acid. In the RNP, the engineered CasX protein of the pre-complexed RNP provides site-specific activity and is guided to (and further stabilized at) a target site within the target nucleic acid sequence that is modified by its association with the ERS. The engineered CasX protein of the RNP complex provides site-specific activity of the complex, such as binding, cleavage, or nicking of the target nucleic acid sequence by the engineered CasX protein. Systems and cells comprising the engineered CasX protein, the ERS, and gene editing pairs of any combination of the engineered CasX and ERS embodiments described herein, and delivery modalities comprising or encoding the engineered CasX and ERS are provided herein. Each of these components, and their use in editing of the target nucleic acid of a gene, are described below in this specification.

[0075] In some embodiments, the present disclosure provides a gene editing pair system comprising an engineered CasX protein selected from any one of the engineered CasX proteins consisting of SEQ ID NOs: 247-294, 24916-49628, 49746-49747, and 49871-49873, or a sequence having at least about 85%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, and an ERS selected from the group consisting of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, and 49719-49735, or a sequence variant having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, wherein the ERS comprises a targeting sequence complementary to the target nucleic acid. In some embodiments, the guide ribonucleic acid is an ERS selected from the group consisting of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, and 49719-49735, and the ERS comprises a targeting sequence complementary to the target nucleic acid or a sequence having at least at least 1, 2, 3, 4, or 5 mismatches thereto.

[0076] In some embodiments, the present disclosure provides a gene editing pair system comprising an engineered CasX protein selected from the group consisting of SEQ ID NOs: 24916-49628, 49746-49747, and 49871-49873, or a sequence having at least about 85%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, wherein the engineered CasX comprises a sequence having one or more mutations compared to the sequence of SEQ ID NO: 228, and the mutation results in an improved characteristic compared to unmodified SEQ ID NO: 228. In some embodiments, the present disclosure provides a gene editing pair system comprising an engineered CasX protein selected from the group consisting of SEQ ID NOs: 49746-49747, and 49871-49873, or a sequence having at least about 85%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, wherein the engineered CasX comprises a sequence having one or more mutations compared to the sequence of SEQ ID NO: 228, and the mutation results in an improved characteristic compared to unmodified SEQ ID NO: 228, and the improved characteristic is one or more of an improvement in the editing activity of the target nucleic acid, an improvement in the editing specificity for the target nucleic acid, an improvement in the editing specificity ratio for the target nucleic acid, a reduction in off-target editing, an increase in the percentage of the eukaryotic genome that can be efficiently edited, an improvement in the ability to form an RNP having ERS and cleavage ability, and an improvement in the stability of the RNP complex.In some embodiments, the ERS for the gene editing pair is selected from the group consisting of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, and 49719-49735, or a sequence variant having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, and the ERS contains a targeting sequence complementary to the target nucleic acid. In some embodiments, the guide ribonucleic acid is an ERS selected from the group consisting of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, and 49719-49735, and the ERS contains a targeting sequence complementary to the target nucleic acid, or a sequence having at least at least 1, 2, 3, 4, or 5 mismatches thereto. In some embodiments, the present disclosure provides a gene editing pair system comprising an engineered CasX protein containing a pair of mutations shown in Table 22 or a further modification thereof. In some embodiments, the present disclosure provides a gene editing pair system comprising an engineered CasX protein containing a pair of mutations shown in Table 22, or a sequence variant having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, and an ERS containing one or more mutations in Tables 44, 45, and 47, or an ERS selected from the group consisting of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, and 49719-49735, or a sequence variant having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto.In some embodiments of the present system, the RNP of the gene editing pair can bind to and cleave a double-stranded target nucleic acid comprising a coding sequence, a complement of the coding sequence, a non-coding sequence, and a regulatory element. In some embodiments of the present system, the RNP of the gene editing pair can bind to a target nucleic acid and generate one or more single-stranded nicks within the target nucleic acid.

[0077] In other embodiments, the present disclosure provides a gene editing pair system comprising an engineered CasX protein, a first ERS having a targeting sequence described herein, and a second ERS, wherein the second ERS has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid as compared to the targeting sequence of the first ERS, and introducing a plurality of cleavages into the target nucleic acid results in permanent indels or mutations in the target nucleic acid, or removal of intervening sequences between the cleavages.

[0078] In some embodiments, the gene editing pair comprising the engineered CasX and ERS has one or more improved characteristics compared to a gene editing pair comprising a CasX variant from which the engineered CasX is derived (e.g., CasX 515, SEQ ID NO: 228) and a gRNA variant from which the ERS is derived (e.g., gRNA scaffold 174, 175, 221, or 235). In the foregoing embodiments, the one or more improved characteristics can be assayed in an in vitro assay under equivalent conditions for the gene editing pair and the CasX variant and gRNA variant from which it is derived, or in vivo in a subject. Exemplary improved characteristics described herein can include, in some embodiments, improved stability of the RNP complex, increased binding affinity between the engineered CasX and the ERS, improved kinetics of RNP complex formation, a higher percentage of RNP having cleavage ability, increased editing activity of the target nucleic acid, increased editing specificity, decreased off-target editing, and enhanced utilization of non-canonical PAM sequences.

[0079] In some embodiments, the present disclosure provides a composition comprising a gene editing pair of any of the embodiments disclosed herein for use in the manufacture of a medicament for the treatment of a subject having a disease.

[0080] In other embodiments, the present disclosure provides vectors encoding or comprising engineered CasX and / or ERS for the generation and / or delivery of the system. Also provided herein are methods of making engineered CasX proteins and ERS, and methods of using engineered CasX and ERS, including gene editing methods and therapeutic methods. The engineered CasX protein and ERS components of the system, their characteristics, as well as delivery modalities and methods of using the system are described more fully below.

[0081] III. Engineered Ribonucleic Acid Scaffolds (ERS) and Targeting Sequences of the System for Gene Editing In another aspect, the present disclosure relates to an engineered guide ribonucleic acid scaffold (ERS) that is useful when complexed with an engineered CasX nuclease protein during in vitro, ex vivo, or in vivo genome editing of a target nucleic acid in a subject when linked to a targeting sequence complementary to (and thus capable of hybridizing with) the target nucleic acid sequence of the gene. The ERS of the present disclosure is a guide ribonucleic acid scaffold that has been modified as compared to a reference gRNA and gRNA variants by the approaches described herein.

[0082] Collectively, the CasX guide ribonucleic acids of the present disclosure, including all ERSs, reference gRNAs, and gRNA variants of the embodiments, include distinct structured regions or domains, namely, RNA triple strands, scaffold stem-loops, extension stem-loops, pseudoknots, and a targeting sequence that is specific for a target nucleic acid in embodiments of the present disclosure and is located at the 3' end of the guide scaffold. The 5' end, RNA triple strand, scaffold stem-loop, pseudoknot, and extension stem-loop, together with an unstructured triple-stranded loop that crosslinks portions of the triple strand, are referred to as the "scaffold" of the guide RNA and ERS. In some cases, the scaffold stem further includes a bubble. In other cases, the scaffold further includes a triple-stranded loop region. In still other cases, the scaffold further includes a 5' unstructured region. In some embodiments, the ERS of the present disclosure for use in the system includes a scaffold stem-loop having the sequence CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 49737).

[0083] The properties and characteristics of CasX guide ribonucleic acids and their domains are described in WO2020 / 247882 (A1), US2022 / 0220508 (A1), and WO2022 / 120095 (A1), which are incorporated herein by reference. Each of the structured domains contributes to the establishment of the overall RNA folding of the guide and retains the functionality of the guide, specifically, the ability to properly complex with the CasX nuclease. For example, while the guide scaffold stem interacts with the helix I domain of the CasX nuclease, residues within the triple strand, triple-stranded loop, and pseudoknot stem interact with the OBD of the CasX nuclease. Collectively, these interactions confer the ability of the guide to bind to CasX and form an RNP with it while maintaining stability, while the spacer (i.e., the targeting sequence) directs and defines the specificity of the RNP for binding to a specific sequence of DNA. The individual domains are described more fully below.

[0084] In an embodiment, the ERS is a single guide construct rather than a double-stranded duplex of wild-type guides, where the "activator" and the "targeting factor" are covalently linked together by intervening nucleotides.

[0085] The targeting sequence linked to the 3'-end of the ERS contains a nucleotide sequence (alternatively called a guide sequence, spacer, targeting factor, or targeting sequence) that is complementary to (and thus hybridizes with) a specific sequence (target site) within a target nucleic acid sequence (e.g., a double-stranded target DNA, a target ssRNA, a strand of target ssDNA, etc.), as fully described below. The targeting sequence linked to the ERS can bind to a target nucleic acid sequence, including, in the context of the present disclosure, a coding sequence, the complement of a coding sequence, a non-coding sequence, and known accessory elements. The protein-binding segment (or "activator" or "protein-binding sequence") interacts with (e.g., binds to) the CasX protein as a complex to form an RNP (as fully described below).

[0086] Site-specific binding and / or cleavage of a target nucleic acid sequence (e.g., genomic DNA) by an engineered CasX protein can occur at one or more positions (e.g., the sequence of the target nucleic acid) determined by base pair complementarity between the targeting sequence of the ERS and the target nucleic acid sequence. Thus, for example, an ERS of the present disclosure having a linked targeting sequence has sequence complementarity to a target nucleic acid adjacent to a sequence complementary to a TC PAM motif or PAM sequence, e.g., ATC, CTC, GTC, or TTC, and can therefore hybridize thereto. Because the targeting sequence of the guide sequence hybridizes to the sequence of the target nucleic acid sequence, the targeting factor can be modified by the user to hybridize to a specific target nucleic acid sequence as long as the position of the PAM sequence is considered. By selecting the targeting sequence of the ERS, a defined region of the target nucleic acid sequence or a sequence surrounding a specific position within the target nucleic acid can be modified or edited using the systems described herein. In some embodiments, the targeting sequence of the ERS has 15 to 20 consecutive nucleotides. In some embodiments, the targeting sequence has 15, 16, 17, 18, 19, or 20 consecutive nucleotides. In some embodiments, the targeting sequence consists of 20 consecutive 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. In some cases, the ERS targeting sequence linked to the ERS scaffold of the present disclosure is complementary to and hybridizes to a gene exon. In some embodiments, the ERS targeting sequence is complementary to and hybridizes to the sequence of the exon splice acceptor site. In other embodiments, the ERS targeting sequence hybridizes to an intron. In other embodiments, the ERS targeting sequence hybridizes to an intron-exon junction. In other embodiments, the ERS targeting sequence hybridizes to an intergenic region of a gene.In other embodiments, the ERS targeting sequence hybridizes to a regulatory region. In some cases, the regulatory region is a promoter or enhancer. In some cases, the regulatory region is located 5’ of the transcription start site or 3’ of the transcription start. In some cases, the regulatory region is within an intron of a gene. In other cases, the regulatory region includes the 5’UTR of the gene. In still other cases, the regulatory region includes the 3’UTR of the gene.

[0087] By selecting the targeting sequence of the gRNA, defined regions of the target nucleic acid sequence can be modified or edited using the CasX:gRNA system described herein. In some embodiments, the gRNA and the linked targeting sequence exhibit a low degree of off-target effect on the cell's DNA. As used herein, "off-target effect" refers to the effect of unintended cleavage such as mutation and indel formation at non-targeted genomic sites that show similar but not identical sequences compared to the target site (i.e., the targeting sequence of the gRNA). In some embodiments, the off-target effect exhibited by the gRNA and the linked targeting sequence is less than about 5%, less than about 4%, less than 3%, less than about 2%, less than about 1%, less than about 0.5%, less than 0.1% in the cell. In some embodiments, the off-target effect is determined in silico. In some embodiments, the off-target effect is determined in an in vitro cell-free assay. In some embodiments, the off-target effect is determined in a cell-based assay.

[0088] In some embodiments, the systems of the present disclosure include a first ERS and further include a second (and optionally, a third, fourth, fifth, or more) ERS, wherein the second ERS or additional ERS has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid sequence as compared to the targeting sequence of the first ERS such that multiple points within the target nucleic acid are targeted, e.g., multiple cuts are introduced into the target nucleic acid by engineered CasX, which are then edited by non-homologous end joining (NHEJ), homology-directed repair (HDR), homology-independent targeted integration (HITI), microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), or base excision repair (BER). In such cases, it will be understood that the second or additional ERS complex forms with an additional copy of the engineered CasX protein. By selecting the targeting sequences linked to the ERS, a defined region of the target nucleic acid sequence surrounding a particular position within the target nucleic acid can be modified or edited using the systems described herein, including promoting the insertion of a donor template containing a mutation of the target gene or the removal of a region or exon containing it by a double-cut mechanism using paired engineered CasX and ERS having different targeting sequences such that intervening nucleotides are removed.

[0089] a. Reference gRNA As used herein, "reference gRNA" refers to a CRISPR guide ribonucleic acid comprising the wild-type sequence of a naturally occurring gRNA. In some embodiments, the CasX reference gRNA comprises a sequence isolated from or derived from Deltaproteobacter. In some embodiments, the CasX reference guide RNA comprises a sequence isolated from or derived from Planctomycetes. In yet other embodiments, the CasX reference gRNA comprises a sequence isolated from or derived from Candidatus Sungbacteria.

[0090] Table 1 provides reference gRNA tracr sequences and scaffold sequences. In some embodiments, the present disclosure provides an ERS sequence having a scaffold that includes an ERS sequence having one or more nucleotide modifications compared to a reference gRNA sequence in which the gRNA has any one of the sequences of SEQ ID NOs: 4-16 in Table 1. [Table 1]

[0091] b. Engineered ribonucleic acid scaffold (ERS) In another aspect, the present disclosure relates to an ERS for use in the systems of the present disclosure, where the ERS comprises a plurality of modifications compared to the gRNA variant scaffold from which it is derived. While forming a complex with CasX engineered as an RNP and retaining the functional property of being able to guide the engineered CasX ribonucleoprotein holocomplex to a target nucleic acid, all ERSs having one or more improved functions, features, or adding one or more new functions when compared to the gRNA variant from which the ERS is derived are contemplated to be within the scope of the present disclosure. Although the present disclosure focuses on ERS and engineered CasX, it is understood that the ERS retains the ability to form a complex with reference CasX and CasX variants to form an RNP, and the engineered CasX retains the ability to form a complex with reference gRNA and gRNA variants to form an RNP. In some embodiments, the ERS has improved features selected from the group consisting of enhanced folding stability of individual regions within the scaffold, enhanced folding stability of the entire scaffold, enhanced transcription efficiency, enhanced binding affinity for the engineered CasX nuclease, increased editing when complexed as an RNP, increased cleavage activity when complexed as an RNP, and increased specificity of the RNP complexed with the target nucleic acid. In some of the foregoing cases, the improved features can be evaluated in in vitro assays, including the assays of the examples. In other foregoing cases, the improved features are evaluated in vivo. In some cases, one or more of the improved features of the ERS are compared to the reference gRNA of SEQ ID NO: 4 or SEQ ID NO: 5, or gRNA variants 174, 175, 221, or 235 (SEQ ID NOs: 17, 18, 61, and 75, respectively).

[0092] In some embodiments, the gRNA variant is subjected to one or more mutagenesis methods, such as Deep Mutational Evolution (DME), Deep Mutational Scanning (DMS), error-prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, domain substitution from one gRNA variant to another, or chemical modifications to generate one or more ERSs having enhanced or altered properties compared to a modified gRNA variant, by subjecting it to the mutagenesis methods described in the examples herein (e.g., Example 11, and PCT / US2021 / 061673 and WO2020 / 247882 (A1), which are incorporated herein by reference), to generate new ERSs. The activity of the gRNA variant from which the ERS is derived is used as a benchmark against which the activity of the ERS is compared, whereby an improvement in the function or other characteristics of the ERS can be measured. In other embodiments, the gRNA variant can be subjected to one or more intentionally specifically targeted mutations to generate a rationally designed variant, such as those described in the examples.

[0093] Table 2 provides exemplary gRNA variant scaffold sequences, which in some cases provided the starting sequences from which the ERSs were derived. In certain embodiments, gRNA variants 174, 175, 221, and 235 were subjected to mutagenesis to obtain ERSs of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, and 49719-49735.

Table 2

[0094] In some embodiments, the ERS of the present disclosure includes a plurality of modifications to the sequence of a previously generated gRNA variant, and this previously generated variant itself functions as the sequence to be modified. In some cases, one or more modifications are introduced into one or more regions of the scaffold, and these regions are selected from the group consisting of the 5'-end, pseudoknot stem I, triple-stranded loop (including triple-stranded regions I and II), pseudoknot stem II, scaffold stem-loop, extension stem-loop, and triple-stranded region III. In some embodiments, one or more modifications are introduced at the 5'-end of the scaffold. In some embodiments, one or more modifications are introduced into the pseudoknot region of the scaffold. In some embodiments, one or more modifications are introduced into the triple-stranded loop region of the scaffold. In some embodiments, one or more modifications are introduced into the scaffold stem-loop region of the scaffold. In some embodiments, one or more modifications are introduced into the extension stem-loop region of the scaffold. In other cases, one or more modifications are introduced into the scaffold bubble. In still other cases, one or more modifications are introduced into two or more of the aforementioned regions. Such modifications can include one or more consecutive nucleotides, i.e., 1, 2, 1-5, 1-10, 1-20, or 1-30 insertions, deletions, or substitutions, or any combination thereof, into the aforementioned regions. Then, by combining the modifications to the aforementioned regions, an ERS having a plurality of modifications can be engineered. Exemplary methods for generating and evaluating modifications are described in Examples 8-12, and representative modifications and the resulting sequences are presented in Tables 29, 30, 37, 38, 40, 43, 44, 45, 46, 47, 50.

[0095] In some embodiments, the ERS comprises a sequence having at least about 70% sequence identity to (i) ACUGGCACUUCUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG (SEQ ID NO: 61), or (ii) ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 156), wherein the sequence contains one or more modifications within the array, and these one or more modifications result in improved characteristics as compared to the unmodified SEQ ID NO: 61 or SEQ ID NO: 156. In some embodiments, the ERS comprises at least two modifications in the sequence of SEQ ID NO: 61 or SEQ ID NO: 156, and these modifications result in improved characteristics as compared to the unmodified SEQ ID NO: 61 or SEQ ID NO: 156. In some embodiments, the modifications include: i) substitution of 1 to 30 consecutive nucleotides in one or more regions of the scaffold; ii) deletion of 1 to 10 consecutive nucleotides in one or more regions of the scaffold; iii) insertion of 1 to 10 consecutive nucleotides in one or more regions of the scaffold; iv) substitution of the scaffold stem-loop from a heterologous RNA source; v) substitution of the extended stem-loop with an RNA stem-loop sequence from a heterologous RNA source; or vi) any combination of (i) to (v). In some embodiments, the modifications include mutations in one or more regions selected from the group consisting of the 5' end, the pseudoknot stem, the triple-stranded loop, the scaffold stem-loop, the extended stem-loop, and the triple-stranded region III. In some embodiments, the modifications include mutations in at least two regions of the ERS, and these regions are selected from the group consisting of the 5' end, pseudoknot stem I, triple-stranded loop, pseudoknot stem II, scaffold stem-loop, extended stem-loop, and triple-stranded region III. In some embodiments, the mutations are selected from the group consisting of the mutations described in any one of Table 44, Table 45, or Table 47.In some embodiments, the ERS comprises individual variant regions selected from the sequences of SEQ ID NOs: 739-753 within the 5' terminal region, the sequences of SEQ ID NOs: 754-772 within the triple-stranded loop region, the sequences of SEQ ID NOs: 773-791 within the triple-stranded region, the sequences of SEQ ID NOs: 792-841 within the pseudoknot region, the sequences of SEQ ID NOs: 842-869 within the scaffold stem region, or the sequences of SEQ ID NOs: 870-907 within the extension stem region. In some embodiments, the ERS comprises combinations of pairs of individual variant sequences from different regions. In some embodiments, the ERS comprises a sequence selected from the group consisting of SEQ ID NOs: 11,568-22,227 and 23,572-24,915, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0096] In some embodiments, the present disclosure provides an ERS having a scaffold of about 85-100 nucleotides or any integer therebetween. In some embodiments, the present disclosure provides an ERS having a scaffold of about 85-95 nucleotides, or about 88-90 nucleotides, or about 89 nucleotides.

[0097] In some embodiments, the present disclosure provides an ERS comprising a sequence selected from the group consisting of SEQ ID NOs: 156, 739-907, 739-907, 11568-22227, 23572-24915, and 49719-49735, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the ERS comprises improved characteristics as compared to the sequence of SEQ ID NO: 17 when assayed in a cell-based in vitro assay under equivalent conditions. In the foregoing, the improved characteristics are one or more functional properties selected from the group consisting of improved binding to the CasX nuclease to form a ribonucleoprotein (RNP), improved folding stability of the ERS, increased half-life of the cell, improved transcriptional efficiency, enhanced ability to synthetically produce the ERS, improved editing activity of the target nucleic acid by the RNP comprising the ERS, and improved editing specificity by the RNP comprising the ERS.

[0098] In some embodiments, the ERS comprises an exogenous extension stem loop having little or no identity with the reference stem loop region disclosed herein (e.g., SEQ ID NO: 15). In some embodiments, the heterologous stem loop improves the stability of the ERS. In some embodiments, the heterologous RNA stem loop can bind to a protein, RNA structure, DNA sequence, or small molecule. In some embodiments, the exogenous stem loop region replacing the stem loop comprises an RNA stem loop or hairpin, where the resulting ERS has improved stability and, depending on the choice of loop, results in non-covalent recruitment to a particular cellular protein or RNA. Non-limiting examples of such non-covalent recruitment components include, respectively, the NCR MS2 coat protein, PP7 coat protein, Qβ coat protein, protein N, protein Tat, phage GA coat protein, iron-responsive binding element (IRE) protein, and U1A signal recognition particle incorporated into the protein-coding nucleic acid used to transfect the packaging host cell, and hairpin RNAs or loops such as the MS2 hairpin, PP7 hairpin, Qβ hairpin, boxB, trans-activation response element (TAR), phage GA hairpin, phage ΛN hairpin, iron-responsive element (IRE), and U1 hairpin II having binding affinity for them.Such exogenous extended stem loops can include thermostable RNAs such as, for example, MS2 hairpin (ACAUGAGGAUCACCCAUGU (SEQ ID NO: 215)), Qβ hairpin (UGCAUGUCUAAGACAGCA (SEQ ID NO: 216)), U1 hairpin II (AAUCCAUUGCACUCCGGAUU (SEQ ID NO: 217)), Uvsx (CCUCUUCGGAGG (SEQ ID NO: 218)), PP7 hairpin (AGGAGUUUCUAUGGAAACCCU (SEQ ID NO: 219)), phage replication loop (AGGUGGGACGACCUCUCGGUCGUCCUAUCU (SEQ ID NO: 220)), kissing loop_a (UGCUCGCUCCGUUCGAGCA (SEQ ID NO: 221)), kissing loop_b1 (UGCUCGACGCGUCCUCGAGCA (SEQ ID NO: 222)), kissing loop_b2 (UGCUCGUUUGCGGCUACGAGCA (SEQ ID NO: 223)), G-quadruplex M3q (AGGGAGGGAGGGAGAGG (SEQ ID NO: 224)), G-quadruplex telomere basket (GGUUAGGGUUAGGGUUAGG (SEQ ID NO: 225)), sarcin-ricin loop (CUGCUCAGUACGAGAGGAACCGCAG (SEQ ID NO: 226)), or pseudoknot (UACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUAUACUUUGGAGUUUUAAAAUGUCUCUAAGUACA (SEQ ID NO: 227)). In some embodiments, one of the aforementioned hairpin sequences is incorporated into the stem loop to assist in transporting the packaging of ERS (associated CasX in the RNP complex) into budding XDP (described more fully below) in the host cell when the corresponding ligand is incorporated into the Gag polyprotein of XDP.

[0099] c. Guide 316 The guide scaffold can be made by several methods including recombinant synthesis or solid-phase RNA synthesis. However, when using solid-phase RNA synthesis, the length of the scaffold can affect manufacturability, and as the length increases, it results in increased manufacturing costs, decreased purity and yield, and a higher synthesis failure rate. When used in lipid nanoparticle (LNP) formulations, solid-phase RNA synthesis of the scaffold is preferred to generate the amounts required for commercial development. In previous experiments, gRNA variant 235 (SEQ ID NO: 75) was identified as having enhanced properties compared to gRNA variant 174 (SEQ ID NO: 17), but its increased length made its use in LNP formulations difficult. Therefore, alternative sequences were sought. In some embodiments, the present disclosure provides an ERS, wherein the ERS scaffold and the linked targeting sequence have a sequence of less than about 115 nucleotides, less than about 110 nucleotides, or less than about 100 nucleotides. In some embodiments, the present disclosure provides an ERS, wherein the ERS scaffold and the linked targeting sequence have a sequence of 100-115 nucleotides or any integer therebetween.

[0100] In some embodiments, an ERS with a modified scaffold 174 (SEQ ID NO: 17) sequence was designed by introducing one, two, three, four or more mutations at positions selected from the group consisting of U11, U24, A29, and A87. In some embodiments, the ERS comprises the extended stem-loop sequence of SEQ ID NO: 49739 and one or more mutations at positions selected from the group consisting of U11, U24, A29, and A87, the sequence of SEQ ID NO: 17, or a sequence having at least about 70% sequence identity thereto. In some embodiments, the ERS comprises the extended stem-loop sequence of SEQ ID NO: 49739 and two mutations at positions selected from the group consisting of U11, U24, A29, and A87, the sequence of SEQ ID NO: 17, or a sequence having at least about 70% sequence identity thereto. In some embodiments, the ERS comprises the extended stem-loop sequence of SEQ ID NO: 49739 and three mutations at positions selected from the group consisting of U11, U24, A29, and A87, the sequence of SEQ ID NO: 17, or a sequence having at least about 70% sequence identity thereto. In some embodiments, the ERS comprises the extended stem-loop sequence of SEQ ID NO: 49739 and four mutations at positions selected from the group consisting of U11, U24, A29, and A87, the sequence of SEQ ID NO: 17, or a sequence having at least about 70% sequence identity thereto. In one of the foregoing embodiments, the mutations consist of U11C, U24C, A29C, and A87G, thereby resulting in the ERS 316 sequence ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 156), or a sequence having at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto.

[0101] In some embodiments, the ERS comprises the sequence of SEQ ID NO: 17, or a sequence having at least about 70% sequence identity thereto, comprising the extended stem-loop sequence of SEQ ID NO: 49739 and one or more mutations at positions selected from the group consisting of U11, U24, A29, and A87, wherein these one or more mutations improve the editing ability of the ERS as compared to SEQ ID NO: 17.

[0102] In one embodiment, an ERS scaffold was designed in which the scaffold 235 sequence (SEQ ID NO: 75) was modified by domain swapping, replacing the extended stem loop of scaffold variant 174 (SEQ ID NO: 49739) with the extended stem loop of scaffold 235. In some embodiments, the present disclosure provides an ERS comprising a sequence of SEQ ID NO: 75 modified to include the extended stem loop sequence of SEQ ID NO: 49739, or a sequence having at least about 70% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments, the ERS modified to include the extended stem loop sequence of SEQ ID NO: 49739 further comprises one or more regions selected from the group consisting of: i) a 5′ end comprising the sequence of AC, ii) a pseudoknot stem I comprising the sequence of UGGCGCU, iii) a triple helix loop comprising the sequence of SEQ ID NO: 49736, iv) a pseudoknot stem II comprising the sequence of AGCGCCA, and a triple helix region III comprising the sequence of CAGAG. In the foregoing embodiment, the modification results in a chimeric ERS 316 (FIGS. 11C and 25) having the sequence of ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 156), having 89 nucleotides within the scaffold compared to 99 nucleotides of gRNA variant 235. In some embodiments, shortening the length of the 316 scaffold sequence results in improved fidelity in the ability to synthetically generate a guide with an accurate and complete sequence, as well as improved ability to be successfully incorporated into an LNP. In addition to improved manufacturability, as described in the examples, the 316 scaffold was determined to function equivalently or preferably to gRNA variant 174 in an editing assay. The resulting 316 scaffold has the further advantage that the extended stem loop did not contain a CpG motif, and the enhanced properties are more fully described below. In some embodiments, the 316 scaffold was subjected to chemical modification to generate additional ERSs as described below. The sequences of the regions of ERS scaffold 316 are presented in Table 3.

Table 3

[0103] d. Chemically modified ERS In some embodiments, the present disclosure provides an ERS having one or more chemical modifications to enhance the chemical stability of the ERS. In some cases, the chemically modified ERS is utilized during LNP formation, and the ability of the RNA incorporated into the LNP is required to fold, assume, and maintain its structural conformation and to resist nuclease degradation or induce an immune response when introduced into the target cell environment. Chemical modification of RNA has been shown to improve stability, increase nuclease resistance to cellular RNases, increase double-stranded binding formation, and decrease immune responses by selective modification of nucleotides, thereby resulting in enhanced editing in the CRISPR system (Basila, M., et al. Minimal 2’-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity. PLoS ONE 12(11):e0188593(2017)). In some embodiments, the chemical modification is the addition of a 2’O-methyl group to one or more nucleotides of the ERS sequence and the linked targeting sequence. In some embodiments, the chemical modification is the addition of a 2’O-methyl group to each of the 5’ and 3’ termini of the ERS. In some embodiments, the chemical modification is the substitution of phosphorothioate linkages between two or more nucleosides of the sequence. In some embodiments, the first 1, 2, or 3 nucleotides at the 5’ terminus of the scaffold (i.e., A, C, and U in the case of scaffolds 174, 235, and 316) are modified by the addition of a 2’O-methyl group, and each of the modified nucleosides is linked to an adjacent nucleoside by a phosphorothioate linkage. Similarly, the last 1, 2, or 3 nucleotides at the 3’ terminus of the targeting sequence linked to the 3’ terminus of the scaffold are similarly modified to generate a terminal protection variant (collectively, the construct having the aforementioned modifications referred to as “v1”).In other embodiments, the nucleotides at the 5' and 3' ends, as well as within selected internal regions, are similarly modified by the addition of 2'O-methyl groups. In another embodiment, a 3' UUU tail was added to the construct in addition to the v1 modification such that the termination sequence used in the cellular transcription system was mimicked and the modified nucleotides of v1 were moved outside the region of the targeting sequence involved in target recognition, and an ERS with a linked targeting sequence was designed (referred to as "v2"). In another embodiment, an ERS was designed (referred to as "v3") in which, in addition to the v1 terminal protection modification, additional 2'OMe modifications were made to nucleotides identified as potentially modifiable based on the scaffold structural analysis. In another embodiment, an ERS was designed (referred to as "v4") in which the 2'OMe modification of the v3 version of the triple-stranded region of the scaffold was removed to reduce disruption of the RNA helix structure and maintain the resulting scaffold backbone flexibility. In another embodiment, an ERS was designed (referred to as "v5") in which the modification included the terminal protection modification of the v1 version and 2'OMe modifications were introduced into the scaffold stem and extension stem regions of the scaffold. In another embodiment, an ERS was designed (referred to as "v6") in which the modification included the terminal protection modification of the v1 version and 2'OMe modifications were introduced only into the extension stem region of the scaffold. Schematic diagrams of these configurations are shown in FIGS. 8A, 8B, 10, 16A, and 16B. In some embodiments, the present disclosure provides an ERS having a v1, v2, v3, v4, v5, v6, v7, v8, or v9 configuration having a sequence selected from the group consisting of the sequences set forth in Table 29 of Example 8 (SEQ ID NOS: 49750-49758, 49760-49768, and 49770-49749) (it is understood that for use in the systems of the present disclosure, the 20 non-targeting nucleotides at the 3' end are replaced with a targeting sequence complementary to the target nucleic acid to be modified). In certain embodiments, the ERS comprises the sequence of SEQ ID NO: 49770 (it is understood that for use in the systems of the present disclosure, the 20 non-targeting nucleotides at the 3' end are replaced with a targeting sequence complementary to the target nucleic acid to be modified).In some embodiments, an ERS having a v1, v2, v3, v4, v5, v6, v7, v8, or v9 configuration and a linked targeting sequence, when evaluated in an equivalent in vitro assay using a CasX nuclease, retains 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% of the editing of the target nucleic acid compared to an unmodified gRNA. In some embodiments, an ERS having a v1, v2, v3, v4, v5, v6, v7, v8, or v9 configuration and a linked targeting sequence exhibits reduced susceptibility of the ERS to degradation by cellular RNases compared to an unmodified ERS. In some embodiments, a chemically modified ERS exhibits reduced susceptibility to degradation by cellular RNases that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% lower compared to an unmodified ERS.

[0104] e. CpG-depleted ERS In connection with the use of a recombinant adeno-associated virus vector (AAV) for delivery of the ERS and engineered CasX of this embodiment, it has been determined that unmethylated CpG dinucleotides in viral DNA can bind to TLR9, an endosomal PRR in plasmacytoid dendritic cells (pDC) and B cells, and can elicit an immune response in mammalian hosts (Faust, SM, et al. CpG-depleted adeno-associated virus vectors evade immune detection. J. Clinical Invest. 123:2294 (2013)). Specifically, the CpG dinucleotide motif in the AAV vector is immunostimulatory because it has a high degree of hypomethylation compared to mammalian CpG motifs with a high degree of methylation. Thus, it is expected that reducing the frequency of unmethylated CpGs in the rAAV vector genome to a level below the threshold for activating human TLR9 will reduce the immune response to exogenously administered rAAV-based biologics.

[0105] In some embodiments, the present disclosure provides an ERS that is codon-optimized for depletion of CpG dinucleotides by substitution of homologous nucleotide sequences from mammalian species, wherein the modified ERS substantially retains the functional properties that drive the expression of ERS when expressed in cells transduced with an AAV comprising the modified ERS. In some embodiments, the present disclosure provides an ERS for inclusion in an rAAV vector, wherein the coding sequence of this ERS comprises less than about 10%, less than about 5%, or less than about 1% CpG dinucleotides and retains the ability to effect transcription of an ERS that can bind to engineered CasX. In some embodiments, the CpG-depleted ERS is encoded by a DNA sequence comprising a sequence selected from the group consisting of the sequences of Table 38 (SEQ ID NOs: 535-556). In some embodiments, the CpG-depleted ERS comprises a sequence selected from the group consisting of the sequences of Table 38 (SEQ ID NOs: 160-181).

[0106] In some embodiments, administration of a therapeutically effective dose of an rAAV vector comprising a CpG-depleted ERS of a transgene results in a reduced immune response as compared to the immune response of an equivalent rAAV vector, where the ERS is not codon-optimized for CpG dinucleotide depletion, and the reduction in response is determined by measurement of one or more parameters such as the generation of antibodies or delayed-type hypersensitivity to the ERS, or the generation of inflammatory cytokines and markers such as TLR9, interleukin-1 (IL-1), IL-6, IL-12, IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ), and granulocyte macrophage colony-stimulating factor (GM-CSF), but not limited thereto. In some embodiments, an rAAV vector comprising a CpG-depleted ERS of a transgene, when assayed in a cell-based in vitro assay, such as a cell-based in vitro assay using cells known in the art suitable for monocytes, macrophages, T cells, B cells, etc., induces a reduction in the production of 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 80%, or at least about 90% of one or more inflammatory markers selected from the group consisting of TLR9, interleukin-1 (IL-1), IL-6, IL-12, IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ), and granulocyte macrophage colony-stimulating factor (GM-CSF), as compared to an equivalent rAAV without CpG depletion. In certain embodiments, an rAAV vector comprising a CpG-depleted ERS of a transgene exhibits a reduction in TLR9 activation in hNPCs of 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 80%, or at least about 90% as compared to an equivalent rAAV without CpG depletion in an in vitro assay.

[0107] f. Complex formation with class 2 type V proteins In some embodiments, upon expression, ERS can complex as an RNP with an engineered CasX protein comprising any one of the sequences of SEQ ID NOs: 247-294, 24916-49628, 49746-49747, and 49871-49873, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0108] In some embodiments, upon expression, ERS can complex as an RNP with an engineered CasX protein comprising a pair of mutations shown in Table 22 or a further variant thereof. In some embodiments, ERS has the ability to form a complex with an engineered CasX protein that is improved as compared to a gRNA variant or a reference gRNA, whereby its ability to form a ribonucleoprotein (RNP) complex having cleavage ability with the engineered CasX protein is improved. Due to the improvement in ribonucleoprotein complex formation, in some embodiments, the efficiency of assembling a functional RNP can be improved. In some embodiments, more than 90%, more than 93%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the RNP comprising ERS and its targeting sequence is capable of gene editing of the target nucleic acid.

[0109] IV. Engineered CasX Proteins for Modifying Target Nucleic Acids The present disclosure provides engineered CasX nuclease proteins that are useful for genome editing of eukaryotic cells. The engineered CasX nucleases used in genome editing systems are class 2 type V nucleases. Although members of the class 2 type V CRISPR-Cas systems differ, they share some common features that distinguish them from the Cas9 system. First, class 2 type V nucleases have a single RNA-guided effector that contains an RuvC domain but not an HNH domain, and they recognize a TC motif PAM upstream of the target region on the non-target strand, which is different from the Cas9 system that depends on a G-rich PAM on the 3′ side of the target sequence. Type V nucleases generate staggered double-strand breaks distal to the PAM sequence, unlike Cas9, which generates blunt ends at a proximal site close to the PAM. In addition, type V nucleases degrade ssDNA in trans when activated by cis-targeted dsDNA or ssDNA binding. In some embodiments, the engineered CasX nucleases of those embodiments recognize a 5′-TC PAM motif and generate staggered ends cleaved only by the RuvC domain. In some embodiments, the present disclosure provides a system (eCasX:ERS system) comprising an engineered CasX protein and one or more ERSs that are specifically designed to edit a target nucleic acid sequence in a eukaryotic cell.

[0110] As used herein, the term "CasX protein" refers to a family of proteins and includes not only all naturally occurring CasX proteins ("reference CasX"), but also engineered CasX proteins having sequence modifications that have one or more improved features compared to the CasX proteins from which they are derived and are more fully described below.

[0111] The reference CasX, CasX variants (e.g., CasX 515), and engineered CasX proteins of the present disclosure include the following domains: a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helix I domain (further classified into a helix I-I subdomain and a helix I-II subdomain), a helix II domain, an oligonucleotide binding domain (OBD, further classified into an OBD-I subdomain and an OBD-II subdomain), and an RuvC DNA cleavage domain (further classified into an RuvC-I subdomain and an RuvC-II subdomain). In some embodiments, the present disclosure contemplates an engineered CasX that has a plurality of mutations within a domain as compared to the CasX from which it is derived, and yet the engineered CasX retains the ability to form an ERS and an RNP and retains nuclease activity. All such engineered CasXs that retain such properties are considered within the scope of the present disclosure. In other embodiments, the RuvC domain may be modified or deleted in a catalytically dead variant.

[0112] a. Reference CasX protein For the purposes of the present disclosure, the sequence of a naturally occurring CasX protein (referred to herein as the "reference CasX protein") is provided for illustrative purposes, for example, for the identification of domains and subdomains and for the ability to refer to selected amino acid positions. For example, a reference CasX protein can be isolated from a naturally occurring prokaryote such as a Deltaproteobacteria species, a Planctomycetes species, or a Candidatus Sungbacteria species. A reference CasX protein is a type II CRISPR / Cas endonuclease belonging to the CasX (alternatively called Cas12e) family of proteins that interact with a guide RNA to form a ribonucleoprotein (RNP) complex.

[0113] In some cases, the reference CasX protein is isolated from or derived from a Deltaproteobacter having the following sequence: 1 MEKRINKIRK KLSADNATKP VSRSGPMKTL LVRVMTDDLK KRLEKRRKKP EVMPQVISNN 61 AANNLRMLLD DYTKMKEAIL QVYWQEFKDD HVGLMCKFAQ PASKKIDQNK LKPEMDEKGN 121 LTTAGFACSQ CGQPLFVYKL EQVSEKGKAY TNYFGRCNVA EHEKLILLAQ LKPEKDSDEA 181 VTYSLGKFGQ RALDFYSIHV TKESTHPVKP LAQIAGNRYA SGPVGKALSD ACMGTIASFL 241 SKYQDIIIEH QKVVKGNQKR LESLRELAGK ENLEYPSVTL PPQPHTKEGV DAYNEVIARV 301 RMWVNLNLWQ KLKLSRDDAK PLLRLKGFPS FPVVERRENE VDWWNTINEV KKLIDAKRDM 361 GRVFWSGVTA EKRNTILEGY NYLPNENDHK KREGSLENPK KPAKRQFGDL LLYLEKKYAG 421 DWGKVFDEAW ERIDKKIAGL TSHIEREEAR NAEDAQSKAV LTDWLRAKAS FVLERLKEMD 481 EKEFYACEIQ LQKWYGDLRG NPFAVEAENR VVDISGFSIG SDGHSIQYRN LLAWKYLENG 541 KREFYLLMNY GKKGRIRFTD GTDIKKSGKW QGLLYGGGKA KVIDLTFDPD DEQLIILPLA 601 FGTRQGREFI WNDLLSLETG LIKLANGRVI EKTIYNKKIG RDEPALFVAL TFERREVVDP 661 SNIKPVNLIG VDRGENIPAV IALTDPEGCP LPEFKDSSGG PTDILRIGEG YKEKQRAIQA 721 AKEVEQRRAG GYSRKFASKS RNLADDMVRN SARDLFYHAV THDAVLVFEN LSRGFGRQGK 781 RTFMTERQYT KMEDWLTAKL AYEGLTSKTY LSKTLAQYTS KTCSNCGFTI TTADYDGMLV 841 RLKKTSDGWA TTLNNKELKA EGQITYYNRY KRQTVEKELS AELDRLSEES GNNDISKWTK 901 GRRDEALFLL KKRFSHRPVQ EQFVCLDCGH EVHADEQAAL NIARSWLFLN SNSTEFKSYK 961 SGKQPFVGAW QAFYKRRLKE VWKPNA (SEQ ID NO: 1).

[0114] In some cases, the reference CasX protein is isolated from or derived from Planctomycetes having the following sequence: 1 MQEIKRINKI RRRLVKDSNT KKAGKTGPMK TLLVRVMTPD LRERLENLRK KPENIPQPIS 61 NTSRANLNKL LTDYTEMKKA ILHVYWEEFQ KDPVGLMSRV AQPAPKNIDQ RKLIPVKDGN 121 ERLTSSGFAC SQCCQPLYVY KLEQVNDKGK PHTNYFGRCN VSEHERLILL SPHKPEANDE 181 LVTYSLGKFG QRALDFYSIH VTRESNHPVK PLEQIGGNSC ASGPVGKALS DACMGAVASF 241 LTKYQDIILE HQKVIKKNEK RLANLKDIAS ANGLAFPKIT LPPQPHTKEG IEAYNNVVAQ 301 IVIWVNLNLW QKLKIGRDEA KPLQRLKGFP SFPLVERQAN EVDWWDMVCN VKKLINEKKE 361 DGKVFWQNLA GYKRQEALLP YLSSEEDRKK GKKFARYQFG DLLLHLEKKH GEDWGKVYDE 421 AWERIDKKVE GLSKHIKLEE ERRSEDAQSK AALTDWLRAK ASFVIEGLKE ADKDEFCRCE 481 LKLQKWYGDL RGKPFAIEAE NSILDISGFS KQYNCAFIWQ KDGVKKLNLY LIINYFKGGK 541 LRFKKIKPEA FEANRFYTVI NKKSGEIVPM EVNFNFDDPN LIILPLAFGK RQGREFIWND 601 LLSLETGSLK LANGRVIEKT LYNRRTRQDE PALFVALTFE RREVLDSSNI KPMNLIGIDR 661 GENIPAVIAL TDPEGCPLSR FKDSLGNPTH ILRIGESYKE KQRTIQAAKE VEQRRAGGYS 721 RKYASKAKNL ADDMVRNTAR DLLYYAVTQD AMLIFENLSR GFGRQGKRTF MAERQYTRME 781 DWLTAKLAYE GLPSKTYLSK TLAQYTSKTC SNCGFTITSA DYDRVLEKLK KTATGWMTTI 841 NGKELKVEGQ ITYYNRYKRQ NVVKDLSVEL DRLSEESVNN DISSWTKGRS GEALSLLKKR 901 FSHRPVQEKF VCLNCGFETH ADEQAALNIA RSWLFLRSQE YKKYQTNKTT GNTDKRAFVE 961 TWQSFYRKKL KEVWKPAV (SEQ ID NO: 2).

[0115] In some cases, the reference CasX protein is isolated from or derived from Candidatus Sungbacteria having the following sequence: 1 MDNANKPSTK SLVNTTRISD HFGVTPGQVT RVFSFGIIPT KRQYAIIERW FAAVEAARER 61 LYGMLYAHFQ ENPPAYLKEK FSYETFFKGR PVLNGLRDID PTIMTSAVFT ALRHKAEGAM 121 AAFHTNHRRL FEEARKKMRE YAECLKANEA LLRGAADIDW DKIVNALRTR LNTCLAPEYD 181 AVIADFGALC AFRALIAETN ALKGAYNHAL NQMLPALVKV DEPEEAEESP RLRFFNGRIN 241 DLPKFPVAER ETPPDTETII RQLEDMARVI PDTAEILGYI HRIRHKAARR KPGSAVPLPQ 301 RVALYCAIRM ERNPEEDPST VAGHFLGEID RVCEKRRQGL VRTPFDSQIR ARYMDIISFR 361 ATLAHPDRWT EIQFLRSNAA SRRVRAETIS APFEGFSWTS NRTNPAPQYG MALAKDANAP 421 ADAPELCICL SPSSAAFSVR EKGGDLIYMR PTGGRRGKDN PGKEITWVPG SFDEYPASGV 481 ALKLRLYFGR SQARRMLTNK TWGLLSDNPR VFAANAELVG KKRNPQDRWK LFFHMVISGP 541 PPVEYLDFSS DVRSRARTVI GINRGEVNPL AYAVVSVEDG QVLEEGLLGK KEYIDQLIET 601 RRRISEYQSR EQTPPRDLRQ RVRHLQDTVL GSARAKIHSL IAFWKGILAI ERLDDQFHGR 661 EQKIIPKKTY LANKTGFMNA LSFSGAVRVD KKGNPWGGMI EIYPGGISRT CTQCGTVWLA 721 RRPKNPGHRD AMVVIPDIVD DAAATGFDNV DCDAGTVDYG ELFTLSREWV RLTPRYSRVM 781 RGTLGDLERA IRQGDDRKSR QMLELALEPQ PQWGQFFCHR CGFNGQSDVL AATNLARRAI 841 SLIRRLPDTD TPPTP (SEQ ID NO: 3).

[0116] b. Engineered CasX protein The present disclosure provides highly modified engineered CasX proteins having multiple mutations as compared to a reference CasX or one or more CasX variant proteins, e.g., CasX 515 or the CasX proteins (SEQ ID NOS: 492-500) of Table 9. These mutations can be present within one or more domains of the parental CasX from which the engineered CasX is derived. The CasX domains and their positions compared to reference CasX SEQ ID NOS: 1 and 2 are presented in Tables 4 and 5.

Table 4

Table 5-1

Table 5-2

[0117] The mutations can be introduced into any one or a combination of the domains of the CasX variant to yield an engineered CasX. These modifications can be amino acid insertions, deletions, substitutions, or any combination thereof. Any amino acid can be substituted with any other amino acid in the substitutions described herein. The substitutions can be conservative substitutions (e.g., a basic amino acid is substituted with another basic amino acid). The substitutions can be non-conservative substitutions (e.g., a basic amino acid is substituted with an acidic amino acid or vice versa). For example, a proline in the CasX protein can be substituted with any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine to generate an engineered CasX protein of the present disclosure. In some embodiments, the engineered CasX contains two mutations compared to the CasX protein from which it is derived. In some embodiments, the engineered CasX contains three mutations compared to the CasX protein from which it is derived. In some embodiments, the engineered CasX contains two, three, four, five, six, seven, eight, nine, ten or more mutations compared to the CasX protein from which it is derived. In some embodiments, the two, three, four, five, six, seven, eight, nine, ten or more mutations are made at positions in the CasX protein sequence that are separated from each other. In other embodiments, the two, three, four, five, six, seven, eight, nine, ten or more mutations can be made in adjacent amino acids within the CasX protein sequence. In some embodiments, the engineered CasX contains two or more mutations compared to two or more different CasX proteins from which they are derived. The methods utilized for the design and production of the engineered CasX are described below, including the methods of the examples.

[0118] Suitable mutagenesis methods for generating engineered CasX proteins of the present disclosure may include, for example, random mutagenesis, site-directed mutagenesis, Markov Chain Monte Carlo (MCMC) directed evolution methods, staggered extension PCR, gene shuffling, rational design, or domain swapping (described in PCT / US2021 / 061673 and WO2020 / 247882 (A1), which are incorporated herein by reference). In some embodiments, the engineered CasX is designed, for example, by selecting a plurality of desired mutations in a CasX variant identified using the approach described in the Examples. In certain embodiments, the activity of the CasX variant protein prior to mutagenesis is used as a benchmark against which the activity of one or more resulting engineered CasXs is compared, whereby an improvement in the function of the engineered CasX is measured.

[0119] In some embodiments of the engineered CasX described herein, the approach for designing the engineered CasX utilizes a directed evolution method adapted from a Markov Chain Monte Carlo (MCMC) directed evolution method simulation (Biswas N., et al. Coupled Markov Chain Monte Carlo for high-dimensional regression with Half-t priors. arViV:2012.04798v2 (2021)) as described in Example 1.

[0120] Upon further iteration of the generation of engineered CasX proteins, arrays can be generated that are screened to mutagenize variant CasX proteins to identify engineered CasX with improved or enhanced properties. Exemplary methods used to generate and evaluate engineered CasX (e.g., CasX 515) derived from other CasX proteins are described in the Examples, which were generated by introducing modifications into the coding sequence to result in amino acid substitutions, deletions, or insertions at one or more positions within one or more domains of the parental CasX protein. In some embodiments, the resulting mutagenized arrays are screened to identify arrays with enhanced nuclease activity. In other embodiments, the mutagenized arrays are screened to identify arrays with enhanced editing specificity and reduced off-target editing. In other embodiments, the mutagenized arrays are screened to identify arrays with enhanced PAM utilization, i.e., the ability to utilize non-standard PAM sequences. In yet other embodiments, the mutagenized arrays are screened to identify arrays with enhanced properties of any two or three of the aforementioned categories, i.e., nuclease activity, specificity (reduced off-target editing), and PAM utilization. In other embodiments, libraries of array variants having one, two, three or more mutations at selected positions compared to the parental CasX protein can be generated and screened using a multiplex pooling approach using an assay such as the E. coli CcdB toxin assay or the PASS assay to identify engineered CasX having enhanced nuclease activity, enhanced specificity, and / or increased PAM utilization compared to cleavage of E. coli nucleic acids as described in Examples 5-7. The domain sequence of CasX 515 is presented in Table 7.

[0121] Any change in the amino acid sequence of a CasX variant protein that is derived from the engineered CasX and that confers improved characteristics on the engineered CasX protein is considered to be an engineered CasX protein of the present disclosure, provided that the engineered CasX retains the ability to form a gRNA or ERS and RNP and retains nuclease activity. In some embodiments, the improved characteristics are one or more of an improvement in the editing activity of a target nucleic acid, an improvement in the editing specificity for a target nucleic acid, an improvement in the editing specificity ratio for a target nucleic acid, a reduction in off-target editing, an increase in the proportion of the eukaryotic genome that can be efficiently edited, an improvement in the ability to form an RNP having ERS and cleavage ability, and an improvement in the stability of the RNP complex. In some embodiments, the improved characteristics are improved by at least about 0.1-fold, at least about 0.5-fold, at least about 1-fold, at least about 1-fold, at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or any integer-fold improvement between the foregoing. In some embodiments, the engineered CasX protein comprises 700 to 1200 amino acids, 800 to 1100 amino acids, or 900 to 1000 amino acids.

[0122] In some embodiments, the present disclosure provides engineered CasX derived from CasX 515 (SEQ ID NO: 49699) that includes two or more modifications of amino acids, i.e., insertions, deletions, or substitutions, within one or more domains (see Table 7 for the CasX 515 domain sequence). In some embodiments, the present disclosure provides an engineered CasX protein that includes a pair of mutations or further variants thereof as compared to CasX 515 (SEQ ID NO: 49699) shown in Table 22. In some embodiments, the engineered CasX comprising two or more modifications comprises a sequence selected from the group consisting of SEQ ID NOs: 247-294, 27857-49628, 49746-49747, and 49871-49873, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto. In a particular approach, as detailed in Example 7, single mutations of CasX 515 (SEQ ID NO: 49699) that exhibited enhanced activity and / or specificity were selected based on positions that were potentially complementary and engineered CasXs having combinations (i.e., having two or three mutations) were generated and screened for activity and specificity in in vitro assays. The positions of the mutations within the domains of CasX are described in detail in Table 21 of the following examples. In some embodiments, the engineered CasX comprises an OBD-I comprising an amino acid sequence that includes one or more mutations as compared to the sequence of SEQ ID NO: 295. In some embodiments, the engineered CasX comprises an OBD-I that includes one or more mutations selected from the group consisting of an I3G substitution, an insertion of G at position 4, a K4G substitution, an insertion of G at position 5, a K8G substitution, an insertion of R at position 26, and an R34P substitution as compared to the sequence of SEQ ID NO: 295. In some embodiments, the engineered CasX comprises an OBD-I comprising a sequence selected from the group consisting of SEQ ID NOs: 295, 49800, 49803-49808, and 49822-49833, or a sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99% sequence identity thereto. In some embodiments,The engineered CasX comprises a helical I-I domain comprising an amino acid sequence that contains one or more mutations as compared to the sequence of SEQ ID NO: 296. In some embodiments, the engineered CasX comprises a helical I-I domain that contains an R7Q substitution as compared to the amino acid sequence of SEQ ID NO: 296. In some embodiments, the engineered CasX comprises a helical I-I domain that contains a sequence selected from the group consisting of SEQ ID NO: 296 and 49809, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered CasX comprises an NTSB domain comprising an amino acid sequence that contains one or more mutations as compared to the sequence of SEQ ID NO: 297. In some embodiments, the engineered CasX comprises an NTSB domain that contains one or more mutations selected from the group consisting of an L68K substitution, an L68Q substitution, an A70Y substitution, an A70D substitution, and an A70S substitution as compared to the sequence of SEQ ID NO: 297. In some embodiments, the engineered CasX comprises an NTSB domain that contains a sequence selected from the group consisting of SEQ ID NO: 297, 49802, 49810, 49811, 49812, 49818, and 49835-49840, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered CasX comprises a helical I-II domain comprising an amino acid sequence that contains one or more mutations as compared to the sequence of SEQ ID NO: 298. In some embodiments, the engineered CasX comprises a helical I-II domain that contains one or more mutations selected from the group consisting of a G32T substitution, an M112T substitution, and an M112W substitution as compared to the sequence of SEQ ID NO: 298. In some embodiments, the engineered CasX comprises a helical I-II domain that contains a sequence selected from the group consisting of SEQ ID NO: 298, 49801, 49813-49814, and 49842, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered CasX comprises a helical II domain comprising an amino acid sequence that contains one or more mutations as compared to the sequence of SEQ ID NO: 299. In some embodiments,The engineered CasX comprises a helical II domain comprising one or more mutations selected from the group consisting of a Y65T substitution and an E148D substitution, as compared to the sequence of SEQ ID NO: 299. In some embodiments, the engineered CasX comprises a helical II domain comprising a sequence selected from the group consisting of SEQ ID NO: 299, 49815 - 49816, and 49843, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered CasX comprises a RuvC-I domain comprising an amino acid sequence comprising one or more mutations as compared to the sequence of SEQ ID NO: 301. In some embodiments, the engineered CasX comprises a RuvC-I domain comprising an S51R substitution as compared to the sequence of SEQ ID NO: 301. In some embodiments, the engineered CasX comprises a RuvC-I domain comprising a sequence selected from the group consisting of SEQ ID NO: 301 and 49821, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered CasX comprises a TSL domain comprising an amino acid sequence comprising one or more mutations as compared to the sequence of SEQ ID NO: 302. In some embodiments, the engineered CasX comprises a TSL domain comprising one or more mutations selected from the group consisting of a V15M substitution, a T76D substitution, and an S80Q substitution, as compared to the sequence of SEQ ID NO: 302. In some embodiments, the engineered CasX comprises a TSL domain comprising a sequence selected from the group consisting of SEQ ID NO: 302, 49817, 49819, 49820, and 49844 - 49846, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered CasX comprises an OBD-II domain comprising the sequence of SEQ ID NO: 300, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered CasX comprises the sequence of SEQ ID NO: 303, or a sequence having at least about 90%, at least about 95%, at least about 98%,It includes an RuvC-II domain that includes a sequence having at least about 99% sequence identity. In some embodiments, the engineered CasX is one of 4.I.G and 64.R.Q, 4.I.G and 169.L.K, 4.I.G and 169.L.Q, 4.I.G and 171.A.D, 4.I.G and 171.A.Y, 4.I.G and 171.A.S, 4.I.G and 224.G.T, 4.I.G and 304.M.T, 4.I.G and 398.Y.T, 4.I.G and 826.V.M, 4.I.G and 887.T.D, 4.I.G and 891.S.Q, 5.-.G and 64.R.Q, 5.-.G and 169.L.K, 5.-.G and 169.L.Q, 5.-.G and 171.A.D, 5.-.G and 171.A.Y, 5.-.G and 171.A.S, 5.-.G and 224.G.T, 5.-.G and 304.M.T, 5.-.G and 398.Y.T, 5.-.G and 826.V.M, 5.-.G and 887.T.D, 5.-.G and 891.S.Q, 9.K.G and 64.R.Q, 9.K.G and 169.L.K, 9.K.G and 169.L.Q, 9.K.G and 171.A.D, 9.K.G and 171.A.Y, 9.K.G and 171.A.S, 9.K.G and 224.G.T, 9.K.G and 304.M.T, 9.K.G and 398.Y.T, 9.K.G and 826.V.M, 9.K.G and 887.T.D, 9.K.G and 891.S.Q, 27.-.R and 64.R.Q, 27.-.R and 169.L.K, 27.-.R and 169.L.Q, 27.-.R and 171.A.D, 27.-.R and 171.A.Y, 27.-.R and 171.A.S, 27.-.R and 224.G.T, 27.-.R and 304.M.T, 27.-.R and 398.Y.T, 27.-.R and 826.V.M, 27.-.R and 887.T.D, 27.-.R and 891.S.Q, 35.R.P and 64.R.Q, 35.R.P and 169.L.K, 35.R.P and 169.L.Q, 35.R.P and 171.A.D, 35.R.P and 171.A.Y, 35.R.P and 171.A.S, 35.R.P and 224.G.T, 35.R.P and 304.M.T, 35.R.P and 398.Y.T, 35.R.P and 826.V.M, 35.R.P and 887.T.D, 35.R.P and 891.S.Q, provided in Table 22.887.T.D and 891.S.Q, 64.R.Q and 169.L.K, 64.R.Q and 169.L.Q, 64.R.Q and 171.A.D, 64.R.Q and 171.A.Y, 64.R.Q and 171.A.S, 64.R.Q and 224.G.T, 64.R.Q and 304.M.T, 64.R.Q and 398.Y.T, 64.R.Q and 826.V.M, 64.R.Q and 887.T.D, 64.R.Q and 891.S.Q, 169.L.K and 171.A.D, 169.L.K and 171.A.Y, 169.L.K and 171.A.S, 169.L.K and 224.G.T, 169.L.K and 304.M.T, 169.L.K and 398.Y.T, 169.L.K and 826.V.M, 169.L.K and 887.T.D, 169.L.K and 891.S.Q, 169.L.Q and 171.A.D, 169.L.Q and 171.A.Y, 169.L.Q and 171.A.S, 169.L.Q and 224.G.T, 169.L.Q and 304.M.T, 169.L.Q and 398.Y.T, 169.L.Q and 826.V.M, 169.L.Q and 887.T.D, 169.L.Q and 891.S.Q, 171.A.D and 224.G.T, 171.A.D and 304.M.T, 171.A.D and 398.Y.T, 171.A.D and 826.V.M, 171.A.D and 887.T.D, 171.A.D and 891.S.Q, 171.A.Y and 224.G.T, 171.A.Y and 304.M.T, 171.A.Y and 398.Y.T, 171.A.Y and 826.V.M, 171.A.Y and 887.T.D, 171.A.Y and 891.S.Q, 171.A.S and 224.G.T, 171.A.S and 304.M.T, 171.A.S and 398.Y.T, 171.A.S and 826.V.M, 171.A.S and 887.T.D, 171.A.S and 891.S.Q, 4.I.G and 35.R.P, 224.G.T and 304.M.T, 224.G.T and 398.Y.T, 224.G.T and 826.V.M, 224.G.T and 887.T.D, 224.G.T and 891.S.Q, 5.-.G and 35.R.P, 4.I.G and 27.-.R, 304.M.T and 398.Y.T, 304.M.T and 826.V.M, 304.M.T and 887.T.D, 304.M.T and 891.S.Q,9.K.G and 35.R.P, 5.-.G and 27.-.R, 4.I.G and 9.K.G, 398.Y.T and 826.V.M, 398.Y.T and 887.T.D, 398.Y.T and 891.S.Q, 27.-.R and 35.R.P, 9.K.G and 27.-.R, 5.-.G and 9.K.G, 4.I.G and 5.-.G, 826.V.M and 8, 87.T.D, 826.V.M and 891.S.Q, 5.K.G and 27.-.R, 5.K.G and 169.L.K, 5.K.G and 171.A.D, 5.K.G and 304.M.T, 5.K.G and 398.Y.T, 5.K.G and 891.S.Q, 6.-.G and 27.-.R, 6.-.G and 169.L.K, 6.-.G and 171.A.D, 6.-.G and 304.M.T, 6.-.G and 398.Y.T, 6.-.G and 891.S.Q, 304.M.W and 27.-.R, 304.M.W and 169.L.K, 304.M.W and 171.A.D, 304.M.W and 398.Y.T, 304.M.W and 891.S.Q, 481.E.D and 27.-.R, 481.E.D and 169.L.K, 481.E.D and 171.A.D, 481.E.D and 304.M.T, 481.E.D and 398.Y.T, 481.E.D and 891.S.Q, 698.S.R and 27.-.R, 698.S.R and 169.L.K, 698.S.R and 171.A.D, 698.S.R and 304.M.T, 698.S.R and 398.Y.T, and 698.S.R and 891.S.Q, comprising two or more mutations selected from, wherein the position of the mutation is compared to the CasX sequence of SEQ ID NO: 49699. In some embodiments, the engineered CasX comprises two or more mutations of Table 22, which result in improved characteristics compared to unmodified CasX 515 (SEQ ID NO: 49699). In some embodiments, the improved characteristics are determined by comparison to unmodified parental CasX 515 in an in vitro assay under equivalent conditions. In some embodiments, the improved characteristics are, for example, a decrease in off-target editing as shown in Table 27. In some embodiments, the improved characteristics are, for example, an increase in on-target editing as shown in Table 25.

[0123] In some embodiments, the engineered CasX contains three mutations in the CasX 515 sequence (SEQ ID NO: 49699), and these three mutations are selected from the group consisting of 27.-.R, 169.L.K, and 329.G.K; 27.-.R, 171.A.D, and 224.G.T; and 35.R.P, 171.A.Y, and 304.M.T, and these mutations result in improved characteristics compared to unengineered CasX 515.

[0124] In some embodiments, SEQ ID NO: 27858, 27859, 27861, 27865, 27866, 27868, 27870, 27871, 27872, 27876, 27877, 27880, 27882, 27889, 27897, 27898, 27903, 27952, 27953, 27954, 27955, 27958, 27959, 27961, 27963, 27969, 27970, 27973, 27975, 27982, 27990, 27991, 27996, 27998, 28003, 28004, 28006, 28008, 28009, 28010, 28014, 28018, 28027, 28035, 28036, 28047, 28048, 28050, 28052, 28053, 28054, 28058, 28062, 28071, 28079, 28080, 28095, 28101, 28105, 28123, 28137, 28143, 28147, 28165, 28253, 28255, 28257, 28258, 28259, 28263, 28267, 28276, 28284, 28285, 28293, 28295, 28296, 28297, 28301, 28305, 28314, 28322, 28323, 28368, 28369, 28370, 28374, 28378, 28387, 28395, 28396, 28438, 28439, 28443, 28444, 28447, 28449, 28456, 28464, 28465, 28470, 28477, 28481, 28490, 28498, 28499, 28511, 28515, 28524, 28532, 28533, 28633, 28635, 28642, 28650, 28651, 28656, 28661, 28679, 28738, 28745, 28753, 28754, 28759, 28799, 28925, 28926, 29011, 29022, 29056, 29098, 29119, 29140, 29245, 29266, 29308, 29371, 29392, 29476, 29560, 29749, 29917, 29938, 30196, 30888, 31244, 31592, 33212, 33512, 34088, 34631, 34870, 35139, 35402, 35422, 35467, 35507, 35512, 43373, 49746, 49747,Engineered CasX selected from the group consisting of 49871-49873 exhibits improved editing activity compared to unmodified parental CasX 515. In some embodiments, the improved characteristics are determined in an in vitro assay under equivalent conditions compared to unmodified parental CasX 515.,

[0125] In some embodiments, SEQ ID NO: 27858, 27859, 27861, 27865, 27866, 27868, 27870, 27871, 27872, 27876, 27877, 27880, 27882, 27889, 27897, 27898, 27903, 27952, 27953, 27954, 27955, 27958, 27959, 27961, 27963, 27969, 27970, 27973, 27975, 27982, 27990, 27991, 27996, 27998, 28003, 28004, 28006, 28008, 28009, 28010, 28014, 28018, 28027, 28035, 28036, 28047, 28048, 28050, 28052, 28053, 28054, 28058, 28062, 28071, 28079, 28080, 28095, 28101, 28105, 28123, 28137, 28143, 28147, 28165, 28253, 28255, 28257, 28258, 28259, 28263, 28267, 28276, 28284, 28285, 28293, 28295, 28296, 28297, 28301, 28305, 28314, 28322, 28323, 28368, 28369, 28370, 28374, 28378, 28387, 28395, 28396, 28438, 28439, 28443, 28444, 28447, 28449, 28456, 28464, 28465, 28470, 28477, 28481, 28490, 28498, 28499, 28511, 28515, 28524, 28532, 28533, 28633, 28635, 28642, 28650, 28651, 28656, 28661, 28679, 28738, 28745, 28753, 28754, 28759, 28799, 28925, 28926, 29011, 29022, 29056, 29098, 29119, 29140, 29245, 29266, 29308, 29371, 29392, 29476, 29560, 29749, 29917, 29938, 30196, 30888, 31244, 31592, 33212, 33512, 34088, 34631, 34870, 35139, 35402, 35422, 35467, 35507, 35512, 43373, 49746, 49747Engineered CasX selected from the group consisting of 49871-49873 exhibits improved editing specificity compared to unmodified parental CasX 515, and in some embodiments, the improved characteristics are determined in in vitro assays under equivalent conditions compared to unmodified parental CasX 515.,

[0126] In some embodiments, SEQ ID NO: 27858, 27859, 27861, 27865, 27866, 27868, 27870, 27871, 27872, 27876, 27877, 27880, 27882, 27889, 27897, 27898, 27903, 27952, 27953, 27954, 27955, 27958, 27959, 27961, 27963, 27969, 27970, 27973, 27975, 27982, 27990, 27991, 27996, 27998, 28003, 28004, 28006, 28008, 28009, 28010, 28014, 28018, 28027, 28035, 28036, 28047, 28048, 28050, 28052, 28053, 28054, 28058, 28062, 28071, 28079, 28080, 28095, 28101, 28105, 28123, 28137, 28143, 28147, 28165, 28253, 28255, 28257, 28258, 28259, 28263, 28267, 28276, 28284, 28285, 28293, 28295, 28296, 28297, 28301, 28305, 28314, 28322, 28323, 28368, 28369, 28370, 28374, 28378, 28387, 28395, 28396, 28438, 28439, 28443, 28444, 28447, 28449, 28456, 28464, 28465, 28470, 28477, 28481, 28490, 28498, 28499, 28511, 28515, 28524, 28532, 28533, 28633, 28635, 28642, 28650, 28651, 28656, 28661, 28679, 28738, 28745, 28753, 28754, 28759, 28799, 28925, 28926, 29011, 29022, 29056, 29098, 29119, 29140, 29245, 29266, 29308, 29371, 29392, 29476, 29560, 29749, 29917, 29938, 30196, 30888, 31244, 31592, 33212, 33512, 34088, 34631, 34870, 35139, 35402, 35422, 35467, 35507, 35512, 43373, 49746, 49747The engineered CasX selected from the group consisting of 49871 to 49873 exhibits improved activity and specificity compared to the unmodified parental CasX 515. In some embodiments, the improved characteristics are determined by comparison with the unmodified parental CasX 515 in an in vitro assay under equivalent conditions.

[0127] In some embodiments, the engineered CasX selected from the group consisting of SEQ ID NOs: 27865, 27952, 27954, 27955, 27958, 27959, 27973, 28009, 28018, 28048, 28101, 28123, 28137, 28285, 28296, 28301, 28305, 28314, 28323, 28368, 28369, 28370, 28378, 28387, 28438, 28447, 28477, 28481, 28498, 28515, 28524, 28532, 28661, 28799, 28925, 29022, 29266, 29308, 29371, 29560, 29749, 29917, 30888, 31244, 33212, 33512, 34088, 34870, 35422, 35507, 43373, 49872, and 49873 exhibits an improved specificity ratio compared to the unmodified parental CasX 515. In some embodiments, the improved characteristics are determined by comparison with the unmodified parental CasX 515 in an in vitro assay under equivalent conditions.

[0128] In some embodiments, the engineered CasX selected from the group consisting of SEQ ID NOs: 27952, 27958, 28101, 28123, 28137, 28285, 28368, 28370, 28378, 28387, 28438, 28799, 28925, 29022, 29308, 29749, 29917, 30888, 34870, 43373, and 49873 exhibits improved editing activity and improved editing specificity compared to the unmodified parental CasX 515. In some embodiments, the improved characteristics are determined by comparison with the unmodified parental CasX 515 in an in vitro assay under equivalent conditions.

[0129] In some embodiments, engineered CasX selected from the group consisting of SEQ ID NOs: 27952, 27958, 28036, 28101, 28123, 28137, 28285, 28368, 28370, 28378, 28387, 28438, 28499, 28799, 28925, 29011, 29022, 29308, 29749, 29917, 30888, 34870, 35402, 35512, 43373, and 49873 exhibits improved editing activity and an improved editing specificity ratio compared to unmodified parental CasX 515. In some embodiments, the improved characteristics are determined in an in vitro assay under equivalent conditions compared to unmodified parental CasX 515.

[0130] In some embodiments, the aforementioned characteristics of engineered CasX are improved compared to unmodified parental CasX 515 and are improved by at least about 0.1-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold.

[0131] In some embodiments, the engineered CasX protein comprises, from the N-terminus to the C-terminus, an OBD-I domain, a helix I-I domain, an NTSB domain, a helix I-II domain, a helix II domain, an OBD-II, a RuvC-I domain, a TSL domain, and a RuvC-II domain, and each domain comprises the sequence set forth in Table 23, or a sequence having at least about 90% or at least about 95% sequence identity thereto. In some embodiments, an engineered CasX protein comprising a pair of mutations shown in Table 22 or a further variant thereof exhibits increased on-target editing activity or decreased off-target activity (specificity) compared to unmodified parental CasX variant 515 when assayed in an in vitro assay under equivalent conditions.

[0132] As described in the examples, an engineered CasX called "CasX 812" was generated. As described in Example 2, CasX 812 was generated by substituting glycine with lysine at position 329 of CasX 515 within the helix I-II domain. CasX 812 exhibited improved specificity compared to CasX 515 in the pooled activity and specificity (PASS) assay described in Examples 2 and 6. The amino acid sequence of the domain of CasX 812 is provided in Table 13 of the examples. Thus, in some embodiments, the present disclosure provides an engineered CasX comprising an amino acid substitution at position 329 compared to the CasX 515 protein comprising the amino acid sequence of SEQ ID NO: 49699. In some embodiments, the engineered CasX comprises a mutation in the helix I-II domain compared to CasX 515. In some embodiments, the engineered CasX comprises a mutation at position G137 compared to the helix I-II domain of CasX 515. In some embodiments, the engineered CasX comprises the helix I-II domain sequence of SEQ ID NO: 298, or a sequence having at least about 90% or at least about 95% sequence identity thereto, including the amino acid substitution at position G137 compared to the sequence of SEQ ID NO: 298. In some embodiments, the substituted position comprises a hydrophilic amino acid residue. In some embodiments, the hydrophilic amino acid residue is a lysine residue. In some embodiments, the hydrophilic amino acid residue is an asparagine residue. In some embodiments, the engineered CasX comprises an OBD-I domain comprising the amino acid sequence of SEQ ID NO: 295, or a sequence having at least about 90% or at least about 95% sequence identity thereto. In some embodiments, the engineered CasX comprises a helix I-I domain comprising the amino acid sequence of SEQ ID NO: 296, or a sequence having at least about 90% or at least about 95% sequence identity thereto. In some embodiments, the engineered CasX comprises an NTSB domain comprising the amino acid sequence of SEQ ID NO: 297, or a sequence having at least about 90% or at least about 95% sequence identity thereto.In some embodiments, engineered CasX comprises a helical I-II domain comprising the amino acid sequence of SEQ ID NO: 49847, or a sequence having at least about 90% or at least about 95% sequence identity thereto. In some embodiments, engineered CasX comprises an OBD-II domain comprising the amino acid sequence of SEQ ID NO: 300, or a sequence having at least about 90% or at least about 95% sequence identity thereto. In some embodiments, engineered CasX comprises a RuvC-I domain comprising the amino acid sequence of SEQ ID NO: 301, or a sequence having at least about 90% or at least about 95% sequence identity thereto. In some embodiments, engineered CasX comprises a TSL domain comprising the amino acid sequence of SEQ ID NO: 302, or a sequence having at least about 90% or at least about 95% sequence identity thereto. In some embodiments, engineered CasX comprises a RuvC-II domain comprising the amino acid sequence of SEQ ID NO: 303, or a sequence having at least about 90% or at least about 95% sequence identity thereto. In another specific embodiment, the present disclosure provides an engineered CasX having the sequence of SEQ ID NO: 266 (CasX variant 812), or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, wherein the engineered CasX exhibits improved specificity as compared to CasX variant 515 (SEQ ID NO: 228).

[0133] The engineered CasX of the present disclosure has one or more improved features compared to the CasX protein from which it is derived, e.g., CasX 515 or the CasX proteins of Table 9 (SEQ ID NOs: 492-500). Exemplary improved features of the engineered CasX embodiments include improved ability to utilize a larger spectrum of PAM sequences upon editing and / or binding of target nucleic acids, increased nuclease activity, improved editing efficiency, improved editing specificity for target nucleic acids, reduced off-target editing or cleavage, increased proportion of eukaryotic genomes that can be efficiently edited, increased nuclease activity, and improved protein:ERS (RNP) complex stability, but are not limited thereto. Specifically, the engineered CasX protein of the present disclosure has enhanced ability to efficiently edit and / or bind to target DNA when complexed with ERS as an RNP, utilizing a PAM TC motif that includes a PAM sequence selected from TTC, ATC, GTC, or CTC. As described above, the PAM sequence is located at least 1 nucleotide on the 5' side of the non-target strand of the protospacer that has identity with the targeting sequence of ERS in an assay system, compared to the editing efficiency and / or binding of an RNP comprising a reference CasX protein and a reference gRNA in an equivalent assay system.

[0134] Additional engineered CasX of the present disclosure includes the sequences of SEQ ID NOs: 247-294 described in Table 6, or sequences having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% sequence identity thereto.

Table 6

[0135] c. Engineered CasX proteins having domains from multiple source proteins Engineered chimeric CasX proteins are also contemplated within the scope of the present disclosure. As used herein, the term "chimeric CasX" protein refers to not only a CasX protein comprising at least two domains from different sources, but also a CasX protein comprising at least one domain that is itself chimeric, i.e., comprising at least one domain. Thus, in some embodiments, an engineered chimeric CasX protein comprises at least two domains isolated from or derived from different sources, such as two different naturally occurring CasX proteins (e.g., two different reference CasX proteins) or two different CasX variant proteins. In the case of split or discontinuous domains such as Helix I, RuvC, and OBD, a portion of the discontinuous domain can be replaced with the corresponding portion from any other source. For example, the Helix I-II domain within SEQ ID NO: 2 can be replaced with the corresponding Helix I-II sequence from SEQ ID NO: 1. In some embodiments, the first domain can be selected from the group consisting of the NTSB, TSL, Helix I-I, Helix I-II, Helix II, OBD-I, OBD-II, RuvC-I, and RuvC-II domains. In some embodiments, the second domain is selected from the group consisting of the NTSB, TSL, Helix I-I, Helix I-II, Helix II, OBD-I, OBD-II, RuvC-I, and RuvC-II domains, and the second domain is different from the aforementioned first domain. Domain sequences from the reference CasX proteins and their coordinates are shown in Table 4.

[0136] In some embodiments, the engineered CasX NTSB domain derived from SEQ ID NO: 2 is replaced with the corresponding NTSB sequence derived from SEQ ID NO: 1, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto, thereby resulting in a chimeric CasX protein. In some embodiments, the engineered CasX coiled coil I-II domain derived from SEQ ID NO: 2 is replaced with the corresponding coiled coil I-II sequence derived from SEQ ID NO: 1, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto, thereby resulting in a chimeric CasX protein. In some embodiments, the engineered CasX coiled coil I-II domain and NTSB domain derived from SEQ ID NO: 2 are replaced with the corresponding coiled coil I-II derived from SEQ ID NO: 1, or a sequence having 1, 2, 3, 4, or 5 mismatches thereto, and the NTSB sequence derived from SEQ ID NO: 1, or a sequence or sequence having 1, 2, 3, 4, or 5 mismatches thereto, thereby resulting in a chimeric CasX protein. Exemplary chimeric CasX includes, but is not limited to, the sequences of SEQ ID NOs: 247-294, 24916-49628, 49746-49747, and 49871, 49873, which have substitutions of the NTSB domain and coiled coil I-II domain derived from SEQ ID NO: 1, while other domains are originally derived from SEQ ID NO: 2 where the engineered CasX has additional amino acid changes (i.e., 1, 2, 3, 4, or 5 mismatches) at selected positions compared to the domains of the reference CasX.

Table 7

[0137] d. Protein affinity for ERS In some embodiments, the engineered CasX protein has an improved affinity for the ERS compared to the CasX protein from which it is derived, thereby resulting in the formation of a ribonucleoprotein complex. Without wishing to be bound by theory, in some embodiments, amino acid changes within the helix I domain can increase the binding affinity of the engineered CasX protein for the ERS sequence, while changes within the helix II domain can increase the binding affinity of the engineered CasX protein for the guide scaffold stem loop, and changes within the oligonucleotide binding domain (OBD) can increase the binding affinity of the engineered CasX protein for the ERS triple strand. The increased affinity of the engineered CasX protein for the ERS can result in, for example, a lower K d that can be achieved, which in some cases can result in more stable RNP complex formation. In some embodiments, the increased affinity of the engineered CasX protein for the ERS results in improved stability of the RNP complex upon delivery to human cells. This improved stability can affect the function and utility of the complex in the cells of the subject and can also result in improved pharmacokinetic properties in the blood upon delivery to the subject. In some embodiments, the increased affinity of the engineered CasX protein and the resulting improved stability of the RNP complex allow for a lower dose of the engineered CasX protein to be delivered to the subject or cells while still having the desired activity, such as in vivo or in vitro gene editing. In some embodiments, the higher affinity (stronger binding) of the engineered CasX protein for the ERS allows for a greater number of editing events to be possible if both the engineered CasX protein and the ERS remain in the RNP complex. The increase in editing events can be evaluated using the editing assays described herein. In some embodiments, the K d of the engineered CasX protein for the ERS is increased compared to the parental CasX protein that was mutagenized to create the engineered CasX. In some embodiments, the K of the engineered CasX for the ERSd is increased by at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100-fold as compared to the CasX from which it is derived. In some embodiments, engineered CasX has a binding affinity for ERS that is increased by about 1.1- to about 100-fold as compared to the CasX from which the ERS is derived, such as CasX 515.

[0138] In some embodiments, the increased affinity of the engineered CasX protein for ERS results in improved stability of the ribonucleoprotein complex upon delivery to mammalian cells, including in vivo delivery to a subject. This improved stability can affect the function and utility of the complex in the cells of the subject and can also result in improved pharmacokinetic properties in the blood upon delivery to the subject. In some embodiments, the increased affinity of the engineered CasX protein and the resulting improved stability of the ribonucleoprotein complex allow for delivery of lower doses of the engineered CasX protein to a subject or cell while still having the desired activity, such as in vivo or in vitro gene editing. The improved ability to form and maintain RNPs in a stable form can be evaluated using assays such as the in vitro cleavage assay described in the examples herein. In some embodiments, an RNP comprising the engineered CasX of the present disclosure achieves at least 2-fold, at least 5-fold, or at least 10-fold higher k 切断 rates when complexed as an RNP as compared to an RNP comprising the CasX from which it is derived, such as CasX 515.

[0139] Methods for measuring the binding affinity of engineered CasX proteins to ERS and for determining the cleavage competent fraction include in vitro methods using purified engineered CasX proteins and ERS, as described in the Examples. When the ERS or the engineered CasX protein is tagged with a fluorophore, the binding affinity for the engineered CasX protein can be measured by fluorescence polarization. Alternatively, or in addition, the binding affinity can be measured by biolayer interferometry, electrophoretic mobility shift assay (EMSA), or filter binding. Additional standard techniques for quantifying the absolute affinity of RNA-binding proteins such as the engineered CasX of the present disclosure for a particular ERS include, but are not limited to, isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR), as well as the methods of the Examples.

[0140] e. Affinity for the target nucleic acid In some embodiments, the engineered CasX protein has an increased binding affinity for the target nucleic acid as compared to the affinity of the CasX protein from which it is derived for the target nucleic acid. Engineered CasX having a higher affinity for those target nucleic acids can, in some embodiments, cleave the target nucleic acid sequence more rapidly than a reference CasX protein that does not have an increased affinity for the target nucleic acid.

[0141] In some embodiments, the improvement in affinity for the target nucleic acid includes an improvement in affinity for the target sequence or protospacer sequence of the target nucleic acid, an improvement in affinity for the PAM sequence, an improvement in the ability to search for DNA for the target sequence, or any combination thereof. Without wishing to be bound by theory, it is believed that CRISPR / Cas system proteins such as CasX can find their target sequences by one-dimensional diffusion along the DNA molecule. This process involves (1) binding of the ribonucleoprotein to the DNA molecule, followed by (2) stalling at the target sequence, both of which, in some embodiments, are affected by the improved affinity of the engineered CasX protein for the target nucleic acid sequence, thereby potentially improving the function of the engineered CasX protein.

[0142] Without wishing to be bound by theory, amino acid changes within the NTSB domain that increase the efficiency of unwinding of the non-target nucleic acid strand in the unwound state or capture it may be able to increase the affinity of the engineered CasX protein for the target nucleic acid. Alternatively, or in addition, amino acid changes within the NTSB domain that improve the ability of the NTSB domain to stabilize the DNA during unwinding may be able to increase the affinity of the engineered CasX protein for the target nucleic acid. Alternatively, or in addition, amino acid changes within the OBD may increase the affinity of the engineered CasX protein binding to the protospacer adjacent motif (PAM), thereby potentially increasing the affinity of the engineered CasX protein for the target nucleic acid. Alternatively, or in addition, amino acid changes within the helix I domain and / or II domain, RuvC domain, and TSL domain that increase the affinity of the engineered CasX protein for the target nucleic acid strand may be able to increase the affinity of the engineered CasX protein for the target nucleic acid.

[0143] In some embodiments, the binding affinity of the engineered CasX protein of the present disclosure for the target nucleic acid molecule is increased compared to the CasX protein from which it is derived. In some embodiments, the engineered CasX protein has a binding affinity for the target nucleic acid that is increased by at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100-fold compared to the CasX 515 variant.

[0144] Methods for measuring the affinity of a CasX protein for target and / or non-target nucleic acid molecules can include electrophoretic mobility shift assay (EMSA), filter binding, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR), fluorescence polarization, and biolayer interferometry (BLI). Further methods for measuring the affinity of a CasX protein for a target include in vitro biochemical assays of the type that measure DNA cleavage events over time.

[0145] In some embodiments, engineered CasX proteins with improved target nucleic acid affinity have an increased affinity for or ability to utilize specific PAM sequences other than the standard TTC PAM recognized by the reference CasX protein of SEQ ID NO: 2 that comprises a PAM sequence selected from the group consisting of ATC, GTC, and CTC, such that the amount of target nucleic acid that can be edited is increased compared to wild-type CasX nuclease or CasX variants 491 or 515. Without wishing to be bound by theory, these engineered CasXs may interact more strongly with DNA overall and may have an improved ability to access and edit sequences within the target nucleic acid due to a stronger binding to or utilization of the PAM sequence than the nuclease sequences of wild-type reference CasX or CasX 491 or 515, thereby potentially enabling a more efficient search process of the CasX protein for the target sequence. A higher overall affinity for DNA can, in some embodiments, increase the frequency with which the CasX protein can effectively initiate and terminate the binding and rewinding steps, thereby facilitating target strand invasion and R-loop formation and ultimately facilitating cleavage of the target nucleic acid sequence.

[0146] f. Improved specificity for the target site In some embodiments, the engineered CasX protein has improved specificity for a target nucleic acid sequence as compared to the CasX protein from which it is derived. As used herein, "specificity," also sometimes referred to as "target specificity," refers to the degree to which a CRISPR / Cas ribonucleoprotein complex cleaves an off-target sequence that is similar but not identical to the target nucleic acid sequence. For example, an engineered CasX RNP with a higher degree of specificity exhibits a reduced off-target effect or cleavage of sequences as compared to the CasX protein from which it is derived. Without wishing to be bound by theory, amino acid changes within the helical I domain and helical II domain that increase the specificity of the engineered CasX protein for the target nucleic acid strand may be able to increase the overall specificity of the engineered CasX protein for the target nucleic acid. In some embodiments, the amino acid changes that increase the specificity of the engineered CasX protein for the target nucleic acid may also result in a decrease in the affinity of the engineered CasX protein for DNA.

[0147] The specificity of CRISPR / Cas proteins and the reduction of potentially harmful off-target effects can be extremely important for achieving a tolerable therapeutic index for use in mammalian subjects. As used herein, "off-target effect" refers to an off-target effect of unintended cleavage and mutation at a non-targeted genomic site that exhibits a similar but not identical sequence compared to the target site. In some embodiments, the off-target effect presented by engineered CasX complexed with an ERS and a linked targeting sequence is less than about 5%, less than about 4%, less than 3%, less than about 2%, less than about 1%, less than about 0.5%, and less than 0.1% in cells. In some embodiments, the off-target effect is determined in silico. In some embodiments, the off-target effect is determined in an in vitro cell-free assay. In some embodiments, the off-target effect is determined in a cell-based assay. In some embodiments, an engineered CasX protein comprising a pair of mutations shown in Table 22 or a further modification thereof exhibits an increase in on-target editing activity, an increase in specificity (or a decrease in off-target activity), an increase in the specificity ratio, or a combination thereof, compared to SEQ ID NO: 228 (CasX variant 515).

[0148] Methods for testing the target specificity of CasX proteins (such as engineered CasX or reference CasX) can include guides and circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) or similar methods. Briefly, in the CIRCLE-seq technique, genomic DNA is sheared and circularized by ligation of a stem-loop adapter that nicks at the stem-loop region to expose a 4-nucleotide palindromic overhang. Subsequently, intramolecular ligation and degradation of the remaining linear DNA are performed. Thereafter, the circular DNA molecules containing the CasX cleavage sites are linearized with CasX, and adapters are ligated to the exposed ends. Subsequently, high-throughput sequencing is performed to generate paired-end reads containing information about off-target sites. Additional assays that can be used to detect off-target events and thus the specificity of the CasX protein include assays used to detect and quantify indels (insertions and deletions) formed at selected off-target sites such as mismatch detection nuclease assays and next-generation sequencing (NGS). Exemplary mismatch detection assays include nuclease assays in which genomic DNA from cells treated with CasX and ERS is PCR amplified, denatured, and re-hybridized to form heteroduplex DNA containing one wild-type strand and one strand with an indel. The mismatch is recognized and cleaved by a mismatch detection nuclease such as Surveyor nuclease or T7 endonuclease I. Methods for evaluating the specificity of engineered CasX are described in the Examples, along with supporting data showing improved specificity of embodiments of engineered CasX.

[0149] g. Protospacer and PAM sequence In this specification, a protospacer is defined as a DNA sequence complementary to the targeting sequence of a guide RNA, called a target strand and a non-target strand, respectively, and DNA complementary to that sequence. As used herein, a PAM is a proximal nucleotide sequence of a protospacer that, together with the targeting sequence of a guide RNA, helps the orientation and placement of CasX for potential cleavage of the protospacer strand.

[0150] The PAM sequences can be degenerate, and certain RNP constructs can have different preferred permissible PAM sequences that support different cleavage efficiencies. Unless otherwise indicated, in accordance with convention, the present disclosure refers to both the PAM sequence and the protospacer sequence, and their orientations according to the orientation of the non-target strand. This is not meant to imply that the PAM sequence of the non-target strand, rather than the target strand, determines cleavage or is mechanically involved in target recognition. For example, when referring to a TTC PAM, it could actually be the complementary GAA sequence required for target binding, or some combination of nucleotides from both strands. In the case of the CasX proteins disclosed herein, the PAM is located 5' of the protospacer where a single nucleotide separates the PAM from the first nucleotide of the protospacer. Thus, for reference CasX, a TTC PAM should be understood to mean a sequence according to the formula 5'-…NNTTCN(protospacer)NNNNNN…3'(SEQ ID NO: 304), where "N" is any DNA nucleotide and "(protospacer)" is a DNA sequence having identity to the targeting sequence of the guide RNA. For engineered CasX with extended PAM recognition, a TTC, CTC, GTC, or ATC PAM should be understood to mean a sequence according to the formula: 5'-…NNTTCN(protospacer)NNNNNN…3'(SEQ ID NO: 304), 5'-…NNCTCN(protospacer)NNNNNN…3'(SEQ ID NO: 305), 5'-…NNGTCN(protospacer)NNNNNN…3'(SEQ ID NO: 306), or 5'-…NNATCN(protospacer)NNNNNN…3'(SEQ ID NO: 307). Alternatively, a TC PAM should be understood to mean a sequence according to the formula 5'-…NNNTCN(protospacer)NNNNNN…3'(SEQ ID NO: 308).

[0151] In some embodiments, the engineered CasX protein of the present disclosure, compared to the RNP of the CasX protein from which it is derived, such as CasX 515 complexed with gRNA 174, utilizes a PAM TC motif that includes a PAM sequence selected from TTC, ATC, GTC, or CTC (in the 5’ to 3’ orientation) to have an improved ability to efficiently edit and / or bind a target nucleic acid when complexed with ERS as an RNP. As described above, the PAM sequence is located at at least 1 nucleotide on the 5’ side of the non-target strand of the protospacer that has identity with the target sequence of ERS in the assay system. In one embodiment, the engineered CasX and ERS RNP exhibits higher editing and / or binding of the target sequence within the target nucleic acid compared to the RNP of the CasX protein from which it is derived, such as CasX 515 and gRNA 174, in an equivalent assay system, where the PAM sequence of the target DNA is TTC. In another embodiment, the engineered CasX and ERS RNP exhibits higher editing and / or binding of the target sequence within the target nucleic acid compared to the RNP that includes the RNP of the CasX protein from which it is derived, such as CasX 515 and gRNA 174, in an equivalent assay system, where the PAM sequence of the target DNA is ATC. In another embodiment, the engineered CasX and ERS RNP exhibits higher editing and / or binding of the target sequence within the target nucleic acid compared to the RNP that includes the RNP of the CasX protein from which it is derived, such as CasX 515 and gRNA 174, in an equivalent assay system, where the PAM sequence of the target DNA is CTC. In another embodiment, the engineered CasX and ERS RNP exhibits higher editing and / or binding of the target sequence within the target nucleic acid compared to the RNP that includes the RNP of the CasX protein from which it is derived and gRNA 174 in an equivalent assay system, where the PAM sequence of the target DNA is GTC.In the foregoing embodiments, the increase in editing and / or binding affinity for one or more PAM sequences is at least about 1.5-fold, at least about 2-fold, at least about 4-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, or at least about 40-fold or more compared to the editing and / or binding affinity of gRNA174 for the RNP and PAM sequences of the CasX protein from which it is derived.

[0152] h. Catalytic activity The ribonucleoprotein complex of the eCasX:ERS system disclosed herein comprises engineered CasX complexed with ERS that binds to and cleaves a target nucleic acid. In some embodiments, the engineered CasX protein has improved catalytic activity compared to the CasX protein from which it is derived. Without wishing to be bound by theory, in some cases, cleavage of the target strand may be a limiting factor for Cas12-like molecules in the generation of dsDNA cleavage. In some embodiments, the engineered CasX protein improves the bending of the target strand of DNA and the cleavage of this strand, thereby resulting in an improvement in the overall efficiency of dsDNA cleavage by the CasX ribonucleoprotein complex.

[0153] Engineered CasX with increased double-stranded nuclease activity can be generated, for example, by amino acid changes within the RuvC nuclease domain. As described above, the engineered CasX generates double-stranded cleavage within 18 - 26 nucleotides on the 5' side of the PAM site on the target strand and within 10 - 18 nucleotides on the 3' side of the non-target strand. Nuclease activity can be assayed by various methods including the methods of the examples. In some embodiments, the engineered CasX has at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% or more improved k 切断 constant compared to the CasX protein from which it is derived.

[0154] In some embodiments, the engineered CasX protein has improved characteristics of forming an ERS and an RNP, thereby resulting in an RNP having a higher cleavage ability compared to the RNP of the CasX protein and gRNA variant from which it is derived. Having cleavage ability means that the formed RNP has the ability to cleave a target nucleic acid. In some embodiments, the RNP of the engineered CasX and ERS exhibits a cleavage rate that is at least 2-fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 10-fold that of the RNP of the CasX protein from which it is derived. In the foregoing embodiments, the improvement in the ability rate can be demonstrated by in vitro assays such as the assays described in the examples.

[0155] In some embodiments, the present disclosure provides an engineered CasX protein that is catalytically dead but retains the ability to bind to a target nucleic acid. Exemplary catalytically dead engineered CasX proteins include one or more mutations at the active site of the RuvC domain of the CasX protein. In some embodiments, the catalytically dead engineered CasX protein includes substitutions at residues 672, 769, and / or 935 compared to the sequence of SEQ ID NO: 1. In one embodiment, the catalytically dead engineered CasX protein includes substitutions of D672A, E769A, and / or D935A compared to the reference CasX protein of SEQ ID NO: 1. In other embodiments, the catalytically dead engineered CasX protein includes substitutions at amino acids 659, 756, and / or 922 compared to the reference CasX protein of SEQ ID NO: 2. In some embodiments, the catalytically dead engineered CasX protein includes substitutions of D659A, E756A, and / or D922A compared to the reference CasX protein of SEQ ID NO: 2. In some embodiments, the present disclosure provides any one of catalytically dead engineered CasXs of SEQ ID NOs: 156, 739-907, 739-907, 11568-22227, 23572-24915, and 49719-49735, which include the aforementioned mutations that render CasX catalytically dead.

[0156] i. Engineered CasX fusion protein In some embodiments, the disclosure provides an engineered CasX protein comprising a heterologous protein fused to CasX, the engineered CasX comprising engineered CasX fused to any of the embodiments described herein. This includes engineered CasX that includes an N-terminal, C-terminal, or internal fusion of CasX to a heterologous protein or a domain thereof.

[0157] In some embodiments, the engineered CasX fusion protein comprises any one of the sequences of SEQ ID NOs: 247-294, 24916-49628, 49746-49747, or 49871-49873 fused to one or more proteins or domains thereof that have different activities or confer different functional properties to result in a fusion protein.

[0158] A variety of heterologous polypeptides are suitable for inclusion in the engineered CasX fusion proteins of the disclosure. In some cases, the fusion partner can regulate the transcription of a target nucleic acid (e.g., inhibit transcription, increase transcription). For example, in some cases, the fusion partner is a protein (or a domain derived from a protein) that inhibits transcription (e.g., a transcriptional repressor that functions by recruitment of a transcriptional inhibitor, modification of the target nucleic acid such as methylation, recruitment of a DNA modification factor, regulation of histones associated with the target nucleic acid, recruitment of histone modification factors such as histone modification factors that modify histone acetylation and / or methylation). In some cases, the fusion partner is a protein (or a domain derived from a protein) that increases transcription (e.g., a transcriptional activator that acts by recruitment of a transcriptional activator, modification of the target nucleic acid such as demethylation, recruitment of a DNA modification factor, regulation of histones associated with the target nucleic acid, recruitment of histone modification factors such as histone modification factors that modify histone acetylation and / or methylation).

[0159] In some cases, the fusion partner has enzymatic activity that modifies a target nucleic acid sequence, such as nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity.

[0160] Examples of proteins (or fragments thereof) that can be used as fusion partners to reduce transcription include transcriptional repressors such as Kruppel-associated box (KRAB or SKD), KOX1 repression domain, Mad mSIN3 interaction domain (SID), ERF repressor domain (ERD), SRDX repression domain (e.g., for repression in plants), etc., histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1, etc., histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID 1C / SMCX, JARID1D / SMCY, etc., histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc., DNA methyltransferases such as HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A) and subdomains such as the DNMT3A catalytic domain and ATRX-DNMT3-DNMT3L domain (ADD), DNMT3L interaction domain (DNMT3L), DNA methyltransferase 3 beta (DNMT3B), Friend of GATA-1 (FOG), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc., and peripheral mobilizing elements such as lamin A, lamin B, etc., but are not limited thereto.

[0161] In some cases, the fusion partner with engineered CasX has enzymatic activity to modify target nucleic acids (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities that can be provided by the fusion partner include nuclease activity, e.g., nuclease activity provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity, e.g., methyltransferase activity provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A) and subdomains, e.g., DNMT3A catalytic domain and ATRX-DNMT3-DNMT3L domain (ADD), DNMT3L interaction domain (DNMT3L), DNA methyltransferase 3 beta (DNMT3B), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), etc.), demethylase activity, e.g., demethylase activity provided by a demethylase (e.g., ten-eleven translocation (TET) dioxygenase 1 (TET 1 CD), TET1, DME, DML1, DML2, ROS1, etc.), DNA repair activity, DNA damage activity, deamination activity, e.g., deamination activity provided by a deaminase (e.g., cytosine deaminase enzyme, e.g., APOBEC protein, e.g., rat apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 {APOBEC1}), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, e.g., integrase activity provided by an integrase and / or resolvase (e.g., Gin invertase, e.g., hyperactive mutant of Gin invertase, GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase, etc.), transposase activity, recombinase activity, e.g., recombinase activity provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity), but are not limited thereto.

[0162] In some cases, the engineered CasX protein of the present disclosure is fused to a polypeptide selected from a domain for increasing transcription (e.g., VP16 domain, VP64 domain), a domain for decreasing transcription (e.g., KRAB domain, e.g., from the Kox1 protein), the core catalytic domain of a histone acetyltransferase (e.g., histone acetyltransferase p300), a protein / domain that provides a detectable signal (e.g., a fluorescent protein such as GFP), a nuclease domain (e.g., Fokl nuclease), a base editing factor (e.g., a cytidine deaminase such as APOBEC1).

[0163] In some cases, the engineered CasX protein of the present disclosure can include an endosomal escape peptide. In some cases, the endosomal escape polypeptide includes the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 309), where each X is independently selected from lysine, histidine, and arginine. In some cases, the endosomal escape polypeptide includes the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 310) or HHHHHHHHH (SEQ ID NO: 311). In some embodiments, the engineered CasX includes any one of the sequences of SEQ ID NOs: 247-294, 24916-49628, 49746-49747, or 49871-49873, and an endosomal escape polypeptide.

[0164] In addition, or alternatively, the engineered CasX protein of the present disclosure can be fused to a polypeptide permeability domain to facilitate cellular uptake. Many permeability domains are known in the art and can be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers. For example, WO2017 / 106569 and US2018 / 0363009(A1), which are hereby incorporated by reference in their entirety, describe the fusion of Cas proteins with one or more nuclear localization sequences (NLSs) to facilitate cellular uptake. In other embodiments, the permeable peptide can be derived from the third alpha helix of Antennapaedia, a Drosophila melanogaster transcription factor called penetratin, which includes the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 312). As another example, the permeable peptide can include the HIV-1 tat basic region amino acid sequence, for example, including amino acids 49-57 of the naturally occurring tat protein. Other permeability domains include polyarginine motifs, such as the region of amino acids 34-56 of the HIV-1 Rev protein, nonaarginine, octaarginine, and the like. The site at which the fusion is made can be selected to optimize the biological activity, secretion, or binding properties of the polypeptide. The optimal site is determined by routine experimentation.

[0165] In some embodiments, the heterologous polypeptide (fusion partner) for use with engineered CasX provides intracellular localization, i.e., the heterologous polypeptide includes an intracellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence for retaining the fusion protein outside the nucleus, e.g., a nuclear export sequence (NES), a sequence for retaining the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to the chloroplast, an ER retention signal, etc.). In some embodiments, the subject RNA-guided polypeptide or conditional active RNA-guided polypeptide and / or the subject CasX fusion protein do not include an NLS, thereby preventing the protein from being targeted to the nucleus, which can be advantageous, for example, when the target nucleic acid sequence is an RNA present in the cytosol. In some embodiments, the fusion partner can provide a tag (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, etc., a histidine tag, e.g., a 6XHis tag, a hemagglutinin (HA) tag, a FLAG tag, a Myc tag, etc.) to facilitate tracking and / or purification (i.e., the heterologous polypeptide is a detectable label).

[0166]

[0167] The present disclosure contemplates a collection of multiple NLSs in various configurations for linking to the engineered CasX protein of the present embodiment. In some embodiments, one or more NLSs are linked to or near the N-terminus of the engineered CasX protein. In other embodiments, one or more NLSs are linked to or near the C-terminus of the engineered CasX protein. In other embodiments, one or more NLSs are linked to or near both the N-terminus and the C-terminus of the engineered CasX protein. In some embodiments, the NLS linked to the N-terminus of the engineered CasX protein is the same as the NLS linked to the C-terminus. In some embodiments, the NLS linked to the N-terminus of the engineered CasX protein is different from the NLS linked to the C-terminus. In some embodiments, the NLS can be linked within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the N-terminus or C-terminus of the engineered CasX protein. In some embodiments, the NLS can be linked to the N-terminus or C-terminus of the engineered CasX protein by a linker peptide, and these embodiments are described herein. In some embodiments, the NLS is linked to another NLS by a linker. In other embodiments, the NLS linked to the N-terminus of the engineered CasX protein is different from the NLS linked to the C-terminus. 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 (SEQ ID NOs: 364-410) described in Table 8. In some embodiments, the NLS linked to the C-terminus of the engineered CasX protein is selected from the group consisting of the C-terminal sequences (SEQ ID NOs: 411-457) described in Table 8.

[0168] Detection of the accumulation of the engineered CasX fusion protein in the nucleus can be performed by any suitable technique. For example, a detectable marker can be fused to the engineered CasX fusion protein such that the intracellular location can be visualized. The cell nucleus may be isolated from the cell and then its contents can be analyzed by any suitable process for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assay. The accumulation in the nucleus may also be determined indirectly.

Table 8-1

Table 8-2

Table 8-3

Table 8-4

[0169] In some cases, the engineered CasX protein comprises a "protein transduction domain" or PTD (also known as CPP, cell - penetrating peptide), which refers to a protein, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates the crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. PTDs conjugated to other molecules, ranging from small polar molecules to large macromolecules and / or nanoparticles, facilitate the movement of the molecule across a membrane, for example, from the extracellular space into the intracellular space or from the cytosol into an organelle. In some embodiments, the PTD is covalently attached to the amino - terminus of the engineered CasX fusion protein. In some embodiments, the PTD is covalently attached to the carboxyl - terminus of the engineered CasX fusion protein. In some cases, the PTD is internally inserted into the sequence of the engineered CasX fusion protein at a suitable insertion site. In some cases, the engineered CasX fusion protein comprises (is conjugated to, is fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, the PTD comprises one or more nuclear localization signals (NLSs).Examples of PTDs include the peptide transduction domain of HIV TAT, including YGRKKRRQRRR (SEQ ID NO: 458), RKKRRQRR (SEQ ID NO: 459), YARAAARQARA (SEQ ID NO: 460), THRLPRRRRRR (SEQ ID NO: 461), and GGRRARRRRRR (SEQ ID NO: 462); polyarginine sequences containing a sufficient number of arginines for direct entry into cells (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10 - 50 arginines, SEQ ID NO: 463), the VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489 - 96), the Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732 - 1737), the truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248 - 1256), polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003 - 13008), RRQRRTSKLMKR (SEQ ID NO: 464), the transporteran GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 465), KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 466), and RQIKIWFQNRRMKWKK (SEQ ID NO: 467), but are not limited thereto. In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5 - 6):371 - 381). The ACPP comprises a polycationic CPP (e.g., Arg9 or "R9") connected via a cleavable linker to a matching polyanion (e.g., Glu9 or "E9") that reduces the net charge to near zero, thereby inhibiting adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally exposing the polyarginine and its inherent adhesiveness, as a result "activating" the ACPP to cross the membrane.

[0170] In some embodiments, the engineered CasX fusion protein can comprise a CasX protein linked to a heterologous polypeptide (heterologous amino acid sequence) via a linker polypeptide (e.g., one or more linker polypeptides). In some embodiments, the engineered CasX fusion protein can be linked to a heterologous polypeptide (fusion partner) at the C-terminus and / or N-terminus via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide can have any of a variety of amino acid sequences. The protein can be linked by a spacer peptide having generally mobile properties, although other chemical linkages are not excluded. Suitable linkers include polypeptides that are 4 to 40 amino acids in length or 4 to 25 amino acids in length. The linking peptide can have substantially any amino acid sequence, bearing in mind that the preferred linker results in a generally mobile peptide. The use of small amino acids such as glycine and alanine is useful in creating mobile peptides. The creation of such sequences is routine for those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. In some embodiments, one or more fusion proteins are linked to an engineered CasX protein or an adjacent fusion protein using a linker peptide, and the linker peptide is selected from the group consisting of RS, (G)n (SEQ ID NO: 468), (GS)n (SEQ ID NO: 469), (GSGGS)n (SEQ ID NO: 470), (GGSGGS)n (SEQ ID NO: 471), (GGGS)n (SEQ ID NO: 472), GGSG (SEQ ID NO: 473), GGSGG (SEQ ID NO: 474), GSGSG (SEQ ID NO: 475), GSGGG (SEQ ID NO: 476), GGGSG (SEQ ID NO: 477), GSSSG (SEQ ID NO: 478), GPGP (SEQ ID NO: 479), GGP, PPP, PPAPPA (SEQ ID NO: 480), PPPG (SEQ ID NO: 481), PPPGPPP (SEQ ID NO: 482), PPP(GGGS)n (SEQ ID NO: 483), (GGGS)nPPP (SEQ ID NO: 484), AEAAAKEAAAKEAAAKA (SEQ ID NO: 485), and TPPKTKRKVEFE (SEQ ID NO: 486), where n is from 1 to 5.One of ordinary skill in the art will recognize that the design of peptides conjugated to any of the above elements can include a linker that is not only a flexible linker but can also include one or more moieties that impart a less flexible structure, such that the linker can include a flexible linker, either in whole or in part.

[0171] V. Methods of Making Engineered CasX Proteins and ERSs The engineered CasX proteins and ERSs of the present disclosure can be designed and constructed by various methods, as described herein. In some embodiments, the method includes designing, constructing, and testing a comprehensive set of mutations to a starting biomolecule to generate a library of biomolecule variants, such as a library of engineered CasX proteins or engineered ERS scaffolds. The methods of the present disclosure can include generating all possible substitutions, as well as all possible small insertions and all possible deletions of amino acids (in the case of proteins) or nucleotides (in the case of RNA or DNA), or swapping domains or subdomains into the starting biomolecule to create a library, after which these libraries are evaluated for functional changes and this information is used to construct one or more additional libraries. Such iterative construction and evaluation of variants can lead to the identification of mutational themes that result in a particular functional outcome, such as a region of a protein or gRNA that confers one or more improved functions when mutated in a particular way. Subsequent layering of such identified mutations can further improve function, for example, through additive or synergistic interactions. The methods of the present disclosure include library design, library construction, and library screening. In some embodiments, multiple rounds of design, construction, and screening are performed.

[0172] a. Library Design In some embodiments, the method of generating a library of mutagenized CasX and ERS is the method of Examples 1-7 and 11. In some embodiments, the biomolecules of the library include protein or ribonucleic acid (RNA) molecules, and the mutagenized monomer units are amino acids or ribonucleotides, respectively. The basic unit of biomolecule mutation includes any one of (1) exchanging a monomer with another monomer having a different identity (substitution), (2) inserting one or more additional monomers into the biomolecule (insertion), or (3) removing one or more monomers from the biomolecule (deletion). A library that includes substitutions, insertions, and deletions to any one or more monomers in any biomolecule described herein, alone or in combination, is considered to be within the scope of the present invention.

[0173] In an exemplary embodiment, as described in Example 1, the present disclosure provides a CasX protein derived from CasX 515 in which the engineered CasX was designed using a Markov Chain Monte Carlo (MCMC) directed evolution method simulation (Biswas S et al. Low-N protein engineering with data-efficient deep learning. Nature Methods. 18(4):389-396(2021)). In this method, codons within CasX 515 were selected and randomly replaced with codons encoding different amino acids such that the probability that the selected amino acid is replaced with any of 19 alternative amino acids is equal. This process was then repeated up to 16 times to obtain simulated mutagenized protein sequences. A machine learning model was then used to determine the predicted fitness of the mutagenized protein sequences to virtually screen the simulated proteins and either discard the simulated proteins or experimentally construct and validate the simulated proteins. In this method, the process of mutagenesis and simulated screening was repeated until the desired number of sequences each containing the desired number of single mutations was obtained, and then this was assayed to identify engineered CasX with improved characteristics.

[0174] In some embodiments, library design includes enumerating all possible mutations of each of one or more target monomers in a biomolecule. As used herein, "target monomer" refers to a monomer in a biomolecule polymer that is the target of mutagenesis by the substitutions, insertions, and deletions described herein. For example, a target monomer can be an amino acid at a specific position in a protein, or a nucleotide at a specific position in an RNA. In some embodiments, a library of variant sequences is created by mutations at each consecutive position of a protein or RNA. In other embodiments, the biomolecule can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more target monomers that are systematically mutated to generate a library of biomolecule variants. In some embodiments, each monomer in the biomolecule is a target monomer. For example, in a parental CasX protein with two target amino acids, library design includes enumerating 40 possible mutations at each of the two target amino acids. In a further example, in a library of RNA with four target nucleotides, library design includes enumerating eight possible mutations at each of the four target nucleotides. In some embodiments, each target monomer of the biomolecule is independently randomly selected or selected thereby by intentional design. Thus, in some embodiments, the library includes random variants, or designed variants, or variants that include random mutations and designed mutations within a single biomolecule, or any combination thereof.

[0175] In some embodiments, the assembled library is then assayed to evaluate a comprehensive set of mutations to the biomolecule that include substitutions, as well as insertions and deletions, of amino acids (in the case of proteins) or nucleotides (in the case of RNA). Construction and functional readout of these mutations can be achieved using a variety of established molecular biology methods. In some embodiments, the library comprises a subset of all possible modifications to the monomer. For example, in some embodiments, the library collectively represents a single modification of one monomer for at least some percentage of the total monomer positions in the biomolecule, and each single modification is selected from the group consisting of a substitution, a single insertion, and a single deletion. In some embodiments, the library collectively represents a single modification of one monomer for at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or up to 100% of the total monomer positions in the starting biomolecule. In certain embodiments, for a particular percentage of the total monomer positions in the starting biomolecule, the library collectively represents each possible single modification of one monomer, e.g., all possible substitutions with 19 other naturally occurring amino acids (in the case of proteins) or 3 other naturally occurring ribonucleotides (in the case of RNA), each insertion of 20 naturally occurring amino acids (in the case of proteins) or 4 naturally occurring ribonucleotides (in the case of RNA), or deletion of the monomer. In still further embodiments, the insertion at each position is independently an insertion of more than one monomer, e.g., two or more, three or more, or four or more monomers, or an insertion of 1-4, 2-4, or 1-3 monomers. In some embodiments, the deletion at a position is independently a deletion of more than one monomer, e.g., two or more, three or more, or four or more monomers, or a deletion of 1-4, 2-4, or 1-3 monomers. Examples of such libraries of engineered CasX and ERS are described in Examples 1-7 and 11.

[0176] In some embodiments, the biomolecule is a protein and the individual monomers are amino acids. In embodiments where the biomolecule is a protein, the number of possible mutations at each monomer (amino acid) position in the protein includes 19 amino acid substitutions, 20 amino acid insertions, and 1 amino acid deletion, resulting in a total of 40 possible mutations per amino acid in the protein.

[0177] In some embodiments, the library of engineered CasX proteins that includes insertions is a 1 - amino acid insertion library, a 2 - amino acid insertion library, a 3 - amino acid insertion library, a 4 - amino acid insertion library, a 5 - amino acid insertion library, a 6 - amino acid insertion library, a 7 - amino acid insertion library, an 8 - amino acid insertion library, a 9 - amino acid insertion library, or a 10 - amino acid insertion library. In some embodiments, the library of engineered CasX proteins that includes insertions includes from 1 to 10 amino acid insertions. In some embodiments, the library of engineered CasX proteins that includes deletions is a 1 - amino acid deletion library, a 2 - amino acid deletion library, a 3 - amino acid deletion library, a 4 - amino acid deletion library, a 5 - amino acid deletion library, a 6 - amino acid deletion library, a 7 - amino acid deletion library, an 8 - amino acid deletion library, a 9 - amino acid deletion library, or a 10 - amino acid deletion library. In some embodiments, the library of engineered CasX proteins that includes deletions includes from 1 to 10 amino acid deletions. In some embodiments, the library of engineered CasX proteins that includes substitutions is a 1 - amino acid substitution library, a 2 - amino acid substitution library, a 3 - amino acid substitution library, a 4 - amino acid substitution library, a 5 - amino acid substitution library, a 6 - amino acid substitution library, a 7 - amino acid substitution library, an 8 - amino acid substitution library, a 9 - amino acid substitution library, or a 10 - amino acid insertion library. In some embodiments, the library of engineered CasX proteins that includes substitutions includes from 1 to 10 amino acid substitutions.

[0178] In some embodiments, the biomolecule is RNA. In embodiments where the biomolecule is RNA, the number of possible DME mutations at each monomer (ribonucleotide) position in the RNA includes three nucleotide substitutions, four nucleotide insertions, and one nucleotide deletion, resulting in a total of eight possible mutations per nucleotide.

[0179] In some embodiments of the method, the mutation is incorporated into the double-stranded DNA encoding the biomolecule. This DNA can be maintained and replicated in a standard cloning vector, such as a bacterial plasmid referred to herein as the target plasmid. Exemplary target plasmids include a DNA sequence encoding the starting biomolecule to be mutagenized, a bacterial origin of replication, and a suitable antibiotic resistance expression cassette. In some embodiments, the antibiotic resistance cassette confers resistance to kanamycin, ampicillin, spectinomycin, bleomycin, streptomycin, erythromycin, tetracycline, or chloramphenicol. In some embodiments, the antibiotic resistance cassette confers resistance to kanamycin.

[0180] Libraries containing the aforementioned variants can be constructed in various ways. In certain embodiments, plasmid recombineering is used to construct the library. Such methods can use DNA oligonucleotides encoding one or more mutations to incorporate those mutations into a plasmid encoding a reference biomolecule. In the case of biomolecule variants having multiple mutations, in some embodiments, two or more oligonucleotides are used. In some embodiments, the DNA oligonucleotide encodes one or more mutations, and the mutation region is flanked by 10 - 100 nucleotides having homology to the target plasmid on both the 5' and 3' sides of the mutation. Such oligonucleotides can, in some embodiments, be commercially synthesized and used for PCR amplification. Exemplary templates for oligonucleotides encoding mutations are provided below. 5’-(N) 10-100 -mutation-(N’)10-100 -3'

[0181] In this exemplary oligonucleotide design, N represents a sequence identical to the target plasmid, herein referred to as the homology arm. When a particular monomer in a biomolecule is targeted for mutation, these homology arms are directly adjacent to the DNA encoding the monomer within the target plasmid. In some exemplary embodiments where the biomolecule undergoing mutagenesis is a protein, the same set of homology arms is used such that 40 different oligonucleotides are used to encode 40 different amino acid mutations listed for each amino acid residue in the protein targeted for mutagenesis. If the mutation is a single amino acid, the region encoding the desired mutation(s) contains three nucleotides encoding the amino acid (in the case of a substitution or single insertion) or zero nucleotides (in the case of a deletion). In some embodiments, the oligonucleotide encodes an insertion of two or more amino acids. For example, if the oligonucleotide encodes an insertion of X amino acids, the region encoding the desired mutation contains 3×X nucleotides encoding the X amino acids. In some embodiments, the mutation region encodes mutations to two or more monomers of a biomolecule, such as two or more monomers that are proximal (e.g., adjacent to each other, or present within one, two, three, four, five, six, seven, eight, nine, or ten or more monomers of each other).

[0182] In some exemplary embodiments where the biomolecule being mutagenized is RNA, eight different oligonucleotides use the same set of homology arms to encode eight different single nucleotide mutations for each nucleotide in the RNA targeted for mutagenesis. When the mutation is of a single ribonucleotide, the region of the oligo encoding the mutation can consist of the following nucleotide sequence: one nucleotide that specifies the nucleotide (in the case of a substitution or insertion) or zero nucleotides (in the case of a deletion). In some embodiments, the oligonucleotide is synthesized as a single-stranded DNA oligonucleotide. In some embodiments, all oligonucleotides targeting a particular amino acid or nucleotide of the biomolecule being mutagenized are pooled.

[0183] b. Library Screening Any suitable method for screening or selecting a library is envisioned within the scope of the present invention as follows. High-throughput methods can be used to evaluate large libraries with thousands of individual mutations. In some embodiments, the throughput of the library screening or selection assay has throughput in millions of individual cells. In some embodiments, assays utilizing live cells are preferred because the phenotype and genotype are physically linked within the same lipid bilayer in live cells. Live cells can also be used to directly amplify subpopulations of the entire library. Exemplary methods for screening a library are described in Examples 1-7 and 11.

[0184] In some embodiments, a library that has been screened or selected for high-function variants is further characterized. In some embodiments, further characterizing the library includes individually analyzing the variants by sequencing, such as Sanger sequencing, to identify the specific mutation(s) that give rise to the high-function variants. Individual mutant variants of the biomolecule can be isolated by standard molecular biology techniques for later functional analysis. In some embodiments, further characterizing the library includes high-throughput sequencing of both the library of high-function variants and one or more of the libraries. This approach can, in some embodiments, enable the rapid identification of mutations that are overrepresented in one or more libraries of high-function variants compared to the naive library. Without wishing to be bound by any theory, mutations that are overrepresented in one or more libraries of high-function variants are likely to be involved in the activity of the high-function variants. In some embodiments, further characterizing the library includes not only sequencing the individual variants, but also high-throughput sequencing of both the naive library and one or more libraries of highly mutagenized variants.

[0185] High-throughput sequencing can generate high-throughput data indicative of the functional effects of library members. In embodiments where one or more libraries represent all possible mutations at all monomer positions, such high-throughput sequencing can evaluate the functional effects of all possible mutations. Such sequencing can be used to evaluate one or more high-function subpopulations of a given library, which can, in some embodiments, lead to the identification of mutations that result in improved function.

[0186] c. Generation of Engineered CasX and ERS The engineered CasX proteins of the present disclosure can be generated in vitro using standard cloning and molecular biology techniques, or as described in the examples, by eukaryotic cells or by prokaryotic cells transformed with a coding vector (described below). The specific sequences and preparation methods are determined by convenience, economics, required purity, etc. In some embodiments, first, a construct containing a DNA sequence encoding the engineered CasX is prepared. Exemplary methods for preparing such constructs are described in the examples. Thereafter, the construct is used to create an expression vector suitable for transforming a host cell, such as a prokaryotic host cell or a eukaryotic host cell, for protein expression and recovery. If desired, the host cell is E. coli. In other embodiments, the host cell is a eukaryotic cell. Eukaryotic host cells can be selected from baby hamster kidney fibroblast (BHK) cells, human embryonic kidney 293 (HEK293), human embryonic kidney 293T (HEK293T), NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, CV-1 (monkey)-derived (COS) cells having SV40 genetic material, HeLa cells, Chinese hamster ovary (CHO) cells, or yeast cells, or other eukaryotic cells known in the art suitable for the production of recombinant products.

[0187] The engineered CasX proteins of the present disclosure may also be isolated and purified according to conventional recombinant synthesis methods. The lysate can be prepared from the expression host, and the lysate can be purified using high performance liquid chromatography (HPLC), size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used contains at least 80% by weight, more usually at least 90% by weight, preferably at least 95% by weight, and usually at least 99.5% by weight of the desired product, in relation to the preparation method of the product and contaminants associated with its purification.

[0188] In the case of generating the ERS (and the linked targeting sequence) of the present disclosure, the recombinant expression vector encoding the ERS can be transcribed in vitro using, for example, the T7 promoter regulatory sequence and T7 polymerase to generate the ERS, which can then be recovered by conventional methods, such as purification by gel electrophoresis described in the examples. Alternatively, the ERS can be prepared synthetically. Once synthesized, the ERS can be utilized in a gene editing system to directly contact and modify the target nucleic acid, or can be introduced into cells by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

[0189] VI. Polynucleotides and Vectors In another aspect, the present disclosure relates to polynucleotides encoding engineered CasX and ERS that are useful for editing target nucleic acids in cells. In some embodiments, the present disclosure provides polynucleotides encoding an engineered CasX protein of any of the system embodiments described herein and a polynucleotide of the ERS. In some embodiments, the present disclosure provides a polynucleotide sequence encoding an engineered CasX of any of the embodiments described herein, comprising an engineered CasX having a sequence identity of at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% with SEQ ID NOs: 247-294, 24916-49628, 49746-49747, or 49871-49873. In some embodiments, the present disclosure provides an isolated polynucleotide sequence encoding an ERS sequence of any of the embodiments described herein, comprising the sequences of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, and 49719-49735, together with a targeting sequence capable of hybridizing with the target nucleic acid to be modified.

[0190] In other aspects, the disclosure relates to methods of generating polynucleotide sequences (including homologous variants thereof) encoding engineered CasX or ERS of any of the embodiments described herein, and methods of expressing a protein or transcribed ERS expressed by the polynucleotide sequences. Generally, the methods include generating a polynucleotide sequence encoding engineered CasX or ERS of any of the embodiments described herein, and incorporating the coding gene into an appropriate expression vector for a host cell. Standard recombinant techniques in molecular biology can be used to generate the polynucleotides and expression vectors of the disclosure. For the generation of any of the encoded reference CasX, engineered CasX, or ERS of the embodiments described herein, the methods include transforming an appropriate host cell with an expression vector containing the coding polynucleotide, and culturing the host cell under conditions that cause or permit any of the resulting reference CasX, engineered CasX, or ERS of the embodiments described herein to be expressed or transcribed in the transformed host cell, whereby an engineered CasX or ERS is produced, and these are recovered by the methods described herein, or by standard purification methods known in the art, or as described in the examples.

[0191] According to the methods of the disclosure, nucleic acid sequences (or their complements) encoding engineered CasX or ERS of any of the embodiments described herein are used to generate recombinant DNA molecules that direct expression in an appropriate host cell. Several cloning strategies are suitable for practicing the disclosure, and many of them are used to generate constructs that contain a gene encoding the composition of the disclosure or its complement. In some embodiments, the cloning strategy is used to generate a gene encoding a construct that contains nucleotides encoding engineered CasX or ERS used to transform a host cell for expression of the composition.

[0192] In some approaches, first, a construct is prepared that contains a DNA sequence encoding the engineered CasX or ERS. Exemplary methods for preparing such constructs are described in the Examples. Thereafter, the construct is used to generate an expression vector suitable for transforming a host cell, such as a prokaryotic or eukaryotic host cell, for protein expression and recovery in the case of the engineered CasX or ERS. If desired, the host cell is E. coli. In other embodiments, the host cell is a eukaryotic cell. The eukaryotic host cell can be selected from baby hamster kidney fibroblast (BHK) cells, human embryonic kidney 293 (HEK293), human embryonic kidney 293T (HEK293T), NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, CV-1 (monkey)-derived (COS) cells having SV40 genetic material, HeLa cells, Chinese hamster ovary (CHO) cells, or yeast cells, or other eukaryotic cells known in the art suitable for the production of recombinant products. Exemplary methods for generating the expression vector, transforming the host cell, and expressing and recovering the engineered CasX or ERS are described in the Examples.

[0193] The gene encoding the engineered CasX or ERS construct can be made in one or more steps either completely synthetically or by synthesis in combination with enzymatic processes such as restriction enzyme-mediated cloning, PCR, and overlap extension, including the methods fully described by the Examples. Using the methods disclosed herein, for example, the sequences of polynucleotides encoding various components of the desired sequence (e.g., the engineered CasX and ERS) genes can be ligated. Genes encoding polypeptide compositions are assembled from oligonucleotides using standard gene synthesis techniques.

[0194] In some embodiments, the nucleotide sequence encoding the engineered CasX protein is codon-optimized. This type of optimization can involve mutations in the coding nucleotide sequence to mimic the codon preference of the intended host organism or cell while encoding the same engineered CasX protein. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell of the engineered CasX protein is a human cell, a human codon-optimized coding nucleotide sequence can be used. As another non-limiting example, if the intended host cell is a mouse cell, a mouse codon-optimized coding nucleotide sequence can be generated. As another non-limiting example, if the intended host cell is a prokaryotic cell (e.g., E. coli), a prokaryotic codon-optimized coding nucleotide sequence can be generated. Gene design can be performed using algorithms that optimize codon usage and amino acid composition appropriate for the host cell utilized for the production of the engineered CasX. In one method of the present disclosure, as described above, a library of polynucleotides encoding the engineered CasX or ERS components is assembled after being prepared, and these variants are assayed to confirm that they retain their functional properties. The resulting gene is then used to transform host cells and produce and recover the engineered CasX or ERS components for evaluation of their properties, as described herein.

[0195] In some embodiments, the nucleotide sequence encoding the engineered CasX protein is depleted of or lacks CpG motifs. In some embodiments, the CpG content of the engineered CasX is less than about 10%, less than about 5%, or less than about 1% CpG. In some embodiments, the sequence encoding the engineered CasX protein depleted of or lacking CpG motifs comprises a sequence selected from the group consisting of SEQ ID NOs: 49850-49861.

[0196] In some embodiments, the nucleotide sequence encoding ERS is depleted of or lacks CpG motifs. In some embodiments, the CpG content of ERS is less than about 10%, less than about 5%, or less than about 1% CpG. In some embodiments, the nucleotide encoding ERS that is depleted of or lacks CpG motifs comprises a sequence selected from the group consisting of SEQ ID NOs: 535-556.

[0197] In some embodiments, the nucleotide sequence encoding ERS is operably linked to a control element, such as a transcriptional control element, for example, a promoter. In some embodiments, the nucleotide sequence encoding the engineered CasX protein is operably linked to a control element, such as a transcriptional control element, for example, a promoter. In some instances, the promoter is a constitutively active promoter. In some instances, the promoter is a regulatable promoter. In some instances, the promoter is an inducible promoter. In some instances, the promoter is a tissue-specific promoter. In some instances, the promoter is a cell-type specific promoter. In some instances, the transcriptional control element (e.g., a promoter) is functional in a targeted cell type or targeted cell population. For example, in some instances, the transcriptional control element can be functional in eukaryotic cells, such as neurons, spinal motor neurons, medium spiny neurons, cortical neurons, striatal neurons, oligodendrocytes, or glial cells.

[0198] Non-limiting examples of Pol II promoters include EF-1 alpha, EF-1 alpha core promoter, Jens Tornoe (JeT), promoters derived from cytomegalovirus (CMV), CMV immediate early (CMVIE), CMV enhancer, herpes simplex virus (HSV) thymidine kinase, early and late simian virus 40 (SV40), SV40 enhancer, long terminal repeat (LTR) derived from retrovirus, mouse metallothionein-I, adenovirus major late promoter (Ad MLP), CMV promoter full-length promoter, minimal CMV promoter, chicken beta-actin promoter (CBA), CBA hybrid (CBh), chicken beta-actin promoter with cytomegalovirus enhancer (CB7), chicken beta-actin promoter and rabbit beta-globin splice acceptor site fusion (CAG), Rous sarcoma virus (RSV) promoter, HIV-Ltr promoter, hPGK promoter, HSVTK promoter, 7SK promoter, mini-TK promoter, human synapsin I (SYN) promoter conferring neuron-specific expression, beta-actin promoter, supercore promoter 1 (SCP1), Mecp2 promoter for selective expression in neurons, minimal IL-2 promoter, Rous sarcoma virus enhancer / promoter (single), spleen focus-forming virus long terminal repeat (LTR) promoter, TBG promoter, promoter derived from the human thyroxine-binding globulin gene (liver-specific), PGK promoter, human ubiquitin C promoter (UBC), UCOE promoter (promoter of HNRPA2B1-CBX3), synthetic CAG promoter, histone H2 promoter, histone H3 promoter, U1a1 small nuclear RNA promoter (226 nt), U1a1 small nuclear RNA promoter (226 nt), U1b2 small nuclear RNA promoter (246 nt) 26, GUSB promoter, CBh promoter, rhodopsin (Rho) promoter, spleen focus-forming virus (SFFV) promoter susceptible to silencing, human H1 promoter (H1), POL1 promoter, TTR minimal enhancer / promoter, b-kinase promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, human eukaryotic initiation factor 4A (EIF4A1) promoter, ROSA26 promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, tRNA promoter, and the truncated versions and sequence variants of the foregoing are included, but not limited to these. In certain embodiments, the Pol II promoter is EF-1 alpha, and this promoter enhances transfection efficiency, transgene transcription or expression of the CRISPR nuclease, the percentage of expression-positive clones, and the copy number of the episomal vector under long-term culture.

[0199] Non-limiting examples of Pol III promoters operably linked to polynucleotides encoding the ERS of the present disclosure include U6, mini-U6, U6 truncated promoters, 7SK, and H1 variants, BiH1 (bidirectional H1 promoter), BiU6, Bi7SK, BiH1 (bidirectional U6, 7SK, and H1 promoters), gorilla U6, rhesus monkey U6, human 7SK, human H1 promoter, and truncated versions and sequence variants thereof, but are not limited thereto. In the foregoing embodiments, the Pol III promoter enhances the transcription of the ERS. In certain embodiments, the Pol III promoter is U6, and this promoter enhances the expression of the CRISPR ERS. In another specific embodiment, the promoter linked to the gene encoding the tropism factor is the CMV promoter. Experimental details and data for the use of such promoters are provided in the examples.

[0200] The recombinant expression vectors of the present disclosure can also include accessory elements that facilitate robust expression of the engineered CasX protein and ERS of the present disclosure. For example, the recombinant expression vector can include one or more of a polyadenylation signal (such as a poly(A) sequence, an intron sequence, or a post-transcriptional regulatory element such as the woodchuck hepatitis post-transcriptional regulatory element (WPTRE)). Exemplary poly(A) sequences include the hGH poly(A) signal (short), HSV TK poly(A) signal, synthetic polyadenylation signal, SV40 poly(A) signal, β-globin poly(A) signal, and the like. In some embodiments, the recombinant expression vector encoding the engineered CasX includes a poly(A) tail having 80 or more adenine nucleotides. Those skilled in the art will be able to select elements suitable for inclusion in the recombinant expression vectors described herein.

[0201] The selection of suitable vectors and promoters is well within the ordinary skill level in the art, for example, because it is related to the control of expression for modifying proteins involved in antigen processing, antigen presentation, antigen recognition, and / or antigen response, and / or its regulatory elements. Expression vectors may also include a ribosome binding site for translation initiation and a transcription terminator. Expression vectors may also include sequences suitable for amplifying expression. Expression vectors may also include a nucleotide sequence encoding a protein tag (e.g., 6xHis tag, hemagglutinin tag, FLAG tag, fluorescent protein, etc.) that can be fused to the engineered CasX protein, thus resulting in a chimeric CasX protein used for purification or detection.

[0202] In some embodiments, provided herein are one or more recombinant expression vectors comprising one or more of: (i) a nucleotide sequence encoding an ERS that hybridizes to a target sequence of a targeted genomic locus operably linked to a promoter operative in a target cell such as a eukaryotic cell (e.g., configured as a single guide or dual guide), and (ii) a nucleotide sequence encoding an engineered CasX protein operably linked to a promoter operative in a target cell such as a eukaryotic cell.

[0203] Polynucleotide sequences are inserted into vectors by a variety of procedures. Generally, DNA is inserted into appropriate restriction endonuclease sites using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art. Such techniques are well known in the art and are fully described in the scientific and patent literature. A variety of vectors are publicly available. The vector may be in the form of, for example, a plasmid, cosmid, viral particle, or phage that is conveniently used in recombinant DNA procedures, and the choice of vector often depends on the host cell into which it is introduced. Thus, the vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be a vector that, when introduced into a host cell, integrates into the host cell genome and is replicated along with the chromosome into which it is integrated. When introduced into a suitable host cell, the expression of proteins involved in antigen processing, antigen presentation, antigen recognition, and / or antigen response can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of engineered CasX can be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array-based technologies (see, e.g., U.S. Patents Nos. 5,405,783, 5,412,087, and 5,445,934) using a probe complementary to any region of the polynucleotide.

[0204] Polynucleotides and recombinant expression vectors can be delivered to target host cells by various methods. Such methods include viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, microinjection, liposome-mediated transfection, particle gun technology, nucleofection, direct addition by a cell-permeable engineered CasX protein that is fused to or mobilizes donor DNA, cell squeezing, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and using commercially available TransMessenger® reagent (Qiagen), Stemfect™ RNA transfection kit (Stemgent), and TransIT® mRNA transfection kit (Mirus Bio LLC), Lonza nucleofection, Maxagen electroporation, and the like, but are not limited thereto.

[0205] In some embodiments, the disclosure provides a vector comprising a polynucleotide encoding engineered CasX or ERS selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a virus-like particle (VLP), a herpes simplex virus (HSV) vector, a plasmid, a minicircle, a nanoplasmid, a DNA vector, an RNA vector, or a CasX delivery particle (XDP). In some embodiments, the disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding an engineered CasX protein and a nucleotide sequence encoding ERS. In other embodiments, the nucleotide sequence encoding the engineered CasX protein and the nucleotide sequence encoding ERS are provided on separate vectors.

[0206] In some embodiments, the recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. AAV is a small (20 nm) non-pathogenic virus useful for the treatment of human diseases in situations where viral vectors for delivery to cells, such as eukaryotic cells, are used either in vivo or ex vivo in the case of cells prepared for administration to a subject. A construct, for example, encoding any of the engineered CasX proteins and ERS embodiments described herein and optionally encoding a donor template, is generated and flanked by AAV inverted terminal repeat (ITR) sequences, thereby enabling packaging of the AAV vector into AAV viral particles.

[0207] The term "AAV" vector may refer to the naturally occurring wild-type virus itself or derivatives thereof. This term encompasses all subtypes, serotypes, and pseudotypes, as well as both naturally occurring and recombinant forms, unless otherwise required. As used herein, the term "serotype" refers to an AAV that is identified and distinguished from other AAVs based on the reactivity of its capsid protein with a defined antiserum. For example, many known serotypes of primate AAVs exist. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAV9.45, AAV9.61, AAV44.9, AAV-Rh74 (AAV derived from rhesus monkeys), and AAVRh10, as well as modified capsids of these serotypes. For example, serotype AAV-2 is used to refer to an AAV that includes the capsid protein encoded by the cap gene of AAV-2 and a genome that includes 5' and 3' ITR sequences from the same AAV-2 serotype. A pseudotype AAV refers to an AAV that includes a capsid protein from one serotype and a viral genome that includes the 5'-3' ITR of a second serotype. Pseudotype rAAV is expected to have gene properties that are consistent with the cell surface binding properties of the capsid serotype and the ITR serotype. Pseudotype recombinant AAV (rAAV) is generated using standard techniques described in the art. As used herein, for example, rAAV1 may be used to refer to an AAV that has both a capsid protein and 5'-3' ITRs from the same serotype, or an AAV that has a capsid protein from serotype 1 and 5'-3' ITRs from a different AAV serotype, such as AAV serotype 2. For each example illustrated herein, the description of vector design and generation describes the serotype of the capsid and 5'-3' ITR sequences.

[0208] "AAV virus" or "AAV virus particle" refers to a virus particle composed of at least one AAV capsid protein (preferably, by all of the capsid proteins of wild-type AAV) and a polynucleotide encapsulated by the capsid. When the particle additionally contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome to be delivered to mammalian cells), it is typically referred to as "rAAV". Exemplary heterologous polynucleotides are polynucleotides comprising an engineered CasX protein and / or ERS of any of the embodiments described herein, and optionally a donor template.

[0209] "Adeno-associated virus inverted terminal repeat" or "AAV ITR" means a region recognized in the art found at each end of the AAV genome that functions cis as an origin of DNA replication and as a packaging signal for the virus. The AAV ITRs, together with the AAV rep coding region, provide for efficient removal and rescue of the nucleotide sequence intervening between two adjacent ITRs, and integration into the mammalian cell genome.

[0210] The nucleotide sequences of the AAV ITR regions are known. See, for example, R.M. (1994) Human Gene Therapy 5:793-801, Berns, K.I. “Parvoviridae and their Replication” in Fundamental Virology, 2nd Edition, (B.N. Fields and D.M. Knipe, eds.). As used herein, the AAV ITR need not have the indicated wild-type nucleotide sequence, but can be modified, for example, by nucleotide insertion, deletion, or substitution. In addition, the AAV ITR can be derived from any of several AAV serotypes, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAV 9.45, AAV 9.61, AAV-Rh74, and AAVRh10, as well as modified capsids of these serotypes. Further, the 5' ITR and 3' ITR flanking the selected nucleotide sequence in the AAV vector need not be identical, nor need they be derived from the same AAV serotype or isolate, so long as they function as intended, i.e., enable removal and rescue of the sequence of interest from the host cell genome or vector and, when the AAV Rep gene product is present in the cell, enable integration of the heterologous sequence into the recipient cell genome. The use of AAV serotypes for integration of heterologous sequences into host cells is known in the art (see, for example, WO2018 / 195555 (A1) and US2018 / 0258424 (A1), which are incorporated herein by reference). In one particular embodiment, the ITR is derived from serotype AAV1.In certain embodiments, the ITR region adjacent to the transgene of this embodiment is derived from AAV2. The 5’ ITR of the transgene of the AAV construct of the present disclosure has the sequence CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 487), and the 3’ ITR of the transgene of the AAV construct of the present disclosure has the sequence AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 488). In other embodiments, the ITR sequence is modified to remove non-methylated CpG motifs to reduce the immunogenic response. In particular, CpG dinucleotide motifs (CpG PAMPs) in AAV vectors are immunostimulatory because they have a high degree of hypomethylation compared to mammalian CpG motifs that have a high degree of methylation. In one embodiment, the modified AAV2 ITR sequence is modified to remove CpG motifs, such that the 5’ ITR has the sequence TGCTCACTCACTCACTCACTGAGGCCTGCAGAGCAAAGCTCTGCAGTCTGGGGACCTTTGGTCCCCAGGCCTCAGTGAGTGAGTGAGTGAGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 489), and the 3’ ITR sequence has the sequence TCTGCTCACTCACTCACTCACTGAGGCCTGCAGAGCAAAGCTCTGCAGTCTGGGGACCTTTGGTCCCCAGGCCTCAGTGAGTGAGTGAGTGAGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT of SEQ ID NO: 490.Similarly, the present disclosure provides an rAAV vector, wherein one or more rAAV transgene construct component sequences selected from the group consisting of a 5' ITR, 3' ITR, Pol III promoter, Pol II promoter, a coding sequence of a CRISPR nuclease, a coding sequence of an ERS, an accessory element, and a poly(A) are codon-optimized to deplete all or a portion of CpG dinucleotides, and the resulting rAAV vector transgene is substantially devoid of CpG dinucleotides. In some embodiments, the present disclosure provides an rAAV vector, wherein one or more rAAV transgene construct component sequences selected from the group consisting of a 5' ITR, 3' ITR, Pol III promoter, Pol II promoter, a coding sequence of a CRISPR nuclease, a coding sequence of an ERS, a 3' UTR, a poly(A) signal sequence, a poly(A), and an accessory element contain less than about 10%, less than about 5%, or less than about 1% CpG dinucleotides. In some embodiments, the present disclosure provides an rAAV vector, wherein one or more rAAV transgene construct component sequences selected from the group consisting of a 5' ITR, 3 ITR, Pol III promoter, Pol II promoter, a coding sequence of a CRISPR nuclease, a coding sequence of an ERS, a 3' UTR, a poly(A) signal sequence, and a poly(A) are devoid of CpG dinucleotides. In some embodiments, the present disclosure provides an rAAV vector, wherein the transgene contains less than about 10%, less than about 5%, or less than about 1% CpG dinucleotides.In some embodiments, the present disclosure provides an rAAV vector, wherein one or more rAAV constituent sequences codon-optimized to deplete CpG dinucleotides are selected from the group consisting of SEQ ID NOs: 489, 490, 535-556, 559-564, and 49850-49861 as set forth in Tables 37, 38, and 51, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, and the resulting AAV exhibits a reduced potential to induce an immune response, either in vivo (when administered to a subject) or in an in vitro mammalian cell assay designed to detect inflammatory response markers, and the reduction in response is determined by measurement of one or more parameters such as production of antibodies or delayed hypersensitivity to rAAV constituents, or production of inflammatory cytokines and markers such as, but not limited to, TLR9, interleukin-1 (IL-1), IL-6, IL-12, IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and granulocyte macrophage colony-stimulating factor (GM-CSF).

[0211] The "AAV rep coding region" means the region of the AAV genome that encodes the replication proteins Rep 78, Rep 68, Rep 52, and Rep 40. These Rep expression products have been shown to have multiple functions including recognition, binding, and nicking of the AAV DNA replication origin, DNA helicase activity, and regulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for AAV genome replication.

[0212] The "AAV cap coding region" means the region of the AAV genome that encodes the capsid proteins VP1, VP2, and VP3, or functional homologs thereof. These Cap expression products provide the packaging functions that are collectively required for packaging of the viral genome.

[0213] In some embodiments, the AAV capsid utilized for delivery of engineered CasX, ERS, and optionally a nucleic acid encoding a donor template nucleotide to a host cell can be derived from any of several AAV serotypes including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 9.45, AAV 9.61, AAV 44.9, AAV-Rh74 (AAV derived from rhesus monkey), and AAVRh10. In some embodiments, the AAV vector and regulatory sequences are selected such that the total size of the vector is about 4.7 - 5 kb or less to allow packaging within the AAV capsid. The AAV vector can be of any AAV serotype, but neurotropism varies among AAV capsid serotypes. Thus, use of an AAV serotype compatible with widespread transgene delivery to astrocytes and motor neurons is preferred. In some embodiments, the AAV vector is of serotype 9 or serotype 6, which have been demonstrated to effectively deliver polynucleotides to motor neurons and glia throughout the spinal cord in preclinical models of ALS (Foust, KD. et al. Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS. Mol Ther. 21(12):2148(2013)). In some embodiments, the method provides for the use of AAV9 or AAV6 for targeting neurons by intracerebral parenchymal injection. In some embodiments, the method is the use of AAV9 for intravenous administration of the vector, wherein AAV9 has the ability to cross the blood-brain barrier and drive gene expression in the nervous system by virtue of both its neuronal and glial tropism. In other embodiments, the AAV vector is derived from serotype 8, which has been demonstrated to effectively deliver polynucleotides to neurons, liver, skeletal muscle, and heart.In other embodiments, the AAV vector is derived from serotype 5, which has been demonstrated to effectively deliver polynucleotides to neurons. In other embodiments, the AAV vector is derived from AAV serotype 2, which has been demonstrated to effectively deliver polynucleotides to retinal cells, skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes.

[0214] To eliminate the virus's integration ability, recombinant AAV vectors can remove rep and cap from the DNA of the viral genome and utilize three plasmid systems to transfect suitable host packaging cells. To generate such vectors, the desired transgene is inserted between the ITRs together with a promoter and any enhancer elements for driving the transcription of those transgenes, and the rep gene and cap gene are provided in trans on a second plasmid. A third plasmid that provides helper genes such as adenovirus E4, E2a, and VA genes is also used. Subsequently, all three plasmids are transfected into appropriate packaging cells using known techniques such as transfection. Alternatively, the host cell genome can contain stably integrated Rep and Cap genes. Suitable packaging cell lines are known to those skilled in the art. See, for example, www.cellbiolabs.com / aav-expression-and-packaging.

[0215] As an advantage of the rAAV constructs of the present disclosure, when the size of the CRISPR type V nuclease, such as the engineered CasX of this embodiment, is reduced, a single rAAV particle can bring about the expression of the CRISPR nuclease and ERS that can effectively modify the target nucleic acid of the target cell, such that all the necessary editing components and auxiliary expression components can be included in the transgene in a form that allows these components to be delivered and transduced into the target cell. This is in marked contrast to other CRISPR systems such as Cas9, which typically use a two-particle system to deliver the necessary editing components to the target cell.

[0216] Thus, in some embodiments of the rAAV system, the present disclosure provides a first plasmid comprising: i) an ITR, a sequence encoding engineered CasX, a sequence encoding one or more ERSs, a first promoter operably linked to CasX, and a second promoter operably linked to the ERS, and optionally a 3’UTR, a poly(A) signal sequence, a poly(A) sequence, and one or more enhancer elements; ii) a second plasmid comprising a rep gene and a cap gene; and iii) a third plasmid comprising a helper gene. Upon transfection of a suitable packaging cell, the cell is capable of producing an rAAV that has the ability to deliver, in a single particle, a sequence capable of expressing an engineered CasX nuclease and an ERS that has the ability to edit a target nucleic acid of a target cell. In some embodiments of the rAAV system, the sequence encoding the CRISPR protein and the sequence encoding at least the first ERS are of a nucleotide length of less than about 3100, less than about 3090, less than about 3080, less than about 3070, less than about 3060, less than about 3050, or less than about 3040 nucleotides such that the first promoter, the second promoter, and optionally the sequence encoding one or more enhancer elements can have a combined nucleotide length of at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or at least about 1900 nucleotides. In some embodiments of the rAAV system, the first promoter and the sequence encoding at least one accessory element have a combined nucleotide length of at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600 nucleotides, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or more than at least about 1900 nucleotides.In some embodiments of the rAAV system, the sequences encoding the first promoter, the second promoter, and at least one accessory element have a combined nucleotide length of at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600 nucleotides, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or at least about 1900 or more. Non-limiting examples of such rAAV systems and coding sequences are disclosed in the following examples.

[0217] Packaging cells are typically used to form virus particles. Eukaryotic packaging host cells can be selected from baby hamster kidney fibroblast (BHK) cells, human embryonic kidney 293 (HEK293), human embryonic kidney 293T (HEK293T), NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, CV-1 (monkey)-derived (COS) cells having SV40 genetic material, HeLa cells, Chinese hamster ovary (CHO) cells, or other eukaryotic cells known in the art suitable for the production of recombinant AAV. Several transfection techniques are generally known in the art; see, for example, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. Particularly suitable transfection methods include calcium phosphate co-precipitation, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transfection, and nucleic acid delivery using a high-speed microprojectile.

[0218] In some embodiments, the host cell transfected with the above AAV expression vector is provided with the ability to provide AAV helper functions to replicate the nucleotide sequence adjacent to the AAV ITR and encapsulate it with a capsid to generate rAAV virus particles. AAV helper functions are generally coding sequences derived from AAV that can be expressed to provide AAV gene products, which then function in trans for productive AAV replication. AAV helper functions are used herein to complement the necessary AAV functions that are missing from the AAV expression vector. Thus, AAV helper functions include one or both of the major AAV ORFs (open reading frames) encoding the rep coding region and the cap coding region, or functional homologs thereof. Using methods known to those of skill in the art, the helper functions can be introduced into the host cell and then expressed in the host cell.

[0219] In other embodiments, suitable vectors may include XDP. XDP particles are particles that are very similar to viruses but do not contain viral genetic material and are therefore non-infectious. In some embodiments, the present disclosure provides in vitro-generated XDPs comprising the eCasX:ERS RNP complex. Non-limiting exemplary XDP systems are described in PCT / US20 / 63488 and WO2021 / 113772(A1), which are incorporated herein by reference. In some embodiments, the present disclosure provides a host cell comprising a polynucleotide or vector encoding any of the foregoing XDP embodiments. Combinations of structural proteins from different viruses are used to generate XDPs comprising components from viral families including Parvoviridae (e.g., adeno-associated virus), Retroviridae (e.g., HIV and alpharetrovirus), Flaviviridae (e.g., hepatitis C virus), Paramyxoviridae (e.g., Nipah), and bacteriophages (e.g., Qβ, AP205). In some embodiments, the present disclosure provides an XDP system designed using components of retroviruses including lentiviruses such as HIV, alpharetrovirus, and other Retroviridae genera, wherein individual plasmids comprising polynucleotides encoding various components are introduced into packaging cells and then XDPs are generated. In some embodiments, the present disclosure provides an XDP comprising polynucleotides encoding one or more components of i) a protease, ii) a protease cleavage site, iii) a Gag polyprotein, or a substrate protein (MA), a nucleocapsid protein (NC), a capsid protein (CA), or a p1-p6 protein, iv) a Gag-pol polyprotein (Gag-TFR-PR) or a cleaved version lacking reverse transcriptase (RT) and integrase but comprising an HIV protease, v) engineered CasX, vi) ERS, and vi) a targeting glycoprotein or antibody fragment, wherein the resulting XDP particles encapsidate multiple eCasX:ERS RNPs with a capsid.The polynucleotide encoding Gag, engineered CasX, and ERS may further comprise paired components designed to assist in the transport of the components from the nucleus of the host cell to budding XDP. Non-limiting examples of such transport components include hairpin RNAs such as MS2 hairpin, PP7 hairpin, Qβ hairpin, and U1 hairpin II, each having binding affinity for MS2 coat protein, PP7 coat protein, Qβ coat protein, and U1A signal recognition particle, respectively. In other embodiments, ERS may comprise a Rev response element (RRE) or a portion thereof having binding affinity for Rev that can be linked to the Gag polyprotein.

[0220] A targeting glycoprotein or antibody fragment on a surface that provides tropism of XDP to target cells, wherein when administered and upon entry into the target cells, the RNP molecule is freely transported into the nucleus of the cell, the targeting glycoprotein or antibody fragment. In other embodiments, the present disclosure provides the aforementioned XDP and further includes a second ERS or donor template. The foregoing provides advantages over other vectors in the art in that viral transduction into dividing and non-dividing cells is efficient and delivers a potent and short-lived RNP that escapes the target's immune surveillance mechanism that would otherwise detect foreign proteins. The present disclosure contemplates a plurality of three-dimensional arrangements of the coded components including replication of some of the coded components. Envelope glycoproteins include Argentine hemorrhagic fever virus, Australian bat lyssavirus, Autographa californica multiple nuclear polyhedrosis virus, avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Borna disease virus, Breda virus, Bunyamwera virus, Chandipura virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus, Duvenhage virus, eastern equine encephalitis virus, Ebola hemorrhagic fever virus, Ebola zaire virus, enteric adenovirus, Ephemerovirus, Epstein-Barr virus (EBV), European bat lyssavirus 1, European bat lyssavirus 2, Fug synthetic gP fusion, gibbon ape leukemia virus, hantavirus, Hendra virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus (GB virus C), herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus (HHV5), human foamy virus, human herpesvirus (HHV), human herpesvirus 7, human herpesvirus 6, human herpesvirus 8, human immunodeficiency virus type 1 (HIV-1), human metapneumovirus, human T-lymphotropic virus type 1, influenza A, influenza B, influenza C virus, Japanese encephalitis virus, Kaposi's sarcoma-associated herpesvirus (HHV8), Kyasanur Forest disease virus, La Crosse virus, Lagos bat virus, Lassa fever virus, lymphocytic choriomeningitis virus (LCMV),It may be derived from any enveloped virus known in the art that confers tropism to XDP, including but not limited to Machupo virus, Marburg hemorrhagic fever virus, measles virus, Middle East respiratory syndrome-related coronavirus, Mokola virus, Moloney murine leukemia virus, monkeypox, mouse mammary tumor virus, mumps virus, murine gammaherpesvirus, Newcastle disease virus, Nipah virus, Nipah virus, Norwalk virus, Omsk hemorrhagic fever virus, papillomavirus, parvovirus, pseudorabies virus, Quaranfil virus, rabies virus, RD114 feline endogenous retrovirus, respiratory syncytial virus (RSV), Rift Valley fever virus, Ross River virus, rotavirus, Rous sarcoma virus, rubella virus, Sabia-related hemorrhagic fever virus, SARS-related coronavirus (SARS-CoV), Sendai virus, Tacaribe virus, Thogoto virus, viruses causing tick-borne encephalitis, varicella-zoster virus (HHV3), varicella-zoster virus (HHV3), smallpox virus, vaccinia virus, Venezuelan equine encephalitis virus, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus (VSV), VSV-G, besivirus, West Nile virus, Western equine encephalitis virus, and Zika virus.

[0221] Upon generation and recovery of XDP comprising eCasX:ERS RNP of any of the embodiments described herein, the XDP can be used in a method of editing target cells of a subject by administration of such XDP, as fully described below.

[0222] In the case of non-viral delivery, vectors can also be delivered, and vectors encoding engineered CasX and ERS (s) are formulated into nanoparticles, and contemplated nanoparticles include but are not limited to nanospheres, liposomes, lipid nanoparticles (LNP), quantum dots, polyethylene glycol particles, hydrogels, and micelles. In some embodiments, the engineered CasX and ERS of the embodiments disclosed herein are formulated in lipid nanoparticles as fully described below.

[0223] VII. Method for Modifying Target Nucleic Acid The engineered CasX proteins, ERSs, nucleic acids, and their variants provided herein, as well as vectors encoding such constructs, are useful for a variety of applications including therapy, diagnosis, and research. To achieve the methods of the present disclosure for gene editing to effect gene modification, programmable systems comprising engineered CasX proteins and ERSs are provided herein. Due to the programmable nature of the systems provided herein, precise targeting is enabled to achieve a desired effect (such as nicking, cleavage, repair, etc.) on one or more regions of interest within the target nucleic acid sequence of a target gene.

[0224] Using a variety of strategies and methods, the systems provided herein can be used to modify target nucleic acid sequences within cells. As described herein, engineered CasX that introduces a double-strand break in a target nucleic acid generates a double-strand break within 18-26 nucleotides 5' of the PAM site on the target strand and within 10-18 nucleotides 3' of the non-target strand. The resulting modifications can lead to random insertions or deletions (indels), substitutions, duplications, frameshifts, or inversions of one or more nucleotides in those regions by the non-homologous DNA end joining (NHEJ) repair mechanism. Alternatively, the editing event can be a cleavage event followed by homology-directed repair (HDR), homology-independent targeted integration (HITI), microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), or base excision repair (BER) that results in modification of the target nucleic acid sequence. In some embodiments of the methods, the modification includes introducing an in-frame mutation into the target nucleic acid. In some embodiments of the methods, the modification includes introducing a frameshift mutation into the target nucleic acid. In some embodiments of the methods, the modification includes introducing a premature stop codon into the coding sequence in the target nucleic acid. As a result of gene knockdown by the aforementioned modifications, protein activity or function may be attenuated, or the protein level may be decreased or eliminated. In some embodiments of the methods, 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% decreased expression of the gene product in the modified cell population compared to cells in which the gene is unmodified. In other embodiments, the present disclosure provides systems and methods for correcting mutations in a gene, wherein a correction sequence is knocked in by introducing a mutation at a selected position by design of a targeting sequence linked to an ERS, such that a wild-type or functional gene product is expressed.

[0225] In some embodiments, the present disclosure provides a method of modifying a target nucleic acid in a cell, the method comprising contacting the target nucleic acid of the cell with: i) an edited pair of an engineered CasX and an ERS, comprising any one of the engineered CasX proteins and the ERS of the embodiments described herein; ii) a nucleic acid encoding an edited pair of an engineered CasX and an ERS; iii) a vector comprising the nucleic acid of (ii) above; iv) an XDP comprising any one of the eCasX:ERS editing pairs of the embodiments described herein; v) an LNP comprising a nucleic acid encoding an ERS and an engineered CasX; or vi) a combination of two or more of (i) to (v), and contacting the target nucleic acid with an edited gene pair of an engineered CasX protein and an ERS, and optionally a donor template, such that the target nucleic acid in the cell is modified. In some cases, the modification results in correction or compensation of a mutation in the cell, thereby generating a cell edited such that expression of a functional gene product can occur. In other embodiments of the method, the modification comprises reducing or eliminating expression of a gene product by gene knockdown or knockout.

[0226] In some embodiments of the method of modifying a target nucleic acid sequence in a cell, the method comprises contacting the target nucleic acid of the cell with an editing pair, the editing pair comprising an engineered CasX selected from the group consisting of the sequences of SEQ ID NOs: 247-294, 24916-49628, 49746-49747, and 49871-49873, or a variant sequence that is 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; the ERS scaffold comprises a sequence selected from the group consisting of the sequences of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, 49719-49735, and 49871-49873, or a sequence that is 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 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 thereto; and the ERS comprises a targeting sequence that is complementary to and capable of hybridizing to the target nucleic acid.

[0227] In cases where engineered CasX is delivered to cells in protein form and ERS is delivered in RNA form, engineered CasX and ERS can be pre-complexed and delivered as an RNP. In cases where engineered CasX and ERS are delivered to target cells as nucleic acids and then expressed intracellularly, engineered CasX and ERS can associate as an RNP. In cases where an LNP delivers ERS and engineered CasX is delivered as mRNA and then expressed intracellularly, engineered CasX and ERS can associate as an RNP. As described above, the engineered CasX protein provides site-specific activity and is guided to (and further stabilized at) the target site within the target nucleic acid sequence that is modified by its association with ERS. The engineered CasX protein of the RNP complex provides site-specific activity of the complex such as binding, and by introducing single-strand or double-strand breaks within or near the gene, permanent indels (deletions or insertions) or other mutations in the target nucleic acid (base changes, inversions, or translocations relative to the genomic sequence) in the target nucleic acid, as described herein, result in corresponding regulation of gene product expression or alteration of function, thereby generating modified cells.

[0228] In other embodiments of the method of modifying a target nucleic acid sequence in a cell, the method comprises contacting the target nucleic acid sequence with a plurality of RNPs having a first ERS and a second ERS, or three ERSs, or four or more ERSs that are targeted to different or overlapping portions of the gene, and the engineered CasX protein introduces multiple single-strand or double-strand breaks in the target nucleic acid, whereby, as described herein, permanent indels (introducing insertions or deletions) or mutations in the target nucleic acid, or removal of intervening sequences between the breaks, result in corresponding regulation of gene product expression or alteration of function, thereby generating modified cells.

[0229] In other embodiments, the present disclosure provides a method of modifying a target nucleic acid sequence of a cell, the method comprising contacting the cell with a vector of any of the embodiments described herein encoding an engineered CasX protein and an ERS of any of the embodiments described herein to form an eCasX:ERS gene editing pair, and optionally a donor template, wherein the ERS comprises a targeting sequence complementary to the target nucleic acid sequence and thus capable of hybridizing therewith, and wherein the contacting results in modification of the target nucleic acid. Introduction of the recombinant expression vector into the cells in vitro can occur in any suitable culture medium and under any suitable culture conditions that promote cell survival. Introduction of the recombinant expression vector into the target cells can be performed in vivo by administration to a subject using the methods and regimens described below.

[0230] In some embodiments, the vector can be provided directly to the target host cell. For example, the cell can be contacted with a vector (e.g., a recombinant expression vector encoding an ERS and an engineered CasX protein) comprising the subject nucleic acid such that the vector is taken up by the cell. Methods of contacting the cell with a nucleic acid vector that is a plasmid include electroporation, calcium chloride transfection, microinjection, and lipofection, which are known in the art. In the case of viral vector delivery, the cell can be contacted with viral particles comprising the subject viral expression vector, e.g., the vector is a viral particle such as an AAV or VLP comprising a polynucleotide encoding the eCasX:ERS components. In the case of non-viral delivery, the vector or eCasX:ERS components can also be formulated into lipid nanoparticles for delivery, as described more fully below.

[0231] In some embodiments, the modification of the target nucleic acid occurs in vitro, within a cell, for example, in a cell culture system. In some embodiments, the modification occurs in vivo, within a cell of a subject, for example, in a cell of an animal. In some embodiments, the cell is a eukaryotic cell. Exemplary eukaryotic cells can include cells selected from the group consisting of mouse cells, rat cells, pig cells, dog cells, and non-human primate cells. In some embodiments, the cell is a human cell. Non-limiting examples of cells include embryonic stem cells, induced pluripotent stem cells, germ cells, fibroblasts, oligodendrocytes, glial cells, hematopoietic stem cells, neuronal progenitor cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, retinal cells, cancer cells, T cells, B cells, NK cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplanted expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow-derived progenitor cells, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, pluripotent progenitor cells, unipotent progenitor cells, monocytes, cardiomyoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, heterologous cells, homologous cells, or postnatal stem cells. In alternative embodiments, the cell is a prokaryotic cell.

[0232] In some embodiments of methods of modifying a target nucleic acid of a cell in vitro or ex vivo to effect cleavage or any desired modification to the target nucleic acid, the ERS and engineered CasX proteins of the present disclosure, and optionally a donor template sequence (whether introduced as a nucleic acid or as a polypeptide), complexed RNP, vector, or XDP are provided to the cell for about 30 minutes to about 24 hours, or at least about 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period of about 30 minutes to about 24 hours, which can be repeated at a frequency of about daily to about every 4 days, for example, every 1.5 days, every 2 days, every 3 days, or any other frequency of about daily to about every 4 days. The agent may be provided to the target cell one or more times, for example, 1, 2, 3, or 4 or more times, such that the cell is able to incubate with the agent for some period of time, for example, 30 minutes to about 24 hours, after each contact event. In the case of in vitro-based methods, after the incubation period with the engineered CasX and ERS (and optionally the donor template), the medium is replaced with fresh medium and the cells are further cultured.

[0233] In some embodiments, the method includes administering to a subject a therapeutically effective dose of a population of cells modified to correct or compensate for a gene mutation. In some embodiments, administration of the modified cells results in expression of a wild-type or functional gene product in the subject. In one embodiment, the cells are autologous to the subject to whom the cells are administered. In another embodiment, the cells are allogeneic to the subject to whom the cells are administered. In some cases, the subject is selected from the group consisting of mice, rats, pigs, and non-human primates. In other cases, the subject is human.

[0234] VIII. Methods of Treatment In another aspect, the present disclosure relates to a method of doing so in a subject in need of treatment of a disease or disorder. Several treatment strategies are used to design a system for use in a method of treating a subject having a disease or disorder associated with a genetic mutation. In some embodiments, the modification of the target nucleic acid occurs in a subject having a mutation that is a mutation in an allele of a gene and that causes the disease or disorder in the subject. In some embodiments, the modification of the target nucleic acid changes a mutation to the wild-type allele of the gene or results in the expression of a functional gene product. In some embodiments, the modification of the target nucleic acid knockdowns or knockouts the expression of an allele of a gene that causes the disease or disorder in the subject.

[0235] In some embodiments, the method comprises administering to a subject a therapeutically effective dose of a system comprising a gene editing pair of an engineered CasX disclosed herein and an ERS having a linked targeting sequence complementary to a target nucleic acid to be modified. In some embodiments, the treatment method comprises administering to a subject a therapeutically effective dose of i) an eCasX:ERS system comprising an engineered CasX and a first ERS (having a targeting sequence complementary to a target nucleic acid to be modified) of any of the embodiments described herein, ii) a nucleic acid encoding the eCasX:ERS system of (i), iii) a vector comprising the nucleic acid of (ii), which can be any AAV of the embodiments described herein, iv) an XDP comprising the eCasX:ERS system of (i), v) an LNP comprising a nucleic acid encoding an ERS and an engineered CasX, or vi) a combination of two or more of (i)-(v), wherein 1) the gene of the subject's cells targeted by the first ERS is modified (e.g., knocked down or knocked out) by the engineered CasX protein (and optionally a donor template), or 2) the gene of the subject's cells targeted by the first ERS is corrected or modified by the engineered CasX protein such that a functional gene product can be expressed. In some embodiments, the treatment method further comprises administering a second, third, or fourth ERS, or a nucleic acid encoding an ERS, or an XDP comprising the second, third, or fourth ERS, wherein the second, third, or fourth ERS has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid sequence as compared to the first ERS. In some cases, use of a second ERS complexed with the engineered CasX results in editing of a gene different from the first ERS. In other cases, use of a second ERS targeting the same gene as the first ERS can result in removal of nucleotides between two cleavage positions. It will be understood that in the foregoing, each different ERS is paired with the engineered CasX protein.In embodiments where two or more gene editing pairs (e.g., including two or more ERSs containing two or more different spacers complementary to different sequences within the same or different target nucleic acids) are provided to a cell, the gene pairs may be provided simultaneously within the same vector (e.g., as two RNPs and / or within a single AAV vector), or may be delivered simultaneously within separate vectors. Alternatively, they may be provided sequentially, e.g., a first gene editing pair may be provided first, followed by a second gene editing pair, or vice versa.

[0236] In some embodiments, the treatment method includes administering a therapeutically effective dose of an AAV vector encoding an eCasX:ERS system, and the capsid of the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 9.45, AAV 9.61, AAV-Rh74, or AAVRh10. In other embodiments, the treatment method includes administering to a subject a therapeutically effective dose of an XDP containing an RNP of an eCasX:ERS system. In other embodiments, the treatment method includes administering a therapeutically effective dose of an LNP containing a nucleic acid encoding an ERS and engineered CasX. The vector, XDP, or LNP can be administered by an administration route selected from the group consisting of intrasubstantially, intravenous, intraarterial, intramuscular, subcutaneous, intraventricular, intracisternal, intrathecal, intracranial, intravitreal, subretinal, intraarticular, and intraperitoneal routes, or combinations thereof, and the administration method is injection, infusion, or transplantation. The administration may be once or twice, or can be administered multiple times using a regimen schedule of once a week, every two weeks, monthly, four times a year, every six months, once a year, or once every two or three years. In some cases, the subject is selected from the group consisting of mice, rats, pigs, and non-human primates. In other cases, the subject is a human.

[0237] In some embodiments of the present method, the modification comprises introducing a single-strand break into the target nucleic acid of the targeted cell of interest. In other cases, the modification comprises introducing a double-strand break into the target nucleic acid of the targeted cell of interest. In some embodiments, the modification introduces one or more mutations into the target nucleic acid, such as insertion, deletion, substitution, duplication, or inversion of one or more nucleotides of a gene, and the expression of the gene product in the modified cell of interest is 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% or more decreased compared to the unmodified cell. In some cases, the gene of the modified cell of interest is modified such that at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the modified cells do not express the gene product at a detectable level. In some embodiments, administration of a therapeutically effective amount of the eCasX:ERS system to a subject having a disease to knockdown or knockout the expression of a gene product results in the prevention or amelioration of the underlying disease, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disease. In some embodiments, administration of a therapeutically effective amount of the eCasX:ERS system to a subject having a disease to correct or compensate for a mutation in a gene product results in the prevention or amelioration of the underlying disease, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disease. In such embodiments, this gene can be modified by the NHEJ host repair mechanism or utilized with a donor template inserted by the HDR mechanism or the HITI mechanism to excise, correct, or compensate for the mutation in the cell of the subject, whereby the expression of the wild-type or functional gene product in the modified cell is 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% increased compared to the unmodified cell.In some embodiments, administration of a therapeutically effective amount of a system of engineered CasX and ERS results in improvement of at least one clinically relevant parameter of a disease.

[0238] IX. eCasX: Particles for Delivery of the eCasX:ERS System In another aspect, the present disclosure provides a particle composition for delivering a repressor system, such as the eCasx:ERS system described herein, for gene repression to a cell or a subject. Particles contemplated within the scope of the present disclosure include, but are not limited to, nanoparticles such as synthetic nanoparticles, polymeric nanoparticles, lipid nanoparticles, viral particles, and virus-like particles. The particles of the present disclosure can optionally encapsulate a payload such as an ERS variant in combination with an mRNA encoding any of the engineered CasX proteins of the embodiments described herein. Alternatively, or in addition, the particles of the present disclosure can encapsulate the payload of the ERS variant and the engineered CasX protein when associated, for example, as a ribonucleoprotein (RNP) complex. In some embodiments, the particles are synthetic nanoparticles that encapsulate the payload of the ERS variant and an mRNA encoding any of the engineered CasXs of the embodiments described herein. In some embodiments, the synthetic nanoparticles include biodegradable polymeric nanoparticles (PNPs). In some embodiments, materials for making biodegradable polymeric nanoparticles (PNPs) include polylactide, poly(lactic-co-glycolic acid) (PLGA), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), and poly(isohexyl cyanoacrylate), polyglutamic acid (PGA), poly(ε-caprolactone) (PCL), cyclodextrin, and natural polymers such as chitosan, albumin, gelatin, alginate, which are the most utilized polymers for the synthesis of PNPs (Production and clinical development of nanoparticles for gene delivery. Molecular Therapy-Methods & Clinical Development 3:16023; doi:10.1038 (2016)). In other embodiments, the particles are lipid nanoparticles that encapsulate the ERS variant and an mRNA encoding any of the engineered CasXs of the embodiments described herein, more fully described below.

[0239] a. Lipid nanoparticles (LNP) The present disclosure provides lipid nanoparticles (LNP) for delivering the eCasX:ERS system described herein to cells or a subject for gene silencing. In some embodiments, the LNP of the present disclosure is tissue-specific, has excellent biocompatibility, and can deliver the eCasX:ERS system with high efficiency, and thus can be used for targeted gene silencing.

[0240] The present disclosure further provides an LNP composition and a pharmaceutical composition comprising a plurality of LNPs described herein.

[0241] Nucleic acid polymers, in their native form, are unstable in biological fluids and cannot pass through the cytoplasm of target cells, and thus require a delivery system. Lipid nanoparticles (LNP) have been proven to be useful not only for protecting nucleic acids but also for delivery to tissues and cells. Furthermore, the use of mRNA in LNP for encoding engineered CasX eliminates the possibility of unwanted genomic integration compared to DNA vectors. Furthermore, since mRNA exerts its function in the cytoplasmic compartment and does not require entry into the nucleus, it is efficiently translated into protein in both mitotic and non-mitotic cells. Therefore, LNP as a delivery platform offers the additional advantage that both mRNA encoding a CRISPR nuclease and ERS can be co-formulated into a single LNP particle.

[0242] Accordingly, in various embodiments, the present disclosure encompasses lipid nanoparticles and compositions that can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells both in vitro and in vivo. In certain embodiments, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof by contacting the subject with lipid nanoparticles encapsulating or associated with a suitable therapeutic agent complexed by various physical, chemical, or electrostatic interactions among one or more of the lipid components used in the composition to make the LNP. In some embodiments, a suitable therapeutic agent includes the eCasX:ERS system described herein.

[0243] In certain embodiments, for example, lipid nanoparticles comprising an mRNA encoding engineered CasX comprising the sequences of SEQ ID NOs: 247-294, 24916-49628, 49746-49747, and 49871-49873, and an ERS variant of the present disclosure comprising the sequences of SEQ ID NOs: 156, 739-907, 11568-22227, 23572-24915, and 49719-49735 are useful for the delivery of nucleic acids. In some embodiments, the present disclosure provides an LNP in which the mRNA encoding ERS and engineered CasX are incorporated into a single LNP particle. In other embodiments, the present disclosure provides an LNP in which the mRNA encoding ERS and engineered CasX are incorporated into separate LNP populations that can be formulated together at various ratios for administration.

[0244] Using the lipid nanoparticles and lipid nanoparticle compositions of certain embodiments of the present disclosure, cells can be contacted with lipid nanoparticles comprising one or more ionizable lipids described herein to suppress the expression of a desired protein both in vitro and in vivo, and the lipid nanoparticles encapsulate or are associated with a nucleic acid expressed to produce the desired protein (e.g., messenger RNA encoding an engineered CasX protein). In some embodiments, lipid nanoparticles and compositions are used to contact cells with lipid nanoparticles comprising one or more novel ionizable cationic lipids or permanently charged cationic lipids described herein to suppress the expression of a target gene both in vitro and in vivo, and the lipid nanoparticles encapsulate or are associated with one or more nucleic acids of the eCasX:ERS system of the present disclosure that suppress the targeted gene. The lipid nanoparticles and compositions of embodiments of the present disclosure may be used separately or in combination for the co-delivery of different nucleic acids (e.g., mRNA, gRNA, siRNA, saRNA, mcDNA, and plasmid DNA), and may be useful, for example, to provide effects that require co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene silencer or enzyme and ERS for gene targeting).

[0245] In some embodiments, the LNPs and LNP compositions described herein comprise at least one cationic lipid, at least one conjugate lipid, at least one steroid or derivative thereof, at least one helper lipid, or any combination thereof. Alternatively, the lipid compositions of the present disclosure can include ionizable lipids such as ionizable cationic lipids, helper lipids (usually phospholipids), cholesterol, and polyethylene glycol-lipid conjugates (PEG-lipids) to, for example, reduce specific absorption of plasma proteins and improve colloidal stability in a biological environment by forming a hydration layer on the nanoparticles. Such lipid compositions can be formulated at typical IL:HL:sterol:PEG-lipid molar ratios of 50:10:37-39:13 or 20-50:8-65:15-70:1-3.0, with variations to adjust individual properties.

[0246] The LNPs and LNP compositions of the present disclosure are configured to protect the encapsulated payload of the present disclosure and deliver it to tissues and cells both in vitro and in vivo. Various embodiments of the LNPs and LNP compositions of the present disclosure are described in further detail herein.

[0247] b. Cationic lipid In some embodiments, the LNPs and LNP compositions of the present disclosure include at least one cationic lipid. The term "cationic lipid" refers to lipid species having a net positive charge. In some embodiments, the cationic lipid is an ionizable cationic lipid having a net positive charge at a selected pH below the pKa of the ionizable lipid. In some embodiments, the ionizable cationic lipid has a pKa of less than 7 such that the LNPs and LNP compositions achieve efficient encapsulation of the payload at a relatively low pH below the pKa of each respective lipid. In some embodiments, the cationic lipid has a pKa of about 5 to about 8, about 5.5 to about 7.5, about 6 to about 7, or about 6.5 to about 7. In some embodiments, the cationic lipid may be protonated at a pH below the pKa of the cationic lipid, which may be substantially neutral at a pH above the pKa. The LNPs and LNP compositions are safely delivered in vivo to target organs (e.g., liver, lung, heart, spleen, and tumor) and / or cells (e.g., hepatocytes, LSECs, heart cells, cancer cells, etc.) and may exhibit a positive charge to release the encapsulated payload by electrostatic interaction with the anionic lipids of the endosomal membrane when the pH decreases below the pKa of the ionizable lipid during endocytosis.

[0248] Initial formulations of LNPs utilizing permanently cationic lipids resulted in LNPs with a positive surface charge that were shown to be toxic in vivo and were additionally rapidly removed by phagocytic cells. Changing to ionizable cationic lipids having a tertiary amine, particularly those having a pKa of less than 7, results in LNPs that achieve efficient encapsulation of nucleic polymers at low pH by electrostatically interacting with the negative charge of the phosphate backbone of the mRNA, thereby also resulting in a system that is predominantly neutral at physiological pH values and thus reducing the problems associated with permanently charged cationic lipids.

[0249] As used herein, "ionizable lipid" means an amine-containing lipid that can be readily protonated, e.g., a lipid whose charge state can vary depending on the ambient pH. The ionizable lipid may be protonated (positively charged) at a pH below the pKa of the cationic lipid, which may be substantially neutral at a pH above the pKa. In one example, the LNP may include protonated ionizable lipid and / or ionizable lipid that exhibits neutralization. In some embodiments, the LNP has a pKa of 5-8, 5.5-7.5, 6-7, or 6.5-7. The pKa of the LNP is important for in vivo stability in the target cell or organ and for the release of the nucleic acid payload of the LNP. In some embodiments, the LNP having the aforementioned pKa range is safely delivered to target organs (e.g., liver, lung, heart, spleen, and tumor) and / or target cells (such as hepatocytes, LSEC, heart cells, cancer cells, etc.) in vivo and can exhibit a positive charge to release the payload encapsulated by electrostatic interaction with the anionic lipid of the endosomal membrane within the endosome.

[0250] An ionizable lipid is an ionizable compound having generally lipid-like characteristics and can play a role in encapsulating a nucleic acid payload in the LNP with high efficiency by electrostatic interaction with a nucleic acid (e.g., the mRNA of the present disclosure).

[0251] Depending on the type of amine group and tail group included in the ionizable lipid, (i) nucleic acid encapsulation efficiency, (ii) PDI (polydispersity index), and / or (iii) nucleic acid delivery efficiency of the LNP to the tissues and / or cells constituting an organ (e.g., hepatocytes or liver sinusoidal endothelial cells in the liver) may vary. In certain embodiments, the ionizable lipid is an ionizable cationic lipid and constitutes about 25 mol% to about 66 mol% of the total lipid present in the particle.

[0252] Lipid nanoparticles (LNPs) containing ionizable lipids containing amines have the following characteristics: (1) the ability to encapsulate nucleic acids with high efficiency, (2) a uniform size of the prepared particles (or having a low PDI value), and / or (3) one or more types of excellent nucleic acid delivery efficiency to organs such as the liver, lung, heart, spleen, bone marrow, and tumors, and / or cells constituting such organs (e.g., hepatocytes, LSEC, heart cells, cancer cells, etc.).

[0253] In certain embodiments, the cationic lipid form plays an important role in both nucleic acid encapsulation by electrostatic interaction and intracellular release by disrupting the endosomal membrane. Nucleic acid payloads are encapsulated within the LNP by the ionic interactions they form with positively charged cationic lipids. Non-limiting examples of ionizable cationic lipid components utilized in the LNPs of the present disclosure include DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), and those selected from TNT (1,3,5-triazinane-2,4,6-trione) and TT (N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide). Non-limiting examples of helper lipids utilized in the LNPs of the present disclosure include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine), and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) DOPG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), sphingolipids, and ceramides. Cholesterol and PEG-DMG ((R)-2,3-bis(octadecyloxy)propyl-1-(methoxypolyethylene glycol 2000) carbamate), PEG-DSG (1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000), or DSPE-PEG2k (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) are components utilized in the LNPs of the present disclosure for LNP stability, circulation, and size.

[0254] In some embodiments, the cationic lipid in the LNP of the present disclosure comprises a tertiary amine. In some embodiments, the tertiary amine comprises an alkyl chain connected to the N of the tertiary amine having an ether bond. In some embodiments, the alkyl chain comprises a C12-C30 alkyl chain having 0 to 3 double bonds. In some embodiments, the alkyl chain comprises a C16-C22 alkyl chain. In some embodiments, the alkyl chain comprises a C18 alkyl chain. Some cationic lipids and related analogs are described in US Patent Publications 2006 / 0083780, 2006 / 0240554, 2011 / 0117125, 2019 / 0336608, 2019 / 0381180, and 2020 / 0121809, US Patents 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; 5,785,992; 9,738,593; 10,106,490; 10,166,298, 10,221,127, and 11,219,634, and PCT Publication WO96 / 10390, the disclosures of which are incorporated herein by reference in their entireties.

[0255] In some embodiments, the cationic lipid in the LNP of the present disclosure may comprise, for example, one or more ionizable cationic lipids, and the ionizable cationic lipid is a dialkyl lipid. In other embodiments, the ionizable cationic lipid is a tetraalkyl lipid.

[0256] In some embodiments, the cationic lipids in the LNP of the present disclosure are 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyoxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.(Cl), 1,2-dilinoleyl-oxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N’,N’-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spemine-carboxamide)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5’-(cholest-5-en-3-beta-oxy)-3’-oxapentoxy)-3-dimethyl-1-(cis,cis-9’,1-2’-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N’-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N’-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), and are selected from any combination of the foregoing.

[0257] In some embodiments, the cationic lipid in the LNP of the present disclosure is selected from heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (1,3,5-triazinane-2,4,6-trione) (TNT), N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT), and any combination of the foregoing.

[0258] In some embodiments, the N / P ratio (nitrogen from cationic / ionizable lipid and phosphate from nucleic acid) in the LNP of the present disclosure is in the range of about 3:1 to 7:1 or about 4:1 to 6:1, or is within the range of 3:1, or 4:1, or 5:1, or 6:1, or 7:1, or 8:1, or 9:1.

[0259] Conjugated lipid In some embodiments, the LNP and LNP compositions of the present disclosure comprise at least one conjugated lipid. In some embodiments, the conjugated lipid can be selected from polyethylene glycol (PEG)-lipid conjugates, polyamide (ATTA)-lipid conjugates, cationic polymer-lipid conjugates (CPL), and any combination of the foregoing. In some cases, the conjugated lipid can inhibit aggregation of the LNP of the present disclosure.

[0260] In some embodiments, the conjugate lipid of the LNP of the present disclosure comprises a pegylated lipid. The terms "polyethylene glycol (PEG)-lipid conjugate", "pegylated lipid", "lipid-PEG conjugate", "lipid-PEG", "PEG-lipid", "PEG-lipid", or "lipid-PEG" are used interchangeably herein and refer to a lipid conjugated to a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer. The pegylated lipid contributes to the stability of the LNP and LNP compositions and reduces the aggregation of the LNP. In other embodiments, the lipid of the LNP comprises a peptide-modified PEG lipid used to target cell surface receptors, such as DSPE-PEG-RGD, DSPE-PEG-transferrin, DSPE-PEG-cholesterol.

[0261] Since the PEG-lipid forms the surface lipid, the size of the LNP can be easily changed by varying the ratio of the surface (PEG) lipid to the core (ionizable cationic) lipid. In some embodiments, the PEG-lipid of the LNP of the present disclosure can be varied by about 1-5 mol% to modify particle properties such as size, stability, and circulation time.

[0262] The lipid-PEG conjugate contributes to the particle stability of the nanoparticles in serum within the LNP and plays a role in preventing aggregation between the nanoparticles. In addition, during in vivo delivery of the nucleic acid, the lipid-PEG conjugate protects the mRNA encoding the engineered CasX protein of the present disclosure or the nucleic acid such as the ERS of the present disclosure from degrading enzymes, enhances the stability of the nucleic acid in vivo, and can increase the half-life of the delivered nucleic acid encapsulated in the nanoparticles. 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 PEG-diacylglycerol (PEG-DAG) conjugates, PEG-dialkyloxypropyl (PEG-DAA) conjugates, PEG-phospholipid conjugates, PEG-ceramide (PEG-Cer) conjugates, and mixtures thereof.

[0263] In some embodiments, the pegylated lipids of the LNPs of the present disclosure are selected from PEG-ceramide, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-dialkoxypropylcarbamate, PEG-phosphatidylethanolamine, PEG-phospholipid, PEG-diacylglycerol succinate, and any combination of the foregoing.

[0264] In some embodiments, the pegylated lipid of the LNP of the present disclosure is PEG-dialkyloxypropyl. In some embodiments, the pegylated lipid is selected from PEG-didecyloxypropyl (C10), PEG-dilauroxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), and any combination of the foregoing.

[0265] In other embodiments, the lipid-PEG conjugate of the LNP of the present disclosure may be PEG conjugated to phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide (PEG-CER, ceramide-PEG conjugate, ceramide-PEG, PEG conjugated to cholesterol or a derivative thereof, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE (DSPE-PEG), and PEG bound to phospholipids such as mixtures thereof, for example, C16-PEG2000 ceramide (N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}), DMG-PEG2000, 14:0 PEG2000 PE.

[0266] In some embodiments, the PEGylated lipid of the LNP of the present disclosure is selected from 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, and any combination of the foregoing.

[0267] In some embodiments, the PEGylated lipid of the LNP of the present disclosure is selected from mPEG2000-1,2-di-O-alkyl-sn3-carbomoyl glyceride (PEG-C-DOMG), 1-[8’-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3’,6’-dioxaoctanyl] carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG), and any combination of the foregoing.

[0268] In some embodiments, PEG is directly attached to the lipid of the PEGylated lipid. In other embodiments, PEG is attached to the lipid of the PEGylated lipid by a linker moiety selected from an ester-free linker moiety or an ester-containing linker moiety. Non-limiting examples of ester-free linker moieties include amide (-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, and combinations thereof. For example, the linker may include a carbamate linker moiety and an amide linker moiety. Non-limiting examples of ester-containing linker moieties include carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0269] The PEG moiety of the pegylated lipid of the LNP of the present disclosure described herein may have an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In certain embodiments, the PEG moiety has an average molecular weight of about 750 Daltons to about 5,000 Daltons, about 1,000 Daltons to about 4,000 Daltons, about 1,500 Daltons to about 3,000 Daltons, about 750 Daltons to about 3,000 Daltons, or about 1750 Daltons to about 2,000 Daltons.

[0270] In some embodiments, the conjugate lipid (e.g., pegylated lipid) constitutes about 1 mol% to about 65 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipids present in the LNP and / or the LNP composition. In certain embodiments, the conjugate lipid constitutes about 0.5 mol% to about 3 mol% of the total lipids present in the particles.

[0271] In additional embodiments, the conjugate lipid (e.g., pegylated lipid) of the LNP of the present disclosure constitutes at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol% of the total lipids present in the LNP and / or the LNP composition, or constitutes any intermediate range among the foregoing.

[0272] In the case of the lipid in the lipid-PEG conjugate of the LNP of the present disclosure, any lipid capable of binding to polyethylene glycol may be used without limitation, and phospholipids and / or cholesterol, which are other elements of the LNP, may also be used. In some embodiments, the lipid in the lipid-PEG conjugate can be, but is not limited to, ceramide, dimyristoyl glycerol (DMG), succinoyl-diacyl glycerol (s-DAG), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine (DSPE), or cholesterol.

[0273] In the lipid-PEG conjugate of the LNP of the present disclosure, PEG can be directly conjugated to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for binding PEG to the lipid, including an ester-free linker moiety and an ester-containing linker moiety, may be used. The ester-free linker moiety includes, but is not limited to, amide (-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, and combinations thereof (e.g., a linker containing both a carbamate linker moiety and an amide linker moiety). The ester-containing linker moiety includes, but is not limited to, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0274] Steroid In some embodiments, the LNP and LNP compositions of the present disclosure include at least one steroid or a derivative thereof. In some embodiments, the steroid includes cholesterol. In some embodiments, the LNP and LNP compositions include a cholesterol derivative selected from cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and any combination of the foregoing.

[0275] In some embodiments, the steroid (e.g., cholesterol) of the LNP of the present disclosure constitutes about 1 mol% to about 60 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipids present in the LNP and / or the LNP composition. In other embodiments, the steroid (e.g., cholesterol) of the LNP of the present disclosure constitutes at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol% of the total lipids present in the LNP and / or the LNP composition, or an intermediate range of any of the foregoing.

[0276] Additional lipid / helper lipid or structural lipid In some embodiments, the LNP and LNP composition of the present disclosure comprise at least one helper lipid. In some embodiments, the helper lipid is a non-cationic lipid selected from anionic lipids, neutral lipids, or both. In some embodiments, the helper lipid comprises at least one phospholipid. In some embodiments, the phospholipid is selected from anionic phospholipids, neutral phospholipids, or both. The phospholipids of the elements of the LNP and LNP composition can play a role in covering and protecting the core of the LNP formed by the interaction between the cationic lipid and the nucleic acid in the LNP, and can facilitate cell membrane permeation and endosomal escape during intracellular delivery of the nucleic acid by binding to the phospholipid bilayer of the target cell. The phospholipids that can promote the fusion of the LNP with the cell can include, but are not limited to, any of the phospholipids selected from the group described below.

[0277] In some embodiments, the LNP comprises helper lipids used to target cell surface receptors, such as DSPE-RGD, DSPE-cRGD, DSPE-Chol. It also includes molecules such as 18:0 Lyso-PC and 18:2 Lyso-PC.

[0278] In some embodiments, the LNPs and LNP compositions comprise at least one phospholipid selected from, but not limited to, dipalmitoyl-phosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylethanolamine (DOPE), dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylglycerol (DOPG), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-phosphatidylglycerol (DPPG), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), phosphatidylethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], and any combination of the foregoing. In one example, LNPs comprising DSPC can be effective for mRNA delivery (excellent drug delivery efficacy).

[0279] In some embodiments, the helper lipid (e.g., phospholipid) of the LNP of the present disclosure constitutes about 1 mol% to about 60 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipids present in the LNP and / or the LNP composition. In other embodiments, the helper lipid (e.g., phospholipid) of the LNP of the present disclosure constitutes at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol% of the total lipids present in the LNP and / or the LNP composition, or an intermediate range of any of the foregoing.

[0280] It will be appreciated that the total lipids present in the LNP and / or the LNP composition include lipids of cationic lipids or ionizable cationic lipids, conjugate lipids (e.g., pegylated lipids), peptide-conjugated PEG lipids, steroids (e.g., cholesterol), peptide-conjugated structural lipids (e.g., DSPE-cRGD), and structural lipids (e.g., phospholipids) individually or in combination, resulting in an LNP formulation containing 1 to multiple components (not limited to 1, 2, 3, 4, or 5 components) in the LNP formulation.

[0281] The LNP and / or the LNP composition can be prepared by dissolving the total lipids (or a portion thereof) in an organic solvent (e.g., ethanol) and subsequently mixing with a payload (e.g., the nucleic acid of the system) dissolved in an acidic buffer (e.g., pH of 1.0 to 6.5) using a micromixer. At this pH, the ionizable cationic lipid is positively charged and interacts with the negatively charged nucleic acid polymer. Subsequently, the resulting nanostructure containing the nucleic acid is converted to a neutral LNP when dialyzed against a neutral buffer including removal of the organic solvent (e.g., ethanol) during exchange of the LNP into a physiologically relevant buffer. The LNP and / or the LNP composition thus formed has a distinct high electron density nanostructured core organized into inverse micelles around the payload encapsulated with the cationic lipid, in contrast to the traditional bilayer liposome structure. In another embodiment, the LNP can form a bleb-like structure having the nucleic acid within an aqueous pocket along a non-electron density lipid core.

[0282] c. Lipid nanoparticle properties In some embodiments, the LNP and / or LNP composition comprises from about 21 mol% to about 85 mol% cationic lipid or ionizable cationic lipid, from about 8 to 65% helper lipid, from about 5 to 79% cholesterol, and from about 0.5 to 10% PEG lipid. In some embodiments, the LNP and / or LNP composition comprises from about 50 mol% to about 85 mol% cationic lipid or ionizable cationic lipid, from about 0.5 mol% to about 5 mol% conjugate lipid (e.g., pegylated lipid), from about 0.5 mol% to about 5 mol% steroid (e.g., cholesterol), and from about 5 mol% to about 20 mol% helper lipid (e.g., phospholipid).

[0283] In some embodiments, the LNP and / or LNP composition of the present disclosure comprises cationic lipid:helper lipid (e.g., phospholipid):steroid (e.g., cholesterol):conjugate lipid (e.g., pegylated lipid) in a molar ratio of 20-50:10-30:30-60:0.5-5, in a molar ratio of 25-45:10-25:40-50:0.5-3, in a molar ratio of 25-45:10-20:40-55:0.5-3, or in a molar ratio of 25-45:10-20:40-55:1.0-1.5.

[0284] In some embodiments, the LNP and / or LNP composition of the present disclosure has a total lipid:payload ratio (mass / mass) of from about 1 to about 100. In some embodiments, the total lipid:payload ratio is from about 1 to about 50, from about 2 to about 25, from about 3 to about 20, from about 4 to about 15, or from about 5 to about 10. In some embodiments, the total lipid:payload ratio is from about 5 to about 15, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or any intermediate range among the foregoing.

[0285] In certain embodiments, the LNPs of the present disclosure comprise a total lipid:nucleic acid mass ratio of from about 5:1 to about 15:1. In some embodiments, the weight ratio of the cationic lipid to the nucleic acid contained in the LNP can be from 1 to 20:1, 1 to...

Claims

1. An engineered ribonucleic acid scaffold (ERS) comprising the sequence of sequence number 17, or a sequence having at least approximately 70% sequence identity thereto, comprising the elongated stem-loop sequence of sequence number 49739 and one or more mutations at positions selected from the group consisting of U11, U24, A29, and A87.

2. The ERS according to claim 1, comprising mutations consisting of U11C, U24C, A29C, and A87G.

3. The ERS according to claim 1, comprising the sequence ACUGGCGCUUCUUCUAUCUGAUUACUCUGAGACAUCAACCAGCGGAACUAUGUUCGUAGUGGGUAAAAGCUCCCUCUUCUUCGGGAGGGAGCCAUCAGAG (Sequence ID 156), or a sequence having at least about 96% sequence identity therewith.

4. The ERS according to claim 3, comprising the sequence of sequence number 156.

5. The ERS according to claim 1, wherein the ERS comprises at least one improved feature compared to the sequence of Sequence ID No. 17, the improved feature being selected from the group consisting of improved binding to CasX nuclease for ribonucleoprotein (RNP) formation, improved folding stability of the ERS, increased intracellular half-life, improved transcription efficiency, enhanced ability to synthetically produce the ERS, improved editing activity of target nucleic acids by the RNP containing the ERS, and improved editing specificity by the RNP containing the ERS.

6. The ERS according to claim 1, comprising a targeting sequence ligated to the 3' end of the ERS, which is complementary to the target nucleic acid sequence.

7. The ERS according to claim 6, wherein the targeting sequence has 15 to 20 nucleotides.

8. The ERS according to claim 7, wherein the targeting sequence has 20 nucleotides.

9. The ERS according to claim 1, wherein the CpG content of the ERS is reduced or depleted, and the CpG content is less than about 10%, less than about 5%, or less than about 1%.

10. The ERS according to claim 1, wherein the ERS comprises one or more chemical modifications to the sequence.

11. The ERS according to claim 10, wherein the chemical modification is the addition of a 2'O-methyl group to one or more nucleotides of the sequence.

12. The ERS according to claim 11, wherein one or more nucleotides at either the 5' or 3' end, or both, of the ERS are modified by the addition of a 2'O-methyl group.

13. The ERS according to claim 10, wherein the chemical modification is the substitution of phosphorothioate bonds between two or more nucleosides of the sequence.

14. The ERS according to claim 13, wherein the chemical modification is the substitution of phosphorothioate bonds between two or more nucleotides at one or both of the 5' and 3' ends of the ERS.

15. The ERS according to claim 10, wherein the chemically modified ERS includes a sequence selected from the group consisting of SEQ ID NOs: 49769 to 49777.

16. The ERS according to claim 15, wherein the chemically modified ERS comprises the sequence of sequence number 49769.

17. The ERS according to claim 15, wherein the chemically modified ERS sequence is modified with a 20-nucleotide targeting sequence complementary to the target nucleic acid.

18. A gene editing pair comprising the ERS described in Claim 1 and an engineered CasX protein.

19. The gene editing pair according to claim 18, wherein the manipulated CasX protein comprises a sequence having at least two mutations in the sequence of CasX 515 (SEQ ID NO: 49699), the mutations resulting in improved characteristics compared to unmodified CasX 515, as determined by an in vitro assay under equivalent conditions.

20. The gene editing pair according to claim 18, wherein the manipulated CasX protein and the RNP of ERS exhibit at least one improved feature compared to the RNP comprising the sequence of SEQ ID NO: 156 and the sequence of SEQ ID NO:

228.

21. A nucleic acid comprising a sequence encoding the ERS described in claim 1.

22. a. The ERS according to claim 1, and b. Nucleic acids encoding the manipulated CasX protein A vector that includes, The vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, CasX delivery particles (XDPs), plasmids, minicircles, nanoplasmids, DNA vectors, and RNA vectors.

23. a. The ERS according to claim 1, and b. Nucleic acids encoding the manipulated CasX protein Lipid nanoparticles (LNPs) containing these.

24. A method for modifying a target nucleic acid in a cell, wherein the modification of the cell occurs in vitro or ex vivo, and the method involves the cell, a. The ERS according to claim 1, and b. Nucleic acids encoding the manipulated CasX protein This includes introducing A method comprising modifying the target nucleic acid of the cells targeted by the ERS with the manipulated CasX.

25. A composition comprising the ERS described in claim 1.

26. A pharmaceutical composition comprising the composition according to claim 25 and a pharmaceutically acceptable excipient.

27. A kit comprising the pharmaceutical composition described in claim 26 and a suitable container.

28. Use of the pharmaceutical composition according to claim 26 for the manufacture of a pharmaceutical for the treatment of a disease.