WO2023240027A1 - Particle delivery systems - Google Patents

Abstract

Provided herein is a particle delivery system (PDS) comprising components selected from: (a) a fusion protein comprising one or more Mononegavirales structural proteins and a heterologous protein; (b) one or more therapeutic payloads; and (c) a tropism factor. The Mononegavirales structural protein can a matrix protein (MA), a nucleocapsid protein (NC), or is both MA and NC. Also provided herein are PDS particles, and methods of making and using same, for example for the delivery of gene editing systems to cells.

Description

PARTICLE DELIVERY SYSTEMS

CROSS-REFERENCE TO RELATED APPLICATIONS plOOt | This application claims priority to, and benefit of, U.S. Provisional Application No. 63/349,999, filed on June 7, 2022, the contents of which are incorporated by reference in their entirety herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SCRB_040_01WO_SeqList_ST26.xml; Size: 14,344,628 bytes; and Date of Creation: May 30, 2023) are herein incorporated by reference in their entirety.

BACKGROUND

100031 The delivery of protein or nucleic acid therapeutics to particular cells or organs of the body generally requires complex systems in which a targeting modality or vehicle is linked to or contains the therapeutic protein or nucleic acid. Even with highly selective targeting modalities, such as monoclonal antibodies, the selectivity of the system for the target cells, tissues, or organs is not absolute, and off-target toxicity can result. The delivery of multicomponent payloads, such as CRISPR gene editing systems, presents additional challenges.

10004] Mononegavirales is an order of negative-strand RNA viruses that encompasses several families that cause chronic and deadly diseases. The order includes eleven virus families: Artoviridae, Bornaviridae, Filoviridae, Lispiviridae, Mymonaviridae, Nyamiviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae. Sunviridae, andXinmoviridae. Well- known members of the order include Ebola virus, human respiratory syncytial virus, measles virus, mumps virus, Nipah virus, and rabies virus. The virions contain one molecule of negative sense single-stranded ribonucleic acid (ssRNA) as a linear sequence coding for five to ten genes in a conserved order. The RNA is not infectious until packaged by the nucleocapsid protein and transcribed by the polymerase and co-factors genes (Pfaller, C.K., et al. Reverse genetics of Mononegavirales: How they work, new vaccines, and new cancer therapeutics. Virology 479:331 (2015)). In the families Filoviridae, Paramyxoviridae, and Rhabdoviridae, the site of multiplication is the cytoplasm, with no involvement of the host cell nucleus, with the exception of viruses classified in the genus Nucleorhabdovirus. The ribonucleoprotein (RNP) is the functional template for replication and transcription, while the matrix (MA) protein organizes the assembly of these genomic RNPs and incorporation of the holo-nucleocapsid and initiation of the budding process (Takimoto T, Portner A. Molecular mechanism of paramyxovirus budding. Virus Res. 106: 133 (2004)). In the Paramyxoviridae viruses, all MA proteins are known to be strongly membrane-associated and have similar hydropathy profiles. The simplicity of the structural organization of the

Mononegavirales offers the possibility for their exploitation as delivery vectors for therapeutic payloads, including gene editing systems.

SUMMARY p)005| The present disclosure provides particle delivers' systems (PDS) having utility for the delivery of therapeutic payloads, including proteins, nucleic acids, small molecules, or combinations thereof, to target cells and tissues.

[0006] In some embodiments, the disclosure provides PDS comprising structural proteins selected I Tom Mononegavirales viruses, a therapeutic payload, and one or more tropism factors wherein the tropism factor (located on the surface of the particle) is selected from a glycoprotein, an antibody fragment, a receptor, a ligand to a target cell marker, or combinations thereof. The structural proteins can be fused to one or more heterologous proteins, such as one or more non-co valent recruitment (NCR) proteins and/or a therapeutic protein. The therapeutic payload can be a protein, a nucleic acid, or both a protein and a nucleic acid. In some embodiments of the PDS, the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNase), a deoxyribonuclease (DNAse), a blood clotting factor, an anticoagulant, granulocyte-macrophage colony-stimulating factor (GMCSF), a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, a transcription factor, a transposon, reverse transcriptase, viral interferon antagonists, a tick protein, and an anti-cancer modality. In some embodiments, the therapeutic pay load is a Class 1 or Class 2 CRISPR protein. In some embodiments, the Class 2 CRISPR protein selected from the group consisting of a Type II, Type V, or Type AT protein. In some embodiments, the Class 2 CRISPR Type V protein is selected from the group consisting of Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Cas 12d (CasY), Casl2e (CasX), Casl2f, Cas 12g, Casl2h, Casl2i, Casl2j, Cas 12k, Cast 4, and Cas®. In some embodiments, the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASO), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, an RNA aptamer, and a CRISPR grade ribonucleic acid (gRNA), or any combination thereof. In some embodiments the CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence. In some embodiments, the therapeutic payload comprises a ribonucleoprotein (RNP) of a CRISPR protein complexed with a gRNA. In a particular embodiment, the therapeutic payload comprises a CasX variant and a guide RNA variant complexed as an RNP; optionally, a donor template is also encapsidated in the PDS.

[0007] In another aspect, the present disclosure provides nucleic acids encoding the components of the PDS, as well as vectors and plasmids comprising the nucleic acids. In some embodiments, the components of the PDS system are encoded on two nucleic acids, on three nucleic acids, on four nucleic acids, or on five nucleic acids.

[0008] In other aspects, the present disclosure provides methods of making a PDS particle comprising a therapeutic pay load. In some embodiments, the method comprises propagating a packaging host cell transfected wi th the encoding vectors of any of the embodiments described herein under conditions such that PDS particles (PDSs) are produced, and harvesting the PDSs produced by the packaging host cell.

[0009] The present disclosure further provides PDS particles produced by the foregoing methods,

[0010] In other aspects, the present disclosure provides methods of modify ing a target nucleic acid sequence in a cell, the methods comprising contacting the cell with a plurality of PDS particles comprising an RNP of any of the embodiments disclosed herein, wherein said contacting comprises introducing into the cell the RNP comprising the CRISPR Class 2 nuclease protein, the guide RNA comprising a targeting sequence capable of binding the target nucleic acid, and, optionally, the donor template nucleic acid sequence, resulting in modification of the target nucleic acid sequence. In one embodiment, the cell is modified in vitro or ex vivo. In another embodiment, the cell is modified in vivo. In the foregoing embodiment, the PDS particles are administered to a subject at a therapeutically effective dose, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

[0011 ] In another aspect, provided herein are PDS particle compositions. In some embodiments, the PDS compositions are for use in the manufacture of a medicament for the treatment of a subject having a disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present 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 of which:

[0013] FIG. 1 is a flow-chart illustrating the qualitative relationship between tested combinations of mutations and their effect on both activity and specificity of the resulting CasX variants.

[0014] FIG. 2 is a schematic of the RNA secondary structure of the Rev Response Element (RRE) with stem II circled and stem IIB boxed (folds predicted using Varna software), as described in Example 2. The sequence shown in FIG. 2 is SEQ ID NO: 7960.

[0015] FIG. 3 shows the results of editing assays (percentage of cells with tdTomato fluorescence) in tdTomato NPCs treated with Mononegavirales-bs&Q^ PDS versions 96 or 170-185, as described in Example I. Results are shown as percent editing for the volume of particles (50 pL) used for treatment. XDP VI 68 was used as the experimental control (a virus-like particle derived from lentiviral-based HIV harboring a Gag-CasX fusion configuration).

[0016] FIG. 4 shows the level of interferon expression in ARPE-19 cells treated with a recombinant lentiviral particle (LV), recombinant AAV particles (AAV2, AAV8, and AAV9), version 206 XDPs, version 329 PDS particles, or version 329 PDS particles with VP35 protein compared to an untreated control, as described in Example 9.

[0017] FIG. 5 shows the level of interferon expression in Jurkat cells treated with a recombinant lentiviral particle (LV), recombinant AAV particles (AAV2, AAV8, and AAV9), version 206 XDPs, version 329 PDS particles, or version 329 PDS particles with VPS 5 protein compared to an untreated control, as described in Example 9.

[0018] FIG. 6 shows the level of interferon expression in K562 cells treated with a recombinant lentiviral particle (LV), recombinant AAV particles (AAV2, AAV 8, and AAV9), version 206 XDPs, version 329 PDS particles, or version 329 PDS particles with VP35 protein compared to an untreated control, as described in Example 9.

[0019] FIG. 7 illustrates the schematics of five configurations of fusion proteins with repressor molecules linked to catalytically-dead CasX ( “dXR’ '). D3A and D3L denote DNA methyltransferase 3 alpha (DNMT3A) and DNMT3A-like protein (DNMT3L), respectively.

L1-L4 are linkers. NLS is the nuclear localization signal.

DETAILED DESCRIPTION

[0020] While preferred embodiments of the present invention have been sho w n and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0021] 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 the present embodiments, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

DEFINITIONS

[0022] The terms “'polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass singlestranded DNA; double-stranded DNA; multi -stranded DNA; single-stranded RNA; doublestranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0023] “Hybridizable” or “complementary⁷” are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalentiy bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary' nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity' and still hybridize to the target nucleic acid. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a 'bulge', ‘bubble’ and the like).

[0024 ] A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (e.g., a protein, RNA), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene may include accessory' element sequences including, but not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary' elements, replication origins, matrix attachment sites and locus control regions. Coding sequences encode a gene product upon transcription or transcription and translation; the coding sequences of the disclosure may comprise fragments and need not contain a full-length open reading frame. A gene can include both the strand that is transcribed as well as the complementary' strand containing the anticodons.

10025 j The term "downstream" refers to a nucleotide sequence that is located 3’ to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[00261 The term "upstream" refers to a nucleotide sequence that is located 5’ to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0027] The term “adjacent to” with respect to polynucleotide or amino acid sequences refers to sequences that are next to, or adjoining each other in a polynucleotide or polypeptide. The skilled artisan will appreciate that two sequences can be considered to be adjacent to each other and still encompass a limited amount of intervening sequence, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or ammo acids.

[0028] The term “regulatory’ element” is used interchangeably’ herein with the term “regulator)-’ sequence,” and is intended to include promoters, enhancers, and other expression regulatory elements. It will be understood that the choice of the appropriate regulatory element will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0029] The term “accessory' element” is used interchangeably herein with the term “accessory sequence,” and is intended to include, inter alia, polyadenylation signals (poly(A) signal), enhancer elements, introns, posttranscnptional regulatory' elements (PTREs), nuclear localization signals (NTS), deaminases, DNA glycosylase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g. in cis or in trans), activators or repressors of transcription, self-cleaving sequences, and fusion domains, for example a fusion domain fused to a CRISPR protein. It will be understood that the choice of the appropriate accessory-- element or elements will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein. 100301 The term "promoter" refers to a DNA sequence that contains a transcription start site and additional sequences to facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as a TATA box, and/or B recognition element (BRE) and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties. A promoter of the present disclosure can include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter can be classified according to criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. A promoter can also be classified according to its strength. As used in the context of a promoter, “strength” refers to the rate of transcription of the gene controlled by the promoter. A “strong” promoter means the rate of transcription is high, while a “weak” promoter means the rate of transcription is relatively low.

[0031] A promoter of the disclosure can be a Polymerase II (Pol II) promoter. Polymerase II transcribes all protein coding and many non-coding genes. A representative Pol II promoter includes a core promoter, which is a sequence of about 100 base pairs surrounding the transcription start site, and serves as a binding platform for the Pol II polymerase and associated general transcription factors. The promoter may contain one or more core promoter elements such as the TATA box, BRE, Initiator (INR), motif ten element (MTE), downstream core promoter element (DPE), downstream core element (DCE), although core promoters lacking these elements are known in the art. All Pol II promoters are envisaged as within the scope of the instant disclosure.

[0032. | A promoter of the disclosure can be a Polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs such as the 5S rRNA, tRNAs, and other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed section of the gene) to support transcription, although upstream elements such as the TATA box are also sometimes used. All Pol III promoters are envisaged as within the scope of the instant disclosure.

[0033] The term “enhancer’ refers to regulatory' DNA sequences that, when bound by specific proteins called transcription factors, regulate the expression of an associated gene. Enhancers may be located in the intron of the gene, or 5’ or 3’ of the coding sequence of the gene. Enhancers may be proximal to the gene (i.e., within a few tens or hundreds ofbase pairs (bp) of the promoter), or may be located distal to the gene (i.e., thousands of bp, hundreds of thousands of bp, or even millions of bp away from the promoter). A single gene may be regulated by more than one enhancer, all of which are envisaged as within the scope of the instant disclosure.

[0034] As used herein, a "post-transcriptional regulatory element (PRE, or PTRE)," such as a hepatitis PRE, refers to a DNA sequence that, when transcribed creates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote expression of an associated gene operably linked thereto.

[0035] “Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see ‘"enhancers” and ’‘promoters”, above).

100361 The term ‘‘recombinant polynucleotide” or “recombinant nucleic acid” refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0037] Similarly, the term “recombinant polypeptide” or “recombinant protein” refers to a polypeptide or protein which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a protein that comprises a heterologous amino acid sequence is recombinant.

[0038] The term “Rev response element” or “RRE” refers to a cis-acting post- transcriptional accessory element that facilitates, in the context of the present disclosure, the transport of a gRNA from the nucleus, across the nuclear membrane, to the cytoplasm of a cell by complexing with factors such as HIV-1 Rev.

[0039] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a target nucleic acid with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid are made to share a physical connection; e.g,, can hybridize if the sequences share sequence similarity.

[0040] “Dissociation constant”, or “Kd”, are used interchangeably and mean the affinity between a ligand “L” and a protein “P”; i.e., how tightly a ligand binds to a particular protein. It can be calculated using the formula Kd= =[ L ] [P|/| LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively.

[0041] The disclosure provides compositions and methods useful for modifying a target nucleic acid. As used herein “editing” is used interchangeably with '‘modify ing” and "modification" and includes but is not limited to cleaving, nicking, editing, deleting, knocking in, knocking out, and the like. Modifying can also encompass epigenetic modifications to a nucleic acid, or chromatin containing the nucleic acid, such as, but not limited to, changes in DNA methylation, and histone methylation and acetylation.

[0042] By “cleavage” it is meant the breakage of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodi ester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and doublestranded cleavage can occur as a result of two distinct single-stranded cleavage events, [0043] The term "knock-out" refers to the elimination of a gene or the expression of a gene. For example, a gene can be knocked out by either a deletion or an addition of a nucleotide sequence that leads to a disruption of the reading frame. As another example, a gene may be knocked out by replacing a part of the gene with an irrelevant sequence. The term "knockdown" as used herein refers to reduction in the expression of a gene or its gene product(s). As a result of a gene knock-down, the protein activity or function may be attenuated or the protein levels may be reduced or eliminated.

[0044] As used herein, "homology-directed repair" (HDR) refers to the form of DNA repair that takes place during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, and uses a donor template to repair or knock-out a target DNA, and leads to the transfer of genetic information from the donor to the target.

Homology-directed repair can result in an alteration of the sequence of the target sequence by insertion, deletion, or mutation if the donor template differs from the target DNA sequence and part or all of the sequence of the donor template is incorporated into the target DNA. [0045] As used herein, "non-homologous end joining" (NHEJ) refers to the repair of double-strand breaks in DNA by direct ligation of the break ends to one another without the need for a homologous template (in contrast, to homology -directed repair, which requires a homologous sequence to guide repair). NHEJ often results in the loss (deletion) of nucleotide sequence near the site of the double-stranded break. [0O46| As used herein "nncro-homoiogy mediated end joining" (MMEJ) refers to a mutagenic DSB repair mechanism, which always associates with deletions flanking the break sites without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). MMEJ often results in the loss (deletion) of nucleotide sequence near the site of the double- strand break.

[0047 j A polynucleotide or polypeptide has a certain percent "sequence similarity" or "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity (sometimes referred to as percent similarity, percent identity, or homolog)') can be determined in a number of different manners. To determine sequence similarity', sequences can be aligned using the methods and computer programs that are known in the art, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol, 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0048] The terms “polypeptide,” and “protein” are used interchangeably herein, and refer to a poly meric form of ammo acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous ammo acid sequence.

[0049 ] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, re., an “insert”, may be attached so as to bring about the replication or expression of the attached segment in a cell.

10050] The term “naturally-occurring” or “unmodified” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. |0051] As used herein, a “mutation” refers to an insertion, deietion, substitution, duplication, or inversion of one or more ammo acids or nucleotides as compared to a wildtype or reference amino acid sequence or to a wild-type or reference nucleotide sequence. [0052] As used herein the term “isolated” is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

10053] A “host cell,” as used herein, denotes a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells are used as recipients for a nucleic acid (e.g., an expression vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology' or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “’genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector.

[0054] The term “tropism” as used herein refers to preferential entry' of the PDS into certain cell, organ or tissue type(s) and/or preferential interaction with the cell surface that facilitates entry into certain cell, organ or tissue types, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the PDS into the cell.

10055] The terms “pseudotype” or “pseudotyping” as used herein, refers to viral envelope proteins that have been substituted with those of another virus possessing preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G- protein (VSV-G) envelope proteins (amongst others, described herein, below'), winch allows HIV to infect a wider range of cells because HIV envelope proteins target the virus mainly to CD4+ presenting cells.

[0056] The term “tropism factor” as used herein refers to components integrated into the surface of an PDS that provides tropism for a certain cell, organ or tissue type. Non-limiting examples of tropism factors include glycoproteins, antibody fragments (e.g., scFv, nanobodies, linear antibodies, etc.), receptors and ligands to target cell receptors or cell markers. [0057] As used herein "non-covalent recruitment protein" or "NCR" means a protein or peptide that has binding affinity for an RNA hairpin. Non-limiting examples of NCR include MS2 coat protein, PP7 coat protein, QP coat protein, protein N, protein Tat, phage GA coat protein, iron-responsive binding element (IRE) protein, or UI A signal recognition particle protein (Ul A) that have affinity to MS2 hairpin, PP7 hairpin, Qp hairpin, boxB, phage GA hairpin, phage AN hairpin, iron response element (IRE), transactivation response element (TAR), or Ul hairpin II, which, upon the interaction of the binding partner and the NCR, can facilitate the non-covalent recruitment and incorporation of the gRNA variant (and CasX variant that complexes to the gRN A) into the budding PDS in the packaging cell..

[0058 j As used herein, "cognate" means components derived from the same species, genus or variety of a virus, while "non-cognate" means components derived from different sources. [0059] A “target cell marker” refers to a molecule expressed by a target cell including but not limited to cell-surface receptors, cytokine receptors, antigens, tumor-associated antigens, glycoproteins, oligonucleotides, enzymatic substrates, antigenic determinants, or binding sites that may be present in the on the surface of a target tissue or cell that may serve as ligands for a tropism factor.

[0060] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the 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, a single domain antibody, a single domain camelid antibody, single-chain variable fragment (scFv) antibody molecules, and multispecific antibodies formed from antibody fragments.

[0061] The term “’conservative ammo acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of ammo acids having aliphatic-hydroxyl side chains consists of serine and threonine: a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of ammo acids having basic side chains consists of lysine, arginine, and histidine; and a group of ammo acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative ammo acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-argmine, alanine- valine, and asparagine-glutamine. [0062J 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 a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can also be achieved with the eradication or amelioration of one or more of the symptoms or an improvement in one or more clinical parameters associated with the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

[0063] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a biologic, alone or as a part of a composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject such as a human or an experimental animal. Such effect need not be absolute to be beneficial.

10064] As used herein, “administering” means a method of giving a dosage of a compound (e.g., a composition of the disclosure) or a composition (e.g., a pharmaceutical composition) to a subject.

[0065] A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats and other rodents.

[0066] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The contents of WO 2020/247882, filed on June 5, 2020, WO 2020/247883, filed June 5, 2020, WO 2021/050593, filed on September 9, 2020, WO 2021/050601, filed on September 9, 2021, WO 2021/142342, filed on January 8, 2021, WO 2021/113763, filed on December 4, 2020, WO 2021/1 13769, filed on December 4, 2020, WO 2021/1 13772, filed on December 4, 2020, WO 2022/120095, filed December 2, 2021, WO 2022/120094, filed on December 2, 2021, WO 2022/261150, filed on June 7, 2022, WO 2023/049742, filed on September 21, 2022, WO 2022/261149, filed on June 7, 2022, and PCT/US2023/067791, filed on June 1, 2023, which disclose CasX variants and gRNA variants, are hereby incorporated by reference in their entirety.

[0067] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

I. General Methods

[0068] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry', chemistry', molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring 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.

[0069] Where a range of values is provided, it is understood that endpoints are included and that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently' be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0070] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly' understood by' one of ordinary' skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0071] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. [0072] It will be appreciated that certain features of the disclosure, which are, for clarity’, described in the context of separate embodiments, may also be provided in combination in a single embodiment. In other cases, various features of the disclosure, which are, for brevity', described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is intended that all combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

II. Particle Delivery Systems for Lise in Targeting Cells

[0073] In a first aspect, the present disclosure relates to particle delivery systems (PDS) designed to self-assemble particles comprising therapeutic payloads, wherein the particles are designed for selective delivery to targeted cells and tissues. Particles produced by the PDS disclosed herein are referred to as PDS particles, which encompass anon-replicating, selfassembling, non-naturally occurring multicomponent structure composed of one or more viral proteins, such as, but not limited to, the nucleocapsid (NC) protein and/or the capsid MA protein of aMononegavirales virus, as well as tropism factors incorporated on the surface of the PDS particle such as envelope glycoproteins derived from viruses or non-viral tropism factors such as antibody fragments, receptors or ligand utilized for tropism to direct the PDS particle to target cells, organ or tissues, with an outer lipid layer (derived from the host cell), wherein the PDS particles are capable of self-assembly in a packaging host cell and encapsidating or encompassing a therapeutic payload and incorporating the tropism factor on the surface the PDS particle upon its release from the cell. Upon release from the packaging host cell, the PDS particle can then be recovered and utilized in the methods of the disclosure. [0074] The PDS of the present disclosure can be created in multiple forms and configurations of PDS particles. These alternative configurations are described more fully, belotv, as well as in the Examples. In some embodiments of the PDS of the disclosure, the therapeutic pay load is multiple particles of RNP of a complexed CRISPR nuclease protein and a gRNA, while the tropism factor is a viral glycoprotein embodiment described herein. In a particular embodiment of the PDS of the disclosure, the therapeutic payload is multiple particles of RNP of a complexed CasX and gRN A embodiment described herein, while the tropism factor is a viral glycoprotein embodiment described herein.

[0075] The PDS particles of present disclosure can be utilized to specifically and selectively deliver therapeutic payloads to target cells, organ or tissues in a subject. The PDS particles of the disclosure have utility in a variety of methods, including, but not limited to, use in delivering a therapeutic in a selective fashion to a target cell, tissue or organ for the treatment of a disease or disorder.

[0076] In some embodiments, the present disclosure provides PDS comprising one or more nucleic acids comprising sequences encoding the viral components of the PDS particle, the therapeutic payload, and tropism factors that, that, when introduced into an appropriate eukaryotic packaging host cell, result in the expression of the individual PDS structural components, therapeutic payloads, and tropism factors that self-assemble into PDS particles that encapsidate the therapeutic payload and incorporate the tropism factor within the membrane envelope upon budding from the packaging host cell. Upon release from the packaging host cell, the PDS particles can be collected and purified for the methods and uses described herein.

[0077] In some embodiments, the therapeutic payloads packaged within the PDS particle comprise therapeutic proteins, described more fully below; In other embodiments, the therapeutic payloads packaged within PDS particle comprise therapeutic nucleic acids or nucleic acids that encode therapeutic proteins. In still other embodiments, the PDS particle comprises both therapeutic proteins and nucleic acids. In some cases, the therapeutic payloads include gene editing systems such as CRISPR nucleases and guide RNA or zinc finger proteins useful for the editing of nucleic acids in target cells. In some embodiments, the therapeutic payloads include Class 2 CRISPR systems. Class 2 systems are distinguished from Class 1 systems in that they have a single multi-domain effector protein and are further divided into a Type II, Type V, or Type VI system, described in Makarova, et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nature Rev. Microbiol. 18:67 (2020), incorporated herein by reference. In some embodiments, the nucleases include Class 2, Type II CRISPR/Cas effector polypeptides such as Cas9. In other cases, the nucleases include Class 2, Type V CRISPR/Cas effector polypeptides such as a Cast 2a (Cpfl ), Cast 2b (C2cl ), Cas t 2c (C2c3), Cas t 2d (CasY), Casl2e (CasX), Casl2f, Cast 2g, Casl2h, Casl2i, Casl2j, Casl2k, Cast 4, and/or Cas<I>. The CRISPR nuclease and guide RN A system payloads can do one or more of the following: (i) modify (e.g., edit) a target ssDNA, dsDNA or RNA (e.g., cleave, nick, or methylate); (ii) modulate transcription of the target nucleic acid; (hi) bind the target nucleic acid (e.g., for purposes of isolation, blocking transcription, labeling, or imaging, etc,); or (v) modify a polypeptide associated with a target nucleic acid. In a particular embodiment, the present disclosure provides PDS compositions, and methods to make the PDS compositions, methods and compositions designed to more effectively package ribonucleic acid particles (RNP) comprising CasX and guide RNA systems (CasX:gRNA system) useful for the editing of nucleic acids in target cells, described more fully, below. Accordingly, the present disclosure pro vides PDS compositions, nucleic acids that encode the components of the PDS (both structural as well as gene-editing components), as well as methods of making and using the PDS. 'The nucleic acids, the components of the compositions, and the methods of making and using them, are described herein, below. a. Mononegavirales Components

[00781 PDS, and the particles produced by the PDS, can be created in multiple forms and configurations utilizing components derived fromMononegavirales viruses.

100791 The structural components of the PDS of the present disclosure are derived from members of the Mononegavirales family of viruses. Mononegavirales is an order of negativestrand RNA viruses. The order includes eleven virus families: Arloviridae, Bornaviridae, Filoviridae, Lispiviridae, Mymonaviridae, Nyamiviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Sunviridae, and Xinmoviridae. Exemplary members of the order include Ebola virus, human respiratory syncytial virus, measles virus, mumps virus, Nipah virus, and rabies virus that result in human disease. In the life-cycle of these viruses, virion attachment is to specific cell-surface receptors, followed by fusion with the cellular membranes and the concomitant release of the virus nucleocapsid into the cytosol. The virus partially uncoats the nucleocapsid and transcribes the genes into positive-stranded mRNAs, which are then translated into structural and nonstructural proteins. Replication results in full-length, positive-stranded antigenomes that are in turn transcribed into negative-stranded vims progeny genome copies. Newly synthesized structural proteins and genomes self-assemble and accumulate near the inside of the cell membrane, which bud off from the cell, gaining their envelopes from the cellular membrane they bud from. The mature progeny particles then infect other cells to repeat the cycle.

[0080] The major structural component of the Mononegavirales is the matrix protein (MA). In the case of several members of the order, formation of the viral particles is driven by the viral MA proteins, which can self-assemble to form ordered arrays. Without wishing to be bound by theory, it is likely that these arrays play key roles in generating the membrane curvature required for budding. For many paramyxoviruses, expression of MA protein in the absence of any other viral components is sufficient to induce the assembly and release of virus-like particles (VLPs) from transfected cells. MA proteins of Sendai virus, measles virus, Nipah virus, Hendra virus, Newcastle disease virus, and human parainfluenza virus 1 are all capable of directing PDS production and release from transfected cells when expressed alone. In these cases, additional viral components including the viral glycoproteins and the nucleocapsid-like structures that form upon expression of paramyxovirus NZNP proteins can be efficiently packaged into the PDS if they are co-expressed along with the MA proteins (Harrison M S, Sakaguchi T, Schmitt A P. 2010. Paramyxovirus assembly and budding: building particles that transmit infections. Int. J. Biochem. Cell. Biol. 42:1416-1429.) 100811 It has been discovered in the context of the present disclosure that incorporation of the sequence encoding the MA protein from each of the orders of the Mononegavirales into encoding sequences for the PDS systems of the embodiments disclosed herein is sufficient for self-assembly of the PDS particle, as it buds from the packaging host cell transfected with the encoding sequences and encapsidates the therapeutic payloads. In some embodiments, a sequence encoding a nucleocapsid from the same or different species is included for the formation of the particle.

10082. | It has been discovered that structural components of PDS can be derived from each of the genera of Mononegavirales , and that the resulting PDS particles are capable selfassembly in a host cell and encapsidating (or encompassing) therapeutic payloads that have utility in the targeted and selective delivery of the therapeutic pay loads to target cells and tissues. In some embodiments, the present disclosure provides PDS comprising one or more structural components derived from a Mononegavirales virus, a therapeutic payload (described more fully, below), and one or more tropism factors (described more fully, below). In some embodiments, the virus structural components are derived from aArtoviridae vims. In some embodiments, the virus structural components are derived from aBornaviridae virus. In some embodiments, the virus structural components are derived from aFiloviridae virus. In some embodiments, the virus structural components are derived from a Lispiviridae virus. In some embodiments, the virus structural components are derived from aMymonaviridae virus. In some embodiments, the virus structural components are derived from a Nyamiviridae vinis. In some embodiments, the virus structural components are derived from a Paramyxovindae virus. In some embodiments, the virus structural components are derived from a Pneumoviridae virus. In some embodiments, the virus structural components are derived from a Rhabdoviridae vims. In some embodiments, the virus structural components are derived from a Sunviridae virus. In some embodiments, the vims structural components are derived from aXinmoviridae virus. In some embodiments, the virus structural components are derived from a Mononegavirales viral genus selected from the group consisting of Carbovirus , Orthobornavirus, Ebolavirus, Berhavirus , Cruslavirus, Formica fusca virus 1, Nyavirus, Orinovirus, Socyvirus, Tapwovirus, Metaavulavirus , Orthoavulavirus, Paraavulavirus , Aquaparamyxovirus , Ferlavirus, Henipavirus, Jeilongvirus,Morbillivirus, Narmovirus, Respirovirus , Orihorubulavirus, Pararubulavirus , Cj.’Hog/ossM.mrws, Hoplichthysvirus, Metapneumovirus, Orthopneumovirus, Almendravirus , Arurhavirus, Barhavirus, Caligrhavirus, Curiovirus, Ephemerovirus , Hapavirus, Ledantevirus, Lyssavirus, Ohlsrhavirus, Perhabdovirus , Sawgrhavirus, Sigmavirus , Sripuvirus, Sunrhavirus, Tibrovirus, Tupavirus, and Vesiculovirus .

[0083] In some embodiments, the MA protein utilized in the PDS of the present disclosure is selected from the group consisting of the sequences of SEQ ID NOS: 1039-1252 as set forth in Table 15 (of Example 2), or a sequence having at least about 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 to a sequence as set forth in Table 15. In some embodiments, the MA protein of the PDS of the present disclosure is selected from the group consisting of the sequences of SEQ ID NOS: 1039-1252 as set forth in Table 15, or a sequence having at least about 85%, at least about 90^i!... 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. In the foregoing embodiments, a PDS comprising the MA protein can retain the ability to self-assemble the PDS components into a PDS particle in a suitable packaging cell.

[00841 hr some embodiments, the disclosure further contemplates incorporation of a nucleocapsid (NC) protein in the PDS. In some embodiments, the NC protein incorporated into the PDS is derived from the same Mononegavirales virus utilized for the MA protein incorporated into the PDS (a "cognate NC"). In other embodiments, the NC protein incorporated into the PDS is derived from a different Mononegavirales virus from that utilized for the MA protein incorporated into the PDS (a "non-cognate NC"). It has been discovered that the incorporation of the NC protein can confer an altered shape on the PDS particle compared to an otherwise equivalent PDS particle in which only a MA protein is used for the structural component. It is contemplated that by the design of the PDS incorporating such NC protein, the size and shape of the resulting PDS particle can effect a selective distribution of the PDS when administered to a subject. In some embodiments, the NC protein utilized in the PDS of the present disclosure is selected from the group consisting of the sequences of SEQ ID NOS: 1597- 1810 as set forth in Table 23 (of Example 6), or a sequence having at least about 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 to a sequence of SEQ ID NOS: 1597- 1810 as set forth in as set forth in Table 23. In some embodiments, the MA protein of the PDS of the present disclosure is selected from the group consisting of the sequences of SEQ ID NOS: 825-1038 as set forth in Table 15. In the foregoing embodiments, a PDS comprising the NC protein can retain the ability to self-assemble the PDS components into a PDS particle in a suitable packaging cell.

10085 j Representative encoding sequences for the viral structural components and the methods to create the encoding plasmids and produce the PDS in host cells are described herein, below.

HI. Protein and Nucleic Acid Therapeutic Payloads of the PDS

[0086] Protein therapeuti c payloads suitable for inclusion in the PDS of the present disclosure include a diversity of categories of protein-based therapeutics, including, but not limited to cytokines (e.g., IFNs a, [3, and y, TNF-a, G-CSF, GM-CSF)), interleukins (e.g., IL- 1 to IL-40), growth factors (e.g., VEGF, PDGF, IGF-1, EGF, and TGF-0), enzymes, receptors, microproteins, hormones (e.g., gro will hormone, insulin), ery thropoietin, RNase, DNase, blood clotting factors (e.g. FVII, FVIII, FIX, FX), anticoagulants, bone morphogenetic proteins, engineered protein scaffolds, thrombolytics (e.g., streptokinase, tissue plasminogen activator, plasminogen, and plasmid), CRISPR proteins (Class 2 Type II, Type V, or Type VI), transcription factors, including repressor factors (such as, but not limited to, Kruppel-associated box (KRAB), DNA methyltransferase 3 alpha (DNMT3A) and subdomains such as DNMT3A catalytic domain and ATRX-DNMT3-DNMT3L domain (ADD), DNMT3L interaction domain (DNMT3L), DNA methyl transferase 3 beta (DNMT3B), DNA methyltransferase 1 (DNMT1), Friend of GATA-1 (FOG), and Mad mSIN3 interaction domain (SID)), transposons, reverse transcriptases, viral interferon antagonists, tick proteins, as well as engineered proteins such as anti-cancer modalities or biologies intended to treat diseases such as neurologic, metabolic, cardiovascular, liver, renal, or endocrine diseases and disorders, or any combination of the foregoing. Nucleic acid pay loads suitable for inclusion in the PDS of the present disclosure include a diversity of categories, including sequences encoding the foregoing protein therapeutic payloads, as well as single-stranded antisense oligonucleotides (ASOs), double-stranded RNA interference (RNAi) molecules, DNA aptamers, RNA aptamers, nucleic acids utilized in gene therapy (e.g., guide RNAs utilized in CRISPR systems and donor templates), micro RNAs, ribozymes, RNA decoys, circular RNAs, or any combination of the foregoing. In some embodiments, the payload of the PDS comprises RNP of a CRISPR Class 2 nuclease and a gRNA. In a particular embodiment, the pay load of the PDS comprises a RNP of a CasX protein of any of the embodiments described herein, including the CasX variants as set forth in Table 3 and a guide RNA of any of the embodiments described herein, including the gRNA variants with a scaffold sequence as set forth in Table 8 and, optionally, a donor template. a CRISPR Proteins of the PDS

[0087] In some embodiments, the present disclosure provides PDS systems, particles and compositions comprising a CRISPR nuclease and one or more guide nucleic acids engineered to bind target nucleic acid that have utility in genome editing of eukaryotic cells.

[0088] The PDS particles, compositions, systems, and methods described in greater detail herein can be designed and adapted for use with Class 2 CRISPR systems. Thus, in some embodiments, the CRISPR system utilized in the PDS is a Class 2 CRISPR system. Class 2 systems are distinguished from Class 1 systems in that they have a single, large, multidomain effector protein. In certain embodiments, the Class 2 system utilized in the PDS can be a Type II, Type V, or Type VI system. Each type of Class 2 system is further divided into subtypes. Class 2, Type II systems can be divided into 4 subtypes: II-A, II-B, II-C1, and II- C2. Class 2, Type V systems can be divided into 17 subtypes: V-A, V-B 1, V-B2, V-C, V-D, V-E, V-FI, V-F1(V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V-U5), V-Ul, V-U2, and V-U4. Class 2, Type VI systems can be divided into 5 subtypes: VI-A, VI-B1, VI-B2, VI-C, and VI- D.

[0089] The nucleases of Type V systems differ from Type II effectors (e.g,, Cas9), which contain two nuclear domains that are each responsible for the cleavage of one strand of the target DNA, with the HNH nuclease inserted inside the Ruv-C like nuclease domain sequence. Although members of Class 2, Type V CRISPR systems have individual differences, they share some common characteristics that distinguish them from the Cas9 systems. Firstly, the Type V nucleases possess a single RNA-guided RuvC domaincontaining effector but no HNH domain, and they recognize T-rich PAM 5’ upstream to the target region on the non-targeted strand, which is different from Cas9 systems which rely on G-ncli PAM at 3 'side of target sequences. Type V nucleases generate staggered doublestranded breaks distal to the PAM sequence, unlike Cast), which generates a blunt end in the proximal site close to the PAM. In addition, Type V nucleases degrade ssDNA in trans when activated by target dsDNA or ssDNA binding in cis. In some embodiments, the Type V nucleases utilized herein recognize a 5’ TC PAM motif and produce staggered ends cleaved by the RuvC domain. The Type V systems (e.g., Casl2) only contain a RuvC-like nuclease domain that cleaves both strands. Type VI (Casl3) are unrelated to the effectors of Type II and V systems and contain two HEPN domains and target RNA.

[0090] In some embodiments, the Class 2 system utilized in the PDS is a Type II system. In some embodiments, the Class 2 system utilized in the PDS is a Type V system. In some embodiments, the Type V CRISPR system utilized in the PDS is selected from the group consisting of Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f, Cast 2g, Casl2h, Casl2i, Casl2j, Cast 2k, Casl 4, and Cas©. In some embodiments the Class 2 system utilized in the PDS is a Type VI system. In some embodiments, the Type VI CRISPR system utilized in the PDS is selected from Casl3a (C2c2), Cast 3b (Group 29/30), Casl3c, Cast 3d, Casl3e and/or Casl 3f.

[0091] In some embodiments, the present disclosure provides PDS and PDS particles comprising a ribonucleoprotein (RNP) of a complexed CRISPR protein and one or more grade ribonucleic acids (gRNA) that are specifically designed to modify a target nucleic acid sequence in eukaryotic cells. In a particular embodiment, the present disclosure provides PDS comprising a ribonucleoprotein (RNP) of a complexed CasX variant protein and a gRNA variant that are specifically designed to incorporate an increased number of RNPs into the PDS particles. In some embodiments, the PDS are configured to include one or more non- covalent recruitment proteins (NCR) having affinity to a cognate ligand in the gRNA so that the individual particles comprise at least about 100 RNP. at least about 200 RNP, at least about 300 RNP, at least about 400 RNP, at least about 500 RNP, at least about 600 RNP, at least about 700 RNP, at least about 800 RNP, at least about 900 RNP, or at least about 1000 RNP. In some embodiments, the PDS are configured so that the individual PDS particles comprise at least about 100 to about 1000 RNP, at least about 200 to about 800 RNP, or at least about 400 to about 600 RNP.

10092. | The term “CasX protein”, as used herein, refers to a family of proteins, and encompasses all naturally occurring CasX proteins (also referred to herein as a “wild-type” or “reference” CasX), as well as CasX variants with one or more modifications, such as amino acid substitutions, deletions, and insertions, in one or more domains relative to a naturally- occurring reference CasX protein.

[0093] The term “CasX variant” is inclusive of variants that are fusion proteins; i.e., the CasX is “fused to” a heterologous sequence. This includes CasX variants comprising CasX variant sequences and N-terminal, C -terminal, or internal fusions of the CasX to a heterologous protein or domain thereof. The term “CasX variant” is also inclusive of variants that are chimeric, i.e., contain domains, or portions of domains, derived from two or more different sources.

10094 j CasX proteins of the disclosure comprise the following domains: a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain (which is further divided into helical I-I and l-II subdomains), a helical II domain, an oligonucleotide binding domain (OBD, which is further divided into OBD-I and OBD-II subdomains), and a RuvC DNA cleavage domain (which is further divided into RuvC-I and II subdomains). The RuvC domain may be modified or deleted in a catalytically dead CasX variant, described more fully, below. The domain sequences, relative to reference CasX, are listed in Tables 1 and 2,

[0095] In some embodiments, a CasX protein can bind and/or modify (e.g., nick, catalyze a double strand break, methylate, demethylate, etc.) a target nucleic acid at a specific sequence targeted by an associated gRNA, which hybridizes to a sequence within the target nucleic acid sequence.

& Reference CasX Proteins

[0096] The disclosure provides naturally-occurring CasX proteins (referred to herein as a "reference CasX protein"), which were subsequently modified to create the CasX variants of the disclosure. For example, reference CasX proteins can be isolated from naturally occurring prokaryotes, such as Deltaproteobacteria, Planctomycetes , or Candidatus Sungbacteria species, A reference CasX protein (interchangeably referred to herein as a reference CasX polypeptide) is a type II CRISPR/Cas endonuclease belonging to the CasX (interchangeably referred to as Casl2e) family of proteins that interacts with a guide RNA to form a ribonucleoprotein (RNP) complex.

[0097] In some cases, a Type V reference CasX protein is isolated or derived from

Deltaproteobacteria having a sequence of:

1 MEKRINKIRK KLSADNATKP VSRSGPMKTL LVRVMTDDLK KRLEKRRKKP EVMPQVI SNN 61 AANNLRMLLD DYTKMKEAIL QVYWQEFKDD HVGLMCKEAQ PASKKIDQNK LKPEMDEKGN 121 LTTAGFACSQ CGQPLFVYKL EQVSEKGKAY TNYFGRCNVA EHEKLT LLAQ LKPEKDSDEA 181 VTYSLGKFGQ RALDFYSIHV TKESTHPVKP LAQZAGNRYA SGPVGKALSD ACMGTIASFL 241 SKYQDZ Z ZEH QK'A/KGNQKR LESLRELAGK ENLEYPSVTL PPQPHTKEGV DAYNEVZARV 301 RNR1VNLNLWQ KLKLSRDDAK PLLRLKGFPS FPWERRENE VDWWNTZNEV KKLZDAKRDM 361 GRVFWSGVTA EKRNTILEGY NYLPNENDHK KREGSLENPK KPAKRQ FGDL LLYLEKKYAG 421 DWGKVFDEAW ERIDKKZAGL TSHIEREEAR NAEDAQSKAV LTDWLRAKAS FVLERLKEMD 481 EKEFYACEIQ LQKWYGDLRG NPFAVEAENR VvDZ SGFSZ G SDGHSZQYRN L1AWKYLENG 541 KREFYLLMNY GKKGR1RFTD GTDIKKSGKW QGLLYGGGKA KV1DLTFDPD DEQLI ILPLA 601 FGTRQGREFI WNDLLSLETG LIKLANGRVI EKTIYNKKIG RDEPALFVAL TFERREWDP 661 SNIKPVNLIG VDRGENIPAV IALTDPEGCP LPEFKDSSGG PTDILRIGEG YKEKQRA1QA 721 AKEVEQRRAG GYSRKFASKS RNLADDMVRN SARDLFYHAV THDAVLVFEN LSRGFGRQGK 781 RTFMTERQYT KMEDWLTAKL AYEGLTSKTY LSKTLAQYTS KTC.SNCGFT1 TTADYDGMLV 841 RLKKTSDGWA TTLNNKELKA EGQITYYNRY KRQTVEKELS AELDRLSEES GNNDZ SKWTK 901 GRRDEALFLL KKRFSHRPVQ EQFVCLDCGH EVHADEQAAL NZARSWLFLN SNSTEFKSYK 961 SGKQPFVGAW QAFYKRRLKE VWKPNA ( SEQ Z D NO : 1 ) .

[0098] In some cases, a Type V reference CasX protein is isolated or derived from

Planctomycetes having a sequence of:

1 MQEIKRINKI RRRLVKDSNT KKAGKTGPMK TLLVRVMTPD LRERLENLRK KPENZ PQPI S

61 NTSRANLNKL LTDYTEMKKA ILHVYWEEFQ KD P VGLM S R V AQPAPKNIDQ RKLZ PVKDGN 121 ERLTSSGEAC SQCCQPLYVY KLEQVNDKGK PHTNYFGRCN VSEHERLILL SPHKPEANDE 181 LVTYSLGKFG QRALDFYS 1H VTRESNHPVK PLEQIGGNSC ASGPVGKALS DACMGAVAS F 241 LTKYQDI ILE HQKVIKKNEK RLANLKDIAS AN GLAFPK1T LPPQPHTKEG Z EAYNNWAQ 301 IVIWVNLNLW QKLKIGRDEA KPLQRLKGFP SFPLVERQAN EVDWWDMVCN VKKLZNEKKE 361 DGKVFWQNLA GYKRQEALLP YLS SEEDRKK GKKFARYQFG DLLLHLEKKH GEDWGKVYDE 421 AWERZ DKKVE GLSKHZ KLEE ERRSEDAQSK AALT DWL RAK AS FVZEGLKE ADKDEFCRCE 481 LKLQKWYGDL RGKPFAIEAE NSILDISGFS KQYNCAFZWQ KDGVKKLNLY LZ ZNYFKGGK 541 LRFKKZKPEA FEANRFYTVZ NKKSGEZVPM EVN FN FD D P N LI ILPLAFGK RQGREFZWND 601 LLSLETGSLK LANG RY’ IE KT LYNRRTRQDE PALFVALTFE RREVLDSSNI KPMNLZGZDR 661 GENIPAVIAL TDPEGCPLSR FKDSLGNPTH 1 R 1 G E S Y K E KQRTT QAAKE V EQ RRAGGY S 721 RKYASKAKNL ADDMVRNTAR DLLYYAVTQD AMLIFENLSR GFGRQGKRTF MAERQYTRME 781 DWLTAKLAYE GLPSKTYLSK TLAQYTSKTC SNCGFTZTSA DYDRVLEKLK KTATGWMTTI 841 NGKELKVEGQ ITYYNRYKRQ NWKDLSVEL D R L S E E S VN N DZ SSWTKGRS GEALSLLKKR 901 FSHRPVQEKF VCLNCGFETH ADEQAALNIA RSWLFLRSQE YKKYQTNKTT GNTDKRAFVE 961 TWQS FYRKKL KEVWKPAV ( SEQ I D NO : 2 )

[0099] In some cases, a Type V reference CasX protein is isolated or derived from

Candidatus Sungbacteria having a sequence of

1 MDNANKPSTK SLVNTTRZ SD HFGVTPGQVT RVFSFGI IPT KRQYAI IERW FAAVEAARER

61 LYGMLYAHFQ ENPPAYLKEK FSYETFFKGR PVLNGLRDZ D PTZMTSAVFT ALRHKAEGAM 121 AAFHTNHRRL FEEARKKMRE YAECLKANFA LLRGAADIDW DK1VNALRTR LNTCLAPEYD 181 AVTADFGALC AFRALTAETN ALKGAYNHAL NQMIJ P AL.VKV DEPEEAEESP RLRFFNGRIN 24 1 DLPKFPVAER ETPPDTETI 1 RQLEDMARVI P D T AE 1 L G Y 1 HRIRHKAARR KPGSAVPLPQ 301 RVALYCAIRM ERNPEEDPST VAGHFLGEID RVCEKRRQGL VRTPFDSQIR ARYMD11 SFR 361 ATLAHPDRWT EIQFLRSNAA SRRVRAETI S APFEGFSWTS NRTNPAPQYG MALAKDANAP 421 ADAPELCICL S P S SAAFS VR EKGGDD1YMR PTGGRRGKDN PGKEITWVPG S FDEYPASGV 481 ALKLRLYFGR SQARRMLTNK TWGLLSDNPR VFAANAELVG KKRNPQDRWK LFFHMVI SGP 541 PPVEYLDFS S DVRSRARTVI GINRGEVNPL AYAVVSVEDG QVLEEGLLGK KEYTDQLIET 601 RRRI SEYQSR EQTPPRDLRQ RVRHLQDTVL GSARAKIHSL 1AFWKGTLAI ERLDDQFHGR 661 EQK1 I PKKTY KANKTGFMNA LSFSGAVRVD KKGNPWGGMT EIYPGGI SRT CTQCGTWLA, 721 RRPKNPGHRD AWVI PDTVD D/AAAT G F DN V DCDAGTVDYG E 1J FT L 3 R E W V RLTPRYSRVM 781 RGTLGDLERA TRQGDDRKSR QMLELALEPQ P Q w GQ F F CH R CGFNGQSDVL AATNLARRAI 841 SLIRRLPDTD TPPTP ( SEQ I' D NO : 3 ) . c. CasX Variant Proteins

In some embodiments of the PDS, the disclosure provides CasX variant proteins for use in the PDS wherein the CasX variants comprise one or more modifications in one or more domains relative to the reference CasX protein, including but not limited to the sequences of SEQ ID MOS: 1-3, or at least one modification relative to another CasX variant from which it was derived. Any change in ammo acid sequence of a reference CasX protein that leads to an improved characteristic of the CasX protein is considered a CasX variant protein of the disclosure. For example, CasX variants can comprise one or more ammo acid substitutions, insertions, deletions, or swapped domains, or any combinations thereof relative to a reference CasX protein sequence. Any permutation of the substitution, insertion and deletion embodiments described herein can be combined to generate a CasX vanant protein of the disclosure.

[0101J The CasX variants of the disclosure have one or more improved characteristics compared to a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO :3, or the variant from which it was derived; e.g. CasX 491 (SEQ ID NO: 190) or CasX 515 (SEQ ID NO: 197). Exemplary improved characteristics of the CasX variant embodiments include, but are not limited to improved folding of the variant, improved binding affinity to the gRN A, improved binding affinity to the target nucleic acid, improved ability to utilize a greater spectrum of PAM sequences in the editing and/or binding of target nucleic acid, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity for the target nucleic acid, decreased off-target editing or cleavage, increased percentage of a eukaryotic genome that can be efficiently edited, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, improved binding of the non-target strand of DNA, improved protein stability, improved solubility', improved protein: gRN A (RNP) complex stability', increased ability to form cleavage-competent RNP, improved fusion characteristics, or a combination thereof. Exemplary improved characteristics are described in WO 2020/247882A1 and WO 2022/120095 Al , incorporated by reference herein. In particular, the CasX proteins of the disclosure have an enhanced ability to efficiently edit and/or bind target DNA, when complexed with a guide RNA scaffold as an RNP, utilizing a PAM TC motif, including PAM sequences selected from TTC, ATC, GTC, or CTC, compared to an RNP of a reference CasX protein and a reference gRNA. In the foregoing, the PAM sequence is located at least 1 nucleotide 5’ to the nontarget strand of the protospacer having identity with the targeting sequence of the gRNA in an assay system compared to the editing efficiency and/or binding of an RNP comprising the reference CasX protein and reference gRNA in a comparable assay system. In the foregoing embodiments, the one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, when assayed in a comparable fashion. In other embodiments, the improvement is at least about 1.1 -fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about. 5000-fold, at least, about 10,000-fold, or at least about 100,000-fold compared to the reference CasX protein of SEQ ID NO: I , SEQ ID NO: 2, or SEQ ID NO: 3, when assayed in a comparable fashion.

[0102] In some embodiments, the modification of the CasX variant is a mutation in one or more amino acids of the reference CasX. Mutations can be introduced in any one or more domains of the reference CasX protein or in a CasX variant to result in a CasX variant, and may include, for example, deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain of the reference CasX protein or the CasX variant from which it was derived. In some embodiments, the CasX variant proteins for use in the PDS of the disclosure comprises one or more modifications in at least 1 domain, in at least each of 2 domains, in at least, each of 3 domains, in at least each of 4 domains or in at least each of 5 domains of the reference CasX protein, including the sequences of SEQ ID NOS: 1-3, or a CasX variant from which it was derived. In other embodiments, the modification is an insertion or substitution of a part or all of a domain from a different CasX protein, resulting in a chimeric CasX variant.

[0103] In other embodiments, the disclosure provides CasX variants for use in the PDS wherein the CasX variants comprise at least one modification relative to another CasX variant; e.g., CasX variant 515 (SEQ ID NO: 197) and 527 (SEQ ID NO: 208) is a variant of CasX variant 491 (SEQ ID NO: 190) and CasX variants 668 (SEQ ID NO: 348) and 672 (SEQ ID NO: 351) are variants of CasX 535 (SEQ ID NO: 216, see, FIG. 1 ).

[0104] The CasX variants of the embodiments described herein have the ability to form an RNP complex with the gRNA disclosed herein, including during the encapsidation process of the PDS particle as the components are expressed in the transfected packaging host cells disclosed herein. The CasX variant proteins of the disclosure have an enhanced ability to efficiently edit and/or bind target DNA, when complexed with a gRNA as an RNP, utilizing a PAM TC motif, including PAM sequences selected from TTC, ATC, GTC, or CTC, compared to an RNP of a reference CasX protein and reference gRNA. In one embodiment, an RNP of a CasX variant and gRNA variant exhibits greater editing efficiency and/or binding of a target sequence in the target DNA compared to an RNP comprising a reference CasX protein and a reference gRNA in a comparable assay system, wherein the PAM sequence of the target DNA is TTC, In another embodiment, an RNP of a CasX variant and gRN A variant exhibits greater editing efficiency and/or binding of a target sequence in the target DNA compared to an RNP comprising a reference CasX protein and a reference gRNA in a comparable assay system, wherein the PAM sequence of the target DNA is ATC. In another embodiment, an RNP of a CasX variant and gRNA variant exhibits greater editing efficiency and/or binding of a target sequence in the target DNA compared to an RNP comprising a reference CasX protein and a reference gRNA in a comparable assay system, wherein the PAM sequence of the target DNA is CTC. In another embodiment, an RNP of a CasX variant and gRNA variant exhibits greater editing efficiency and/or binding of a target sequence in the target DNA compared to an RNP comprising a reference CasX protein and a reference gRNA in a comparable assay system, wherein the PAM sequence of the target DNA is GTC. In the foregoing embodiments, the increased editing efficiency and/or binding affinity for the one or more PAM sequences is at least 1.5 -fold greater or more compared to the editing efficiency and/or binding affinity of an RNP comprising any one of the CasX proteins of SEQ ID NOS: 1-3 and the gRNA of Table 8 for the PAM sequences.

[0105] In some embodiments, the CasX variant protein comprises between 500 and 1500 amino acids, between 700 and 1200 amino acids, between 800 and 1100 ammo acids or between 900 and 1000 ammo acids. In some embodiments, the CasX variant protein is less than 1500 amino acids, less than 1200 amino acids, less than 1100 amino acids, or less than 1000 amino acids in length. d. CasX Variant Proteins with Domains from Multiple Source Proteins [0106] Also contemplated within the scope of the disclosure are PDS comprising chimeric CasX variant proteins. As used herein, a “chimeric CasX protein” refers to a CasX containing at least two domains isolated or derived from different sources, such as two naturally occurring proteins, which may, in some embodiments, be isolated from different species. Alternatively, a chimeric CasX protein may contain protein sequences from two or more CasX variant proteins. In a particular embodiment, the CasX variants of 491, 514-791 (SEQ ID NOS: 190, 196-458 and 1905) have aNTSB and Helical I-II domain derived from the sequence of SEQ ID NO: 1, while the other domains are derived from SEQ ID NO: 2, it being understood that the variants have 1, 2, 3 or 4 additional amino acid changes at select locations. In other embodiments, the CasX variant of 494 has a NTSB domain derived from the sequence of SEQ ID NO: 1 , while the other domains are derived from SEQ ID NO: 2. [0107] In some embodiments, a CasX variant protein comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second, different CasX protein. As used herein, a “chimeric domain” refers to a domain containing at least two parts isolated or derived from different sources, such as two naturally occurring proteins or portions of domains from two reference CasX proteins. The at least one chimeric domain can be any of the NTSB, TSL, helical I, helical II, OBD or RuvC domains as described herein. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 660 to 823 of SEQ ID NO: 1 and amino acids 921 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, a chimeric RuvC domain comprises amino acids 647 to 810 of SEQ ID NO: 2 and amino acids 934 to 986 of SEQ ID NO: 1. In the case of split or non -contiguous domains such as helical I, RuvC and OBD, a portion of the non-contiguous domain can be replaced with the corresponding portion from any other source. For example, the helicai I-I domain (sometimes referred to as helical I-a) in SEQ ID NO: 2 can be replaced with the corresponding helical I-I sequence from SEQ ID NO: 1, and the like. Domain sequences from reference CasX proteins, and their coordinates, are shown in Table 1, Representative exampies of chimeric CasX proteins include the variants of CasX 472-483, 485-491 and 515, the sequences of which are set forth in Table 3.

[0108] Exemplary domain sequences are provided in Table 2 below'. The person of skill in the art will understand that the boundaries of the domain sequences provided in Table I below' may be approximate, and that domains whose boundaries differ by, e.g., 1, 2, 3, 4 or 5 amino acids may have the same activity of the sequences provided in Table 1. Table 1: Domain coordinates in Reference CasX proteins

Table 2: Exemplary Domain Sequences in Reference CasX proteins

e. Exemplary CasX Variants

[0109] In some embodiments, a CasX variant protein utilized in the PDS comprises a sequence selected from the group consisting of SEQ ID NOS: 136-176, 180-506, 1905, 7731-7891 and 7978-7980. In other embodiments, a CasX variant protein utilized in the PDS comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91 % identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to a sequence as set forth in SEQ ID NOS: 136-176, and 180-506, 1905, 7731-7891 and 7978- 7980, wherein the variant retains the functional properties of the ability^ to form an RNP with a gRNA and to bind, and optionally, to cleave a target nucleic acid. In a particular embodiment, a CasX variant protein utilized in the PDS is selected from CasX variants 491, 515, 593, 668, 672, 676, and 812, corresponding to SEQ ID NOS: 190, 197, 273, 348, 351, 355, and 478 respectively. As the results of the Examples demonstrate, despite changes in amino acid composition amongst the variants, the CasX variants retain the functional properties of the ability to form an RNP with a gRNA and to bind and cleave a target nucleic acid at diverse loci, underscoring that the variants collectively have the ability to be utilized for a common use; the genetic editing of DNA.

[0110] In still other embodiments, a CasX variant protein utilized in the PDS comprises a sequence set forth in Table 3 and further comprises one or more heterologous proteins or peptides disclosed herein at or near either the N-terminus, the C-terminus, or both. It will be understood that in some cases, the N-terminal methionine of the CasX variants of the Tables is removed from the expressed CasX variant during post-translational modification.

Table 3: CasX Variant Sequences

[0111] Additional CasX variants are provided as SEQ ID NOS: 101-135, 7731-7891 and 7978-7980. Accordingly, in some embodiments, a CasX variant comprises a sequence of 101-176, 180-506 or 1905, or a sequence having 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% sequence identity thereto, wherein the CasX variant is capable of forming a an RNP with a guide ribonucleic acid (gRNA). In some embodiments, the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 101-176, 180-506, 1905, 7731- 7891 and 7978-7980. In some embodiments, the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 190 and 197. In some embodiments, the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 7731-7891 and 7978-7980. In some embodiments, the engineered CasX protein comprises a sequence of SEQ ID NOS: 7731-7891 and 7978-7980, and two or more modifications relative to the CasX 515 protein, and the two or more modifications act to increase activity, specificity, or both, of the engineered CasX protein. In some embodiments, the two or more mutati ons act additively or synergistically. In some embodiments, the engineered CasX protein comprising a sequence selected from the group consisting of SEQ ID NOS: 7731-7891 and 7978-7980, and exhibits greater editing activity, editing specificity, specificity ratio, or a combination thereof, compared to CasX 515 when assayed under equivalent conditions. In some embodiments, the improved characteristics is determined compared to the unmodified parental CasX 515 in an in vitro assay under comparable conditions. In some embodiments, the engineered CasX protein comprises a P at position 793 (corresponding to SEQ ID NO: 2). f. CasX Fusion Proteins

[0112] Also contemplated within the scope of the disclosure are PDS particles and systems comprising CasX variant proteins comprising a heterologous protein fused to the CasX. In the context of a CasX, as used herein, a heterologous protein fused to the CasX comprises a protein that has a different activity of interest, or confers a property on the resulting fusion protein. For example, in some embodiments, the CasX variant protein is fused to a protein (or domain thereof) that inhibits transcription, modifies a target nucleic acid, or modifies a polypeptide associated with a nucleic acid (e.g., histone modification), or facilitates the transit of the CasX (such as an NLS). This includes CasX variants comprising N-terminal, C- terminal, or internal fusions of the CasX to a heterologous protein or domain thereof. |0113] A variety of heterologous poly peptides are suitable for inclusion in a CasX variant fusion protein utilized in the PDS particles and systems of the disclosure. In some cases, the fusion partner can recruit a gRNA in order to facilitate the formation of the RNP complex between the CasX variant and the guide nucleic acid, as well as facilitate the trafficking of the RNP into the budding PDS particle assembling in the packaging host cell (such fusion partners are referred to herein as “non-covalent recruitment protein” or “NCR protein”). Such fusion partners include RNA binding proteins such as MS2 coat protein, PP7 coat protein, QP, boxB, phage GA hairpin, phage AN hairpin, iron response element (IRE), transactivation response element (TAR), U1 A protein, or phage R-loop (encoding DNA sequences in Table 27 as SEQ ID NOS: 1821-1830), which can facilitate the binding of gRNA comprising the corresponding ligands of the fusion partners to CasX; i.e., MS2 hairpin, PP7 hairpin, Qp hairpin, boxB, phage GA hairpin, phage AN hairpin, iron response element (IRE), transactivation response element (TAR), or UI hairpin II.

[0114] In some cases, a CasX fusion partner utilized in the PDS particles and systems has enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity', methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity', dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity', integrase activity, transposase activity, recombinase activity', polymerase activity', ligase activity', helicase activity', photolyase activity or glycosylase activity). In some embodiments, a CasX variant comprises any one of the sequences of SEQ ID NOS: 136-176, 180-506 or 1905 as set forth in Table 3, SEQ ID NOS: 7731-7891, or SEQ ID NOS: 7978-7980, and a polypeptide with methyltransferase activity, demethylase activity', acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity', SUMOylating activity, deSUMOylaling activity, ribosylation activity, deribosylation activity', myristoylation activity' or demyristoylation activity'.

|0115] In some cases, a CasX fusion partner utilized in the PDS has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid (e.g., methyltransferase activity, demethylase activity', acetyltransferase activity’, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity'). JOI 16] Examples of proteins (or fragments thereof) that can be used as a CasX fusion partner utilized in the PDS particles and systems to increase transcription include but are not limited to: transcriptional activators such as VP16, VP64. VP48, VP160, p65 subdomain (e.g., fromNFkB), and activation domain of EDLL and/or TAL activation domain (e.g., for activity in plants); histone lysine methyl transferases such as SET! A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a/b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACER, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TETl, DME, DML1, DM1.2. ROSE and the like.

[0117] Examples of proteins (or fragments thereof) that can be used as a CasX fusion partner (or fused to a dCasX) in the PDS particles and systems to decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); K0X1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyl transferases such as Pr-SET7/8, SUV4- 20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2 A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1 A/RBP2, JARID1B/PLU-1, JARID 1C/SMCX, JARTD1D/SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HD AC 11 , and the like; DNA methylases such as Hhal DMA mSc-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMTl), DNA methyltransferase 3 alpha (DNMT3A) and subdomains such as DNMT3A catalytic domain and ATRX-DNMT3-DNMT3L domain (ADD), DNMT3L interaction domain (DNMT3L), DNA methyltransferase 3 beta (DNMT3B), DNA methyltransferase 1 (DNMTl), Friend of GATA-1 (FOG), Mad mSIN3 interaction domain (SID), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[0118] In some cases, the CasX fusion partner utilized in the PDS particles and systems has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the fusion partner include but are not limited to: nuclease activity' such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity' such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMTl), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), DNA methyltransferase 3 Like (DNMT3L), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g,, Ten-Eleven Translocation (TET) dioxygenase 1 (TET 1 CD), TET1, DME, DML1, DML2, ROS1, and the like), DNA repair activity, DNA damage activity', deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme, e.g,, an APOBEC protein such as rat APOBEC1), dismutase activity', alkylation activity', depurination activity', oxidation activity', pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and/or resol vase (e.g.. Gin invertase such as the hyperactive mutant of the Gin invertase, GinHIOoY; human immunodeficiency virus type 1 integrase (IN); Tn3 resol vase; and the like), transposase activity, recombinase activity' such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity).

[0119] In some embodiments, a CasX variant utilized in the PDS particles and systems of the disclosure comprises a sequence of any one of the sequences of Table 3 and an endosomal escape peptide or polypeptide to facilitate its transit out of an endosome of a host target cell. In some cases, a CasX variant polypeptide of the present disclosure can include an endosomal escape peptide. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 94), wherein each X is independently selected from lysine, histidine, and arginine. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 95), or HHHHHHHHH (SEQ ID NO: 96).

[0120] In some cases, a heterologous polypeptide (a fusion partner) provides for subcellular localization of the CasX to which it is fused, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like). In some embodiments, a subject RNA-guided polypeptide does not include a NLS so that the protein is not targeted to the nucleus (which can be advantageous, e.g., when the target nucleic acid is an RNA that is present in the cytosol). In some embodiments, a fusion partner can provide a tag (i.e., the heterologous polypeptide is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, and the like; a histidine tag, e.g,, a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).

[0121] In some cases, a CasX variant protein for use in the PDS particles and systems includes (is fused to) a nuclear localization signal (NLS). In some cases, a CasX variant protein is fused to 2 or more, 3 or more, 4 or more, or 5 or more 6 or more, 7 or more, 8 or more NLSs. Non-limiting examples of NLSs suitable for use with a CRISPR protein, such as an CasX variant, include sequences having at least about 80%, at least about 90%, or at least about 95% identity or are identical to sequences derived from: the NLS of the SV40 virus large T-antigen, having the ammo acid sequence PKKKRKV (SEQ ID NO: 7589); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 7590); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 7591) or RQRRNELKRSP (SEQ ID NO: 7592); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 7593); the sequence RMRTZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:

7594) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:

7595) and PPKKARED (SEQ ID NO: 7596) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 7597) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 7598) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 7599) and PKQKKRK (SEQ ID NO: 7600) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 7601) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 7602) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 7603) of the human poiy(ADP-ribose) polymerase; the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 7604) of the steroid hormone receptors (human) glucocorticoid; the sequence PRPRKIPR (SEQ ID NO: 7605) of Boma disease virus P protein (BDV-P1); the sequence PPRKKRTVV (SEQ ID NO: 7606) of hepatitis C virus nonstructural protein (HCV-NS5A); the sequence NLSKKKKRKREK (SEQ ID NO: 7607) of LEF1; the sequence RRPSRPFRKP (SEQ ID NO: 7608) of ORF57 simirae; the sequence KRPRSPSS (SEQ ID NO: 7609) of EBV LANA; the sequence KRGINDRNFWRGENERKTR (SEQ ID NO: 7610) of Influenza A protein; the sequence PRPPKMARYDN (SEQ ID NO: 7611) of human RNA helicase A (RHA); the sequence KRSFSKAF (SEQ ID NO: 7612) of nucleolar RNA helicase II; the sequence KLKIKRPVK (SEQ ID NO: 7613) of TUS -protein; the sequence PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 7614) associated with importm-alpha; the sequence PKTRRRPRRSQRKRPPT (SEQ ID NO: 7615) from the Rex protein in HTLV-1; the sequence SRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 7616) from the EGL-13 protein of Caenorhabditis elegans; and the sequences KTRRRPRRSQRKRPPT (SEQ ID NO: 7617), RRKKRRPRRKKRR (SEQ ID NO: 7618), PKKKSRKPKKKSRK (SEQ ID NO: 7619), HKKKHPDASVNFSEFSK (SEQ ID NO: 7620), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 7621), LSPSLSPLLSPSLSPL (SEQ ID NO: 7622), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 7623), PKRGRGRPKRGRGR (SEQ ID NO: 7624), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 7625), PKKKRKVPPPPKKKRKV (SEQ ID NO: 7626), PAKRARRGYKC (SEQ ID NO: 7627), KLGPRKATGRW (SEQ ID NO: 7628), PRRKREE (SEQ ID NO: 7629), PYRGRKE (SEQ ID NO: 7630), PLRKRPRR (SEQ ID NO: 7631), PLRKRPRRGSPLRKRPRR (SEQ ID NO: 7632), PAAKRVKLDGGKRTADGSEFESPKKKRKV (SEQ ID NO: 7633), PAAKRVKLDGGKRTADGSEFESPKKKRKVGIHGVPAA (SEQ ID NO: 7634), PAAKRVKLDGGKRTADGSEFESPKKKRKVAEAAAKEAAAKEAAAKA (SEQ ID NO: 7635), PAAKRVKLDGGKRTADGSEFESPKKKRKVPG (SEQ ID NO: 7636), KRKGSPERGERKRIIW (SEQ ID NO: 7637), KRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO: 7638), and PKKKRKVGGSKRTADSQHSTPPKTKRKVEFEPKKKRK.V (SEQ ID NO: 7639). In some embodiments, the one or more NLS are linked to the CRISPR protein or to adjacent NLS with a linker peptide wherein the linker peptide is selected from the group consisting of SR, RS, (G)n (SEQ ID NO: 7640), (GS)n (SEQ ID NO: 7641 ), (GSGGS)n (SEQ ID NO: 7642), (GGSGGS)n (SEQ ID NO: 7643), (GGGS)n (SEQ ID NO: 7644), GGSG (SEQ ID NO: 7645), GGSGG (SEQ ID NO: 7646), GSGSG (SEQ ID NO: 7647), GSGGG (SEQ ID NO: 7648), GGGSG (SEQ ID NO: 7649), GSSSG (SEQ ID NO: 7650), GPGP (SEQ ID NO: 7651), GGP, PPP, PPAPPA (SEQ ID NO: 7652), PPPG (SEQ ID NO: 7653), PPPGPPP (SEQ ID NO: 7654), PPP(GGGS)n (SEQ ID NO: 7655), (GGGS)nPPP (SEQ ID NO: 7656), AEAAAKEAAAKEAAAKA (SEQ ID NO: 7657), TPPKTKRKVEFE (SEQ ID NO: 7658) and GP AEAAAKEAAAKEAAAKA (SEQ ID NO: 1932), where n is 1 to 5. In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of a CasX variant fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to a CasX variant fusion protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.

[0122] The disclosure contemplates assembly of multiple NLS in various configurations for linkage to the CRISPR protein utilized in the PDS particles and systems of the embodiments described herein. In some embodiments, 1, 2, 3, 4 or more NLS are linked by linker peptides to the N-terminus of the CRISPR protein. In other embodiments, 1, 2, 3, 4 or more NLS are linked by linker peptides to the C -terminus of the CRISPR protein. In some embodiments, the NLS linked to the N-terminus of the CRI SPR protein utilized in the PDS are identical to the NLS linked to the C-terminus. In other embodiments, the NLS linked to the N-terminus of the CRISPR protein utilized in the PDS are different to the NLS linked to the C-terminus. In some embodiments, the NLS linked to the N-terminus of the CRISPR protein utilized in the PDS are selected from the group consisting of the N-terminal sequences of SEQ ID NOS: 507-553 as set forth in Table 4. In some embodiments, the NLS linked to the C-terminus of the CRISPR protein utilized in the PDS are selected from the group consisting of the C- terminal sequences of SEQ ID NOS: 554-600 as set forth in Table 4. The person of ordinary skill in the art will understand that an NLS at or near the N- or C-terminus of a protein can be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the N- or C-terminus. In some embodiments, the NLS linked to the N-terminus of the CasX protein are identical to the NLS linked to the C-terminus. In other embodiments, the NLS linked to the N-terminus of the CasX protein are different to the NLS linked to the C-terminus. Detection of accumulation in the nucleus of the CasX protein enhanced by the addition of NLS may be performed by any suitable technique. For example, a detectable marker may be fused to a reference or CasX variant fusion protein such that location within a cell may be vi suali zed. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry’, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly. Table 4: NLS Sequences

[0123] In some embodiments, the CasX protein of the PDS particles and systems comprises one or more nuclear export signal (NES) sequences as a fusion partner to facilitate the export of the expressed CasX through the nuclear pore complex and into the cytoplasm, facilitating its incorporation into the budding PDS. By incorporation of the NES as a fusion partner, it can counteract the sequestering of the CasX that also comprise one or more NLS. In some embodiments, the NES comprises a sequence selected from the group of sequences set forth in Table 28. In some embodiments, the NES is linked to a C -terminal NLS by a linker and a cleavage sequence capable of being cleaved by a protease such that the NES can be released from the CasX upon its export to the cytoplasm of the packaging host cell. In a particular embodiment, the linker comprises the sequence GPAEAAAKEAAAKEAAAKA (SEQ ID NO: 97) and the cleavage sequence is SQNYPIVQ (SEQ ID NO: 100), which is cleavable by the HIV-1 protease. Alternatively, the TEV protease cleavage site (ENLYFQS; SEQ ID NO: 98) may be used in place of the aforementioned HIV protease cleavage sequence. In some embodiments, the sequence encoding the protease is linked to the sequence encoding the MA or the MA-NC at the 3' end, wherein upon expression, it is capable of cleaving the protease cleavage sequence and releasing the NES from the CasX.

[0124] In some cases, a CasX variant fusion protein includes a "Protein Transduction Domain" or PTD (also known as a CPP - cell penetrating peptide), which refers to a protein, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from an extracellular space to an intracellular space, or from the cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the amino terminus of a reference or CasX variant fusion protein. In some embodiments, a PTD is covalently linked to the carboxyl terminus of a CasX variant fusion protein. In some cases, the PTD is inserted internally in the sequence of a CasX variant fusion protein at a suitable insertion site. In some cases, a CasX variant fusion protein includes (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, a PTD includes one or more nuclear localization signals (NLS). Examples of PTDs include but are not limited to peptide transduction domain of HIV TAT comprising YGRKKRRQRRR (SEQ ID NO: 7659), RKKRRQRR (SEQ ID NO: 7660); ¥ ARAA ARQARA (SEQ ID NO: 7661); THRLPRRRRRR (SEQ ID NO: 7662); and GGRRARRRRRR (SEQ ID NO: 7663); a poly arginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines, SEQ ID NO: 7664); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a 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: 7665); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 7666); KALAWEAKLAKALAKALAKI-ILrkKALAKALKCEA (SEQ ID NO: 7667); and RQIKIWFQNRRMKWKK (SEQ ID NO: 7668). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise a poly cationic CPP (e.g., Arg9 or ”R9") connected via a cleavable linker to a matching polyanion (e.g., Glu9 or "E9"), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the poly anion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus "activating" the ACPP to traverse the membrane. In some embodiments, a CasX variant comprises any one of the sequences of Table 3 and a PTD.

[0125] In some embodiments, a CasX variant fusion protein is linked to the heterologous polypeptide via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. The use of small amino acids, such as gly cine, serine, proline and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. Example linker polypeptides include glycine polymers (G)n (SEQ ID NO: 7640), glycine-senne polymer (including, for example, (GS)n (SEQ ID NO: 7641), (GSGGS)n (SEQ ID NO: 7642), (GGSGGS)n (SEQ ID NO: 7643), and (GGGS)n (SEQ ID NO: 7644), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, glycine-proline polymers, proline polymers and proline-alanine poly mers. Example linkers can comprise amino acid sequences including, but not limited to SR, RS, (G)n (SEQ ID NO: 7640), (GS)n (SEQ ID NO: 7641), (GSGGS)n (SEQ ID NO: 7642), (GGSGGS)n (SEQ ID NO: 7643), (GGGS)n (SEQ ID NO: 7644), GGSG (SEQ ID NO: 7645), GGSGG (SEQ ID NO: 7646), GSGSG (SEQ ID NO: 7647), GSGGG (SEQ ID NO: 7648), GGGSG (SEQ ID NO: 7649), GSSSG (SEQ ID NO: 7650), GPGP (SEQ ID NO: 7651), GGP, PPP, PPAPPA (SEQ ID NO: 7652), PPPG (SEQ ID NO: 7653), PPPGPPP (SEQ ID NO: 7654), PPP(GGGS)n (SEQ ID NO: 7655), (GGGS)nPPP (SEQ ID NO: 7656), AEAAAKEAAAKEAAAKA (SEQ ID NO: 7657), and TPPKTKRKVEFE (SEQ ID NO: 7658), where n is 1 to 5. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure. g. CatalytfcaHy-dead CasX variants

[0126] The present disclosure provides catalytically-dead variants (referred to herein as ‘"dCasX”) for use as therapeutic payloads in the PDS systems as a component of the dXR fusion proteins for repression of expression of a target nucleic acid. An exemplary catalytically dead CasX protein comprises one or more mutations in the active site of the RuvC domain of the CasX protein. In some embodiments, a cataly tically dead reference CasX protein comprises substitutions at residues 672, 769 and/or 935 with reference to SEQ ID NO: 1. In one embodiment, a catalytically-dead reference CasX protein comprises substitutions of D672A, E769A and/or D935 A with reference to SEQ ID NO: 1. In other embodiments, a catalytically-dead reference CasX protein comprises substitutions at amino acids 659, 756 and/or 922 with reference to SEQ ID NO: 2. In some embodiments, a catalytically-dead reference CasX protein comprises D659A, E756A and/or D922A substitutions with reference to of SEQ ID NO: 2. An exemplary RuvC domain comprises amino acids corresponding to 660-823 and 934-986 of SEQ ID NO: 1, or amino acids 647- 810 and 921-978 of SEQ ID NO: 2, with the foregoing mutations to render it catalytically dead, as well as mutations at positions utilized to create the base variant; e.g., 491, to enhance the characteristics of the dCasX relative to a reference dCasX. It will be understood that the same foregoing substitutions or deletions can similarly be introduced into any of the CasX variants of the disclosure, relative to the corresponding positions (allowing for any insertions or deletions) of the starting variant, resulting in a dCasX variant.

[0127} In some embodiments, a dCasX variant protein utilized in the gene repressor PDS particles or systems of the disclosure comprises the sequence selected from the group consisting of SEQ ID NOS: 7716 and 7937-7959, or a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 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 that retains the ability to form an RNP with a gRNA of the disclosure. In a particular embodiment, a dCasX variant protein utilized in the gene repressor PDS particles or systems of the disclosure comprises the sequence of SEQ ID NO: 7716 of Table 5, or a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81 % identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 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 that retains the ability to form an RNP with a gRNA of the disclosure. In some embodiments, the dCasX of the disclosure and linked repressor domain(s) ("dXR") are utilized with the gRNA of any of the embodiments described herein, wherein the dXR and gRNA are able to form a ribonucleoprotein (RNP) complex and bind to the target nucleic acid to affect the repression of transcription of the gene.

Table 5: dCasX Variant Sequence

g. dXR Fusion Proteins

[0128] In some embodiments, the disclosure provides PDS particles and systems comprising RNP of catalytically-dead CRISPR proteins linked to one or more repressor domains as a fusion protein in designed configurations complexed with a guide ribonucleic acid (gRNA) compri sing a targeting sequence complementary to a target nucleic acid sequence that, upon delivery of the PDS particle to a cell and binding to the target nucleic acid, have utility in the repression of transcription of the target nucleic acid. In some embodiments, the catalytically-dead CRISPR protein for use in the fusion protein with linked repressor domain(s) can be a Class 2, Type II, Type V, or Type VI CRISPR nuclease protein. In some embodiments, the catalytically-dead CRISPR protein for use in the fusion protein is a Type V catalytically-dead CasX protein (the fusion protein of dCasX and linked repressor domain(s) is referred to herein as "dXR"). In a particular embodiment, the catalytically dead CasX protein utilized in the dXR fusion proteins of the disclosure comprises the sequence of SEQ ID NO: 7716. Additional representative dCasX sequences are provided as SEQ ID NOS: 7937-7959. Exemplary' dXR fusion proteins are described in International Publication No. WO2023049742A2, hereby incorporated by reference in its entirety'.

[0129] In the context of the present disclosure and with respect to a gene, “repression”, “repressing”, “inhibition of gene expression”, “downregulation”, and “silencing” are used interchangeably herein to refer to the inhibition or blocking of transcription of a gene or a portion thereof. Accordingly, repression of a gene can result in a decrease in production of a gene product. Examples of gene repression processes which decrease transcription include, but are not limited to, those which inhibit formation of a transcription initiation complex, those which decrease transcription initiation rate, those which decrease transcription elongation rate, those which decrease processivity of transcription, and those which antagonize transcriptional activation (by, for example, blocking the binding of a transcriptional activator). Gene repression can constitute, for example, prevention of activation as well as inhibition of expression below an existing level. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription. In some embodiments, repression by the systems of the disclosure comprises any detectable decrease in the production of a gene product in cells, preferably a decrease in production of the gene product by at least about 10 % 20%, 30%, 40%, 50*%, 60%, 70%, 80%, 90%, 95%, or 99%, or any integer there between, when compared to untreated cells or cells treated with a comparable system comprising a non-targeting spacer. Most preferably, gene repression results in complete inhibition of gene expression, such that no gene product is detectable. In some embodiments, the repression of transcription by the systems of the embodiments is sustained for at least about 8 hours, at least about 1 day, at least about 1 week, at least about 1 month, or at least about 3 months, or at least about 6 months when assessed in an in vitro assay, including cell-based assays. In some embodiments, the repression of transcription by the gene repressor systems of the embodiments is sustained for at least about 1 day, at least about 1 w'eek, at least about 1 month, or at least about 3 months, or at least about 6 months when assessed in a subject that has been administered a therapeutically -effective dose of PDS particles comprising RNP of dXR and gRNA of the embodiments described herein. In some embodiments, gene repression by the system results in no or minimal detectable off-target binding or off-target activity, when assessed in an in vitro assay. In other embodiments, gene repression by the system results in no or minimal detectable off-target binding or off-target activity, when assessed in a subject that has been administered a therapeutical ly-effective dose of a system of the embodiments described herein.

[0130] In the RNP, the dCasX protein and linked repressor domains of the pre-complexed dXR:gRNA provides the site-specific activity and is guided to a target site (and further stabilized at a target site) within a target nucleic acid sequence to be repressed by virtue of its association with the gRNA, In some embodiments, the gene target nucleic acid sequence complementary to the targeting sequence of the gRNA is within 1 kb of a transcription start site (TSS) in the targeted gene. In some embodiments, the gene target nucleic acid sequence target nucleic acid sequence complementary to the targeting sequence of the gRNA is within 500 bps upstream to 500 bps downstream of a TSS of the gene. In some embodiments, the gene target nucleic acid sequence target nucleic acid sequence complementary to the targeting sequence of the gRNA is within 300 bps upstream to 300 bps downstream of a TSS of the gene. In some embodiments, the gene target nucleic acid sequence target nucleic acid sequence complementary to the targeting sequence of the gRNA is within 1 kb of an enhancer of the gene. In some embodiments, the gene target nucleic acid sequence target nucleic acid sequence complementary-’ to the targeting sequence of the gRNA is within the 3’ untranslated region of the gene. In some embodiments, the gene target nucleic acid sequence target nucleic acid sequence complementary to the targeting sequence of the gRNA is within an exon of the gene. In some embodiments, the gene target nucleic acid sequence target nucleic acid sequence complementary' to the targeting sequence of the gRNA is within exon 1 of the gene. In some embodiments, the gRNA is designed with a targeting sequence complementary' to a sequence within 1 kb of a 3’ or a 5’ untranslated region of a gene. In some embodiments, the gRNA is designed with a targeting sequence complementary to a sequence within the open reading frame of the gene, inclusive of exons and introns. In other embodiments, the gRN A is designed with a targeting sequence complementary’ to a regulatory element of the gene.

[0131] In some embodiments, the disclosure provides systems comprising a first repressor domain operably’ linked to the catalytically-dead Class 2, Type V CRISPR protein as a dXR fusion protein, wherein the catalytically-dead Class 2, Type V CRISPR protein is a dCasX of SEQ ID NO: 7716 as set forth in Table 5, or a sequence variant having at least about 65%, at least about 75%, 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, and wherein the repressor domain is selected from the group of sequences consisting of SEQ ID NOS: 7720- 7728 or a sequence having at least about 65%, at least about 75%, 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, wherein the fusion protein retains the ability^ to repress transcription of the target nucleic acid. In some embodiments, the fusion proteins comprise a single repressor domain operably linked by a peptide linker to a dCasX selected from the group of sequences of SEQ ID NOS: 7712, 7714-7715, 7717, and 7730.

[0132] In some embodiments, the fusion proteins comprise 1 , 2, 3 or 4 repressor domains operably linked to the dCasX, each independently selected from the group consisting of SEQ ID NOS: 7712, 7714-7715, 7717, and 7730. In some embodiments, the disclosure provides systems comprising a first, second, third, and fourth repressor domain operably linked to the dCasX protein. In some embodiments, each repressor domain is independently selected from the group consisting of SEQ ID NOS: 7712, 7714-7715, 7717, and 7730. In some embodiments, the repressor domains are linked to the dCasX and/or adjacent domains by a linker peptide.

[0133] In some embodiments, the disclosure provides systems comprising a first, second, third, and fourth repressor domain operably linked to the dCasX protein as a dXR fusion protein wherein the dCasX comprises the sequence of SEQ ID NO: 7716 as set forth in Table 5, or a sequence variant having at least about 65%, at least about 75%, 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, the first repressor domain comprises a sequence selected from the group consisting of SEQ ID NOS: 7720-7728, or a sequence having 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, the second repressor domain is a DNMT3A catalytic domain comprising the sequence of SEQ ID NO: 7711, or a sequence having 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, the third repressor is a DNMT3L interaction domain comprising the sequence of SEQ ID NO: 7713, or a sequence having 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, and the fourth repressor domain is a DNMT3A ADD domain of SEQ ID NO: 7710, or a sequence variant having 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. The ADD domain is known to have two key functions: 1) it allosterically regulates the catalytic activity of DNMT3A by serving as a methyl transferase auto-inhibitory domain, and 2) it recognizes unmethylated H3K4 (H3K4meO). Without wishing to be bound by theory, it is thought that the interaction of the ADD domain with the H3K4meO mark unveils the catalytic site of DNMT3A, thereby recruiting an active DNMT3A to chromatin to implement de novo methylation at these sites. In a surprising finding, it has been discovered that the addition of the DNMT3A ADD domain to the dXR constructs comprising the DNMT3A catalytic and DNMT3L interaction domains greatly enhances the repression of the target nucleic acid in comparison to dXR constructs lacking the ADD domain, resulting in epigenetic long-term, heritable repression of transcription of the target nucleic acid. In the foregoing, the DNMT3L helps maintain the methylation pattern after DNA replication. In some embodiments, upon binding of an RNP of the dXR fusion protein and the gRNA to the target nucleic acid, the inclusion of the ADD domain enhances the repression of the target nucleic acid compared to a dXR not comprising the ADD domain by 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%. at least about 90%, at least about 100%, or any integer in between, when assayed in an in vitro assay under comparable conditions, including cell-based assays. In some embodiments, the dXR fusion protein of the PDS systems comprising four repressor domains has a configuration of, N-terminal to C-terminal of configuration 1 (NLS-DNMT3A ADD domain- DNMT3A catalytic domain-Linker2-DNMT3L interaction domain-Linkerl -Linker3-dCasX- Lmker3-RD1-NLS), configuration 2 (NLS-Linker3-dCasX-Linker3- RD 1-NLS -Linker 1- DNMT3A ADD domain-DNMT3A catalytic domain-Linker2-DNMT3L interaction domain), configuration 3 (NLS-Linker3-dCasX-Linkerl-DNMT3A ADD domain-DNMT3A catalytic domain-Linker2-DNMT3Lmteraction domain-Linker3~ RD 1-NLS), configuration 4 (NLS- RD1-Linker3-DNMT3A ADD domain-DNMT3A catalytic domain-Linker2-DNMT3L interaction domain-Linkerl -dCasX-Linker3-NLS), or configuration 5 (NLS-DNMT3A ADD domain-DNMT3A catalytic domain-Linker2-DNMT3L interaction domain-Linker3- RD1-

Linker! -dCasX-Linker3-NLS), wherein “RD!” indicates a first repressor domain comprising one or more of the 9 motifs as described herein. In some embodiments of the system, the fusion protein components of configurations 1-5 are configured as schematically portray ed in

FIG. 7 and representative sequences of configurations 1, 4 and 5 are presented in Table 6 (it being understood that where alternative domain sequences are presented, the sequence can be selected from any one of the alternatives).

Table 6: dXR Configurations and sequences

[0134] In some embodiments, the repression of transcription of the gene by the system compositions is sustained for at least about 8 hours, at least about 1 day, at least about 7 days, at least 2 weeks, at least about 3 weeks, at least about I month, or at least about 2 months. when assayed in an in vitro assay, including cell-based assays. In a particular embodiment, dXR configurations 4 and 5, when used in the dXR:gRNA system, result in less off-target methylation or off-target activity' in an in vitro assay compared to configuration 1. In some embodiments, use of the dXR configurations 4 and 5, when used in the dXR:gRNA system, results in off-target methylation or off-target activity' that is less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% in the cells.

IV. Guide Nucleic Acids of PDS Systems

[0135] In another aspect, the disclosure relates to PDS components that encode or incorporate specifically-designed CRISPR Class 2. guide ribonucleic acids (gRNA) wherein the gRNA comprises a targeting sequence complementary' to a target nucleic acid sequence of a gene. Such gRNAs, when complexed with a CRISPR nuclease, or when complexed with a dXR, have utility' in the genome editing or modification of specific locations in the target nucleic acid in a cell, or repression of transcription of a gene, respectively.

[0136] As used herein, the term "gRNA” covers naturally-occurring reference gRNA as well as gRNA variants, including chimeric gRNA variants comprising domains from different gRNA. The gRNA of the systems of the disclosure are capable of forming a complex with a CasX nuclease or a dXR; a ribonucleoprotein (RNP) complex, described more fully, below.

[0137] In certain embodiments, the Class 2 system utilized in the PDS systems and particles can be a Type II, Type V, or Type VI system. It is envisioned that in some embodiments, multiple gRNAs are delivered by the PDS particles for the modification of a target nucleic acid. For example, a pair of gRNAs with targeting sequences to different or overlapping regions of the target nucleic acid sequence can be used, when each is complexed with a CRISPR nuclease, in order to bind and cleave at two different or overlapping sites within the gene, which is then edited by non-homologous end joining (NHEJ), homology- directed repair (HDR ), homology-independent targeted integration (HITI), micro-homology' mediated end joining (MMEJ), single strand annealing (SSA) or base excision repair (BER), winch can result in excision of the nucleotides located between the two cleavage sites. a. Reference gRNA

[0138] As used herein, a "reference gRNA" refers to a CRISPR guide ribonucleic acid comprising a wild-type sequence of a naturally -occurring gRNA. In some embodiments, a reference gRNA of the disclosure may be subjected to one or more mutagenesis methods, such as the mutagenesis methods described in WO2022120095 Al and WO2020247882A1, incorporated by reference herein, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, domain swapping, or chemical modification to generate one or more gRNA variants with enhanced or varied properties relative to the gRNA scaffold that was modified. The activity of the gRNA scaffold from which a gRNA variant was derived may be used as a benchmark against which the activity of the gRNA variant is compared, thereby measuring improvements in function or other characteristics of the gRNA scaffold.

[01391 Table 7 provides the sequences of reference gRN A tracr and scaffold sequences. In some embodiments, the disclosure provides gRNA sequences wherein the gRNA has a scaffold comprising a sequence having one or more nucleotide modifications relative to a reference gRNA sequence of any one of SEQ ID NOS: 4-16 of Table 7. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gRNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gRNA sequence embodiments described herein, including the sequences of Table 7 and Table 8.

Table 7: Reference gRNA tracr and scaffold sequences

gRNA Domains and their Function

[0140] The gRNAs of the disclosure comprise two segments: a targeting sequence and a protein-binding segment. The targeting segment of a gRNA includes a nucleotide sequence (referred to interchangeably as a spacer, a targeter, or a targeting sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within the target nucleic acid sequence (e.g., a strand of a double stranded target DNA, a target ssRNA, a target ssDNA, etc.), described more fully below. The targeting sequence of a gRNA is capable of binding to a target nucleic acid sequence, including, in the context of the present disclosure, a coding sequence, a complement of a coding sequence, a non-coding sequence, and to accessory elements. The protein-binding segment (or “activator” or “protein-binding sequence”) of the gRNA interacts with (e.g., binds to) a CasX protein as a complex, forming an RNP (described more fully, below). As used herein, “scaffold” refers to all parts to the guide with the exception of the targeting sequence, which is comprised of several regions, described more fully, below. The properties and characteristics of CasX gRNA, both wild-type and variants, are described in W02020247882A1, US20220220508A1, and W02022120095A1, incorporated by reference herein.

[0141] In the case of a reference gRNA, the gRNA occurs naturally as a dual guide RNA (dgRNA), wherein the targeter and the activator portions each have a duplex-forming segment that have complementarity with one another and hybridize to one another to form a double stranded duplex (dsRNA duplex for a gRNA). The term “targeter” or “targeter RNA” is used herein to refer to a crRNA-like molecule (crRNA: "CRISPR RNA") of a CasX dual guide RNA (and therefore of a CasX single guide RNA when the “activator" and the "targeter” are linked together, e.g., by intervening nucleotides). The crRNA has a 5' region that anneals with the tracrRNA followed by the nucleotides of the targeting sequence. In the case of the gRNA for use in the systems of the disclosure, the scaffolds are designed such that the activator and targeter portions are covalently linked to one another (rather than hybridizing to one another) and comprise a single molecule, and can be referred to as a “single-molecule gRNA,” “single guide RNA”, a “single-molecule guide RNA,” a “one- molecule guide RNA”, or a “sgRNA” The gRN A variants of the disclosure for use in the systems are all single molecule versions. [0142] Collectively, the gRNAs of the disclosure comprise distinct structured regions, or domains: the RNA triplex, the scaffold stem loop, the extended stem loop, the pseudoknot, and the targeting sequence that, in the embodiments of the disclosure is specific for a target nucleic acid and is located on the 3’ end of the gRNA. The RNA triplex, the scaffold stem loop, the pseudoknot and the extended stem loop, together with the unstructured triplex loop that bridges portions of the triplex, together, are referred to as the “scaffold” of the gRNA. In some cases, the scaffold stem further comprises a bubble. In other cases, the scaffold further comprises a triplex loop region. In still other cases, the scaffold further comprises a 5’ unstructured region. In some embodiments, the gRNA scaffolds of the disclosure for use in the systems of the disclosure comprise a scaffol d stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1907), or a sequence with at least at least 1 , 2, 3, 4, or 5 mismatches thereto.

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

[0144] Site-specific binding and/or cleavage of a target nucleic acid sequence (e.g., genomic DNA) by the CasX protein can occur at one or more locations (e.g., a sequence of a target nucleic acid) determined by base-pairing complementarity between the targeting sequence of the gRNA and the target nucleic acid sequence. Thus, for example, the gRNA of the disclosure have sequences complementarity' to and therefore can hybridize with the target nucleic acid that is adjacent to a sequence complementary to a TC protospacer adjacent motif (PAM) motif or a PAM sequence, such as ATC, CTC, GTC, or TTC. Because the targeting sequence of a guide sequence hybndiz.es with a sequence of a target nucleic acid sequence, a targeting sequence can be modified by a user to hybridize with a specific target nucleic acid sequence, so long as the location of the PAM sequence is considered. By selection of the targeting sequences of the gRNA, defined regions of the target nucleic acid sequence or sequences bracketing a particular location within the target nucleic acid can be modified or edited using the CasX:gRNA systems described herein. In some embodiments, the targeting sequence of the gRNA has between 15 and 20 consecutive nucleotides. In some embodiments, the targeting sequence has 15, 16, 17, 18, 19, and 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. By selection of the targeting sequences of the gRNA, defined regions of the target nucleic acid sequence can be modified or edited using the CasXigRNA systems described herein. In some embodiments, the gRNA and linked targeting sequence exhibit a low degree of off-target effects to the DNA of a cell. As used herein, "off-target effects" refers to off-target effects of unintended cleavage and mutations at untargeted genomic sites showing a similar but not an identical sequence compared to the target site. In some embodiments, the off-target effects exhibited by the gRNA and 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 cells. In some embodiments the off-target effects are determined in silico. In some embodiments the off-target effects are determined in an in vitro cell-free assay. In some embodiments the off-target effects are determined in a cell-based assay.

[0145] In some embodiments of the gRNAs of the disclosure utilized in the PDS particles and systems, the extended stem loop is followed by a region that forms part of the triplex, and then the targeting sequence (or “spacer”) linked at the 3’ end of the gRNA scaffold. The targeting sequence targets the CasX ribonucleoprotein holo complex to a specific region of the target nucleic acid sequence of the gene to be modified. Thus, for example, gRNA targeting sequences of the disclosure have sequences complementarity to, and therefore can hybridize to, a portion of the target gene in a nucleic acid in a eukaryotic cell (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.) as a component of the RNP when the TC PAM motif or any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5’ to the non-target strand sequence complementary to the target sequence. The targeting sequence of a gRNA can be modified so that the gRNA can target a desired sequence of any desired target nucleic acid sequence, so long as the PAM sequence location is taken in to consideration. In some embodiments, the gRNA scaffold is 5’ of the targeting sequence, with the targeting sequence on the 3’ end of the gRNA. In some embodiments, the PAM motif sequence recognized by the nuclease of the RNP is TC. In other embodiments, the PAM sequence recognized by the nuclease of the RNP is NTC. [0146] In some embodiments, the gRNA of the PDS comprises a targeting sequence (a) complementary to a nucleic acid sequence encoding i) a target protein, which may be a wildtype sequence or may comprise one or more mutations or ii) a protein accessory element, which may be a wild-type sequence; or (b) complementary' to a complement of a nucleic acid sequence encoding a protein or its accessory' element, which may comprise one or more mutations. In some embodiments, the targeting sequence of the gRNA is specific for a portion of a gene encoding a target protein comprising one or more mutations. In some embodiments, the targeting sequence of a gRNA is specific for a target gene exon. In some embodiments, the targeting sequence of a gRNA is specific for a target gene intron. In some embodiments, the targeting sequence of the gRNA is specific for a target gene intron-exon junction. In some embodiments, the targeting sequence of the gRNA is complementary' to a sequence comprising one or more single nucleotide polymorphisms (SNPs) of the target gene or its complement. In other embodiments, the targeting sequence of the gRNA is complementary to a sequence of an intergenic region of the target gene or a sequence complementary to an intergenic region of the target gene. In other embodiments, the targeting sequence of the gRNA is complementary to a non-coding sequence. In some embodiments, the targeting sequence of a gRNA is specific for an accessory element that regulates expression of a target gene. Such accessory' elements include, but are not limited to promoter regions, enhancer regions, intergenic regions, 5’ untranslated regions (5‘ UTR), 3‘ untranslated regions (3’ UTR), intergenic regions, gene enhancer elements, conserved elements, and regions comprising cis-accessory elements. The promoter region is intended to encompass nucleotides within 5 kb of the target gene initiation point or, in the case of gene enhancer elements or conserved elements, can be 1 Mb or more distal to the target gene. In some embodiments, the targeting sequence of a gRNA is specific for a target gene sequence adjacent to polynucleotide repeat region, wherein the repeat results in a dysfunctional gene product associated with a disease.

[0147] In some embodiments, the CasX:gRNA of the PDS system comprises a first gRNA and further comprises a second (and optionally a third, fourth or fifth) gRNA, wherein the second gRNA has a targeting sequence complementary' a different portion of the target nucleic acid or its complement compared to the targeting sequence of the first gRN A, resulting in double-stranded cleavage at each target nucleic acid location that can, in some cases, result in excision of the intervening nucleotides, for example an expanded polynucleotide repeat region or aberrantly spliced region. By selection of the targeting sequences of the gRNA, defined regions of the target nucleic acid can be modified or edited using the CasX:gRNA systems described herein. c. gRNA Variants

[0148] In another aspect, the disclosure provides guide nucleic acid variants (referred to herein as ‘'gRNA variant” when the nucleic acid variant comprises RNA), which comprise one or more modifications relative to a reference gRNA scaffold.

[0149] gRNA variants can be produced by subjecting a reference gRNA of the to one or more mutagenesis methods, such as the mutagenesis methods described herein. gRNA variants also include variants comprising one or more exogenous sequences, for example fused to either the 5’ or 3’ end, or inserted internally. The activity of reference gRNAs maybe used as a benchmark against which the activity of gRNA variants are compared, thereby- measuring improvements in function or other characteristics of the gRNA variants. In other embodiments, a reference gRNA may be subjected to one or more deliberate, specifically- targeted mutations in order to produce a gRNA variant, for example a rationally designed variant. Exemplary gRNA variants produced by such methods are described in the Examples and representative sequences of gRNA scaffolds are presented in Table 8.

[0150] In some embodiments, a gRNA variant comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced regions relative to a reference gRN A sequence of the disclosure that improve a characteristic relative to the reference gRNA. A representative example of such a gRNA variant is guide 235 (SEQ ID NO: 2293). Exemplary regions for modification include the RNA triplex, the pseudoknot, the scaffold stem loop, and the extended stem loop. In some cases, the variant scaffold stem further comprises a bubble. In other cases, the variant scaffold further comprises a triplex loop region. In still other cases, the variant scaffold further comprises a 5’ unstructured region. In one embodiment, the gRNA variant scaffold comprises a scaffold stem loop having at least 60% sequence identity to SEQ ID NO: 14. In another embodiment, the gRNA variant comprises a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1907). In another embodiment, the disclosure provides a gRNA scaffold comprising, relative to SEQ ID NO:5, a C 18G substitution, a G55 insertion, a U1 deletion, and a modified extended stem loop in which the original 6 nt loop and 13 most-loop-proximal base pairs (32 nucleotides total) are replaced by a Uvsx hairpin (4 nt loop and 5 loop- proximal base pairs; 14 nucleotides total) and the loop-distal base of the extended stem was converted to a fully base-paired stem contiguous with the new Uvsx hairpin by deletion of the A99 and substitution of G64U. In the foregoing embodiment, the gRNA scaffold comprises the sequence

ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA GUGGGUAAAGCDCCCDCUUCGGAGGGAGCAUCAAAG (SEQ ID NO; 2238).

[0151] In other cases, one or more mutations can be introduced in any region of a gRNA variant to produce another gRNA variant. All gRNA variants that have one or more improved functions or characteristics, or that add one or more new functions when the variant gRNA is compared to the gRNA variant from which it was derived are envisaged as within the scope of the disclosure. In some embodiments, the gRN A variant has an improved characteristic selected from the group consisting of increased editing activity, increased pseudoknot stem stability, increased triplex region stability, increased scaffold stem stability, extended stem stability, reduced off-target folding intermediates, and increased binding affinity to a Class 2, Type V CRISPR protein, or any combination thereof. In some cases of the foregoing, the improved characteristic is assessed in an in vitro assay, including the assays of the Examples. In other cases of the foregoing, the improved characteristic is assessed in vivo.

[0152] In exemplary embodiments, a gRNA variant for use in the PDS particles and systems comprises one or more modifications relative to gRNA scaffold variant 174 (SEQ ID NO;2238), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 174, when assessed in an in vitro or in vivo assay under comparable conditions.

[0153] In exemplary embodiments, a gRNA variant for use in the PDS particles and systems comprises one or more modifications relative to gRNA scaffold variant 215 (SEQ ID NO:2.2.75), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 215, when assessed in an in vitro or in vivo assay under comparable conditions,

[0154] In exemplary’ embodiments, a gRNA variant for use in the PDS particles and systems comprises one or more modifications relative to gRNA scaffold variant 221 (SEQ ID NO: 2281 ), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 221, when assessed in an in vitro or in vivo assay under comparable conditions. [0155] In exemplar}-’ embodiments, a gRNA variant for use in the PDS particles and systems comprises one or more modifications relative to gRNA scaffold variant 225 (SEQ ID NO: 2285), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 225, when assessed in an in vitro or in vivo assay under comparable conditions.

[0156] In exemplary' embodiments, a gRNA variant for use in the PDS particles and systems comprises one or more modifications relative to gRNA scaffold vanant 235 (SEQ ID NO: 2293), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 225, when assessed in an in vitro or in vivo assay under comparable conditions.

[0157] In exemplary embodiments, a gRNA variant for use in the PDS particles and systems comprises one or more modifications relative to gRNA scaffold variant 251 (SEQ ID NO: 2309), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 251, when assessed in an in vitro or in vivo assay under comparable conditions.

[0158] In some embodiments, the gRNA variant comprises an exogenous extended stem loop, with such differences from a reference gRNA described as follows. In some embodiments, an exogenous extended stem loop has little or no identity to the reference stem loop regions disclosed herein (e.g., SEQ ID NO: 15). In some embodiments, an exogenous stem loop is at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, at least 100 bp, at least 2.00 bp, at least 300 bp, at least 400 bp, or at least 500 bp. In some embodiments, the heterologous stem loop increases the stability of the gRNA. In some embodiments, the heterologous RNA stem loop is capable of binding a protein, an RNA structure, a DNA sequence, or a small molecule. In some embodiments, an exogenous stem loop region replacing the stem loop comprises an RNA stem loop or hairpin in which the resulting gRNA has increased stability and, depending on the choice of loop, can interact with certain cellular proteins or RNA. Such exogenous extended stem loops can comprise, for example a thermostable RN A such as MS2 hairpin (ACAUGAGGAUCACCCAUGU (SEQ ID NO: 1908)), Qp hairpin (AUGCAUGUCUAAGACAGCAU (SEQ ID NO: 1909)), IJ1 hairpin II (GGAAUCCAUUGCACUCCGGAUUUCACUAG (SEQ ID NO: 1910)), Uvsx (CCUCUUCGGAGG (SEQ ID NO: 1911)), PP7 hairpin (AAGGAGUUUAUAUGGAAACCCUU (SEQ ID NO: 1912)), Phage replication loop (AGGUGGGACGACCUCUCGGUCGUCCUAUCU (SEQ ID NO: 1913)), Kissing loop., a (UGCUCGCUCCGUUCGAGCA (SEQ ID NO: 1914)), Kissing loop_bl (UGCUCGACGCGUCCUCGAGCA (SEQ ID NO: 1915)), Kissing loop_b2 (UGCUCGUUUGCGGCUACGAGCA (SEQ ID NO: 1916)), G quadriplex M3q (AGGGAGGGAGGGAGAGG (SEQ ID NO: 1917)), G quadriplex telomere basket (GGUUAGGGUUAGGGlJUAGG (SEQ ID NO: 1918)), Sarcin-ricin loop (CUGCUCAGUACGAGAGGAACCGCAG (SEQ ID NO: 1919)), Pseudoknots (UACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUAUACUUU GGAGUUUUAAAAUGUCUCUAAGUACA (SEQ ID NO: 1920)), transactivation response element (TAR) (GGCUCGUGUAGCUCAUUAGCUCCGAGCC (SEQ ID NO: 1921)), iron responsive element (IRE) CCGUGUGCAUCCGCAGUGUCGGAUCCACGG (SEQ ID NO: 1922)), phage GA hairpin (AAAACAUAAGGAAAACCUAUGUU (SEQ ID NO: 1923)), phage AN hairpin (GCCCUGAAGAAGGGC (SEQ ID NO: 1924)), or sequence variants thereof. In some embodiments, one of the foregoing hairpin sequences is incorporated into the stem loop of the gRNA scaffold to help traffic the incorporation of the gRNA (and an associated CasX in an RNP complex) into a budding PDS (described more fully, below) when the counterpart ligand is incorporated into the MA fusion protein of the PDS (i.e., the NCR protein and its binding partner element).

[0159] Table 8 provides exemplary gRNA variant scaffold sequences of the disclosure. In some embodiments, the gRNA variant scaffold for use in the PDS of the disclosure comprises any one of the sequences listed in Table 8, wherein the gRNA variant retains the ability to form an RNP with a CasX of the disclosure and to bind a target nucleic acid, whereupon the RNP modifies the target nucleic acid. In other embodiments, the gRNA variant scaffold for use in the PDS of the disclosure comprises a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to a sequence of Table 8, wherein the gRNA variant retains the ability to form an RNP with a CasX of the disclosure and to bind a target nucleic acid, whereupon the RNP modifies the target nucleic acid. In a particular embodiment, a gRNA variant scaffold utilized in the PDS is selected from gRNA variants 174, 215, 221, 235, and 251. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gRN A, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gRNA sequence embodiments described herein. Table 8: gRNA Variant Scaffold Sequences

[0160] Additional sgRNA variants are presented in the attached sequence listing, as SEQ ID NOS: 2101 -2237.

[0161] In some embodiments, a sgRNA variant of the disclosure comprises one or more additional changes to a previously generated variant, the previously generated variant itself serving as the sequence to be modified. In some embodiments, a sgRNA variant comprises one or more additional changes to a sequence of SEQ ID NO: 2238, SEQ ID NO: 2239, SEQ ID NO: 2240, SEQ ID NO: 2241, SEQ ID NO: 2241, SEQ ID NO:2274, SEQ ID NO:2275, SEQ ID NO: 2281, SEQ ID NO: 2285, SEQ ID NO: 2293, or SEQ ID NO: 2309 of Table 8.

«/. Transport of CRISPR Components: gRNA Binding Partners and Packaging Elements

[0162] In some embodiments of the PDS particles and systems, in addition to the RNA stem loops with affinity to NCR, the disclosure provides gRNA variants that comprise additional domains that facilitate the transport of the gRN A, and any CRISPR nuclease complexed with the gRNA, out of the nucleus and facilitates the transport of the gRNA and complexed CRISPR nuclease (e.g., CasX) to the budding PDS particle, thereby enhancing the ability of the packaging host cell to package the gRNA and CRISPR nuclease complexed as an RNP into the PDS, In some embodiments, the gRNA-encoding plasmid comprises a sequence for one or more RRE or components of RRE, described below, incorporated into the extended stem region of the gRNA. The term “Rev response element’’ or “RRE” refers to a cis-acting post-transcriptional accessory element that, in the context of retroviral reproduction, serves as a specific RNA scaffold that coordinates the assembly of a unique homo-oligomeric ribonucleoprotein (RNP) complex to mediate the nuclear export of essential, intron-containing, viral messages. It has been discovered, however that incorporation of certain RNA sequences capable of binding a retroviral Rev protein into the gRNA facilitates the export of a gRNA from the nucleus by interaction with multiple molecules of Rev, across the nuclear membrane, to the cytoplasm of a cell. Examples of RNA binding partners include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE.) (see e.g., Cullen et al. (1991) J. Virol. 65: 1053; and Cullen et al. (1991) Cell 58: 423-426), the constitutive transport element (CTE) of the simian retrovirus (Giulietti, M., et al. Export Aid: database of RNA elements regulating nuclear RNA export in mammals. Bioinformatics 31:246 (2015)), the hepatitis B virus post-transcriptional regulatory element (PRE) (see e.g., Huang et al. (1995) Molec. and Cell. Biol. 15(7): 3864-3869; Huang et al. (1994) J. Virol. 68(5): 3193-3199; Huang et al. (1993) Molec. and Cell. Biol 13(12): 7476-7486), and U.S. Pat. No. 5,744,326, and heterogeneous nuclear ribonucleoparticle protein (hnRNP)(Lei, E. et al. Protein and RNA Export from the Nucleus. Develop. Cell 2:261 (2002)), which are all hereby incorporated by reference). In some embodiments of the PDS system, the nucleic acid encoding the guide RNA variants comprises one or more NES components selected from the group consisting of Stem IIB of Rev response element (RRE), Stem II-V of RRE, Stem II of RRE, Rev -binding element (RBE) of Stern IIB, and full-length RRE. In the foregoing embodiment, the components include sequences of

UGGGCGCAGCGUCAAUGACGCUGACGGUACA (Stem IIB, SEQ ID NO: 1925), GCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAGACAAUUAUUG UCUGGUAUAGUGC (Stem II, SEQ ID NO: 1941), GCUGACGGUACAGGC (RBE, SEQ ID NO: 1926),

CAGGAAGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAGACAA UUAUUGUCUGGUAUAGUGCAGCAGCAGAACAAUUUGCUGAGGGCUAUUGAGG CGC AACAGCAUC UGUUGCAACUCAC AGUCUGGGGCAUCAAGC AGCUCCAGGC A AGAAUCCUG (Stem II-V, SEQ ID NO: 1927), and

AGGAGCUUUGUUCCUUGGGUUCUUGGGAGCAGCAGGAAGCACUAUGGGCGCA G C GU C A A U GAC GC Li GAC GG Li AC AGG CCA GAC A A UU A Li LJ GU C U GGU A LJ AGU G C AGCAGC AGAAC AAUUU GC UGAGGGCU ALi UGAGGCGCAAC AGCAU CU GUU GCA ACUCACAGUCUGGGGCAUCAAGCAGCUCCAGGCAAGAAUCCUGGCUGUGGAAA GAUACCUAAAGGAUCAACAGCUCCU (full-length RRE, SEQ ID NO: 1928). In some embodiments, the gRNA variant comprises one RRE component selected from RBE, Stem IIB, Stem II-V, Stem II, and full-length RRE, wherein the RRE component is incorporated in the extended stem of the guide RNA. In other embodiments, the gRNA variant comprises two RRE components selected from RBE, Stem IIB, Stem II-V, Stem II, and full-length RRE, which may be identical or may be different, wherein the RRE component is incorporated in the extended stem of the guide RNA. In other embodiments, the gRNA variant comprises three RRE components selected from RBE, Stern IIB, Stem II-V, Stern II, and full-length RRE, which may be identical or may be different, wherein the RRE component is incorporated in the extended stem of the guide RNA. In other embodiments, the gRNA variant, comprises four RRE components selected from RBE, Stem IIB, Stern II-V, Stem II, and full-length RRE, which may be identical or may be different, wherein the RRE component is incorporated in the extended stern of the guide RNA. In some embodiments, the disclosure provides gRNA variants comprising a Rev-binding element (RBE) of Stem IIB, depicted in FIG. 2. In other embodiments, the disclosure provides gRNA variants comprising two or more (e.g., 2, 3, 4, 5 or more) RBE as concatenates in the extended stem of the gRNA. In the foregoing embodiments, a sequence encoding lentiviral Rev protein can be incorporated into the nucleic acid encoding the MA protein of the PDS system such that upon expression, the Rev can bind with the RRE or RBE elements of the gRNA and facilitate the transport, of the CasX:gRNA RNP complex into the budding PDS. Non-limiting representative gRNA sequences comprising RBE include gRNA scaffolds 226, 243, 249-254, and 256 and 264 of Table 8, corresponding to SEQ ID NOS: 2286, 2301, 2307-2312, 2314 and 2322. It will be further appreciated that the inclusion of the RRE in the gRNA serves to coun teract the effects of the NLS incorporated into CRISPR nuclease of the RNP that “drives” entry of the CRISPR nuclease into the nucleus, thereby contributing to the ability’ of the packaging host cell to package the RNP into the PDS. In other embodiments, the gRNA can comprise one or more RRE and one or more MS2 hairpin sequences wherein the PDS comprises a Rev element and an MS2 coat protein fused to the MA protein such that the gRNA has enhanced affinity for both the ligand and exhibits improved incorporation for the CRISPR nuclease.’gRNA complex into the budding PDS in the producing host cell. In some embodiments, the gRNA comprises one or more binding partner elements to facilitate the recruitment of the gRNA and any associated CRISPR nuclease into the budding PDS in the packaging host cell, wherein the ligand of the binding partner is fused to the MA protein component incorporated into the PDS. In some embodiments, the disclosure provides sequences encoding a gRNA comprising one or more binding partner elements listed in Table 36, with the sequences encoding the binding partner elements listed in Tables 20 and 27. In some embodiments, the sequence encoding the gRNA is selected from the group consisting of SEQ ID NOS: 2860-2880. In some embodiments, the disclosure provides sequences of MA linked to NCR selected from the group consisting of SEQ ID NOS: 2.881-5234.

J0163] In some embodiments, one or more Mononegavirales structural proteins (e.g., MA) in the PDS particle is a fusion protein comprising an NCR protein, and the therapeutic payload comprises gRNA (e.g., as part of a CasX:gRNA RNP) comprising a binding partner element that binds to the NCR protein. In some embodiments, the binding partner element is selected from the group consisting of a MS2, Phage AN hairpin, a PP7 hairpin, a Trans- activation response element (TAR), an Iron response element (IRE), a U1 hairpin II, a Qp hairpin and a Phage GA hairpin, and the NCR protein is selected from the group consisting of MS2 coat protein, protein N, a PP7 coat protein, a TAT protein, iron-responsive binding element protein 1 (IRP1), IRP2, U1A signal recognition particle, a QP coat protein and a Phage GA coat protein.

[0164] In some embodiments, the NCR protein comprises protein N, and the binding partner element comprises a Phage AN hairpin. In some embodiments, the protein N comprises a sequence of SEQ ID NO : 1821 or 1822, and the Phage AN hairpin is encoded by a sequence cotnpri sing SEQ ID NO: 1831. In some embodiments, the NCR protein comprises a PP7 coat protein, and the binding partner element comprises a PP7 hairpin. In some embodiments, the PP7 coat protein comprises a sequence of SEQ ID NO: 1823, and the PP7 hairpin is encoded by a sequence comprising SEQ ID NO: 1832. In some embodiments, the NCR protein comprises a TAT protein, and the binding partner element comprises a Trans-activation response element (TAR). In some embodiments, the TAT protein comprises a sequence of SEQ ID NO: 1824, and the TAR is encoded by a sequence comprising SEQ ID NO: 1833. In some embodiments, the NCR protein comprises iron-responsive binding element protein I (IRP1), and the binding partner element comprises an Iron response element (IRE). In some embodiments, IRP1 comprises a sequence of SEQ) ID NO: 1825, and the IRE is encoded by a sequence comprising SEQ ID NO: 1834. In some embodiments, the NCR protein comprises iron-responsive binding element protein 2 (IRP2), and the binding partner element comprises an Iron response element (IRE). In some embodiments, IRP2 comprises a sequence of SEQ ID NO: 1826, and the IRE is encoded by a sequence comprising SEQ ID NO: 1834. In some embodiments the NCR protein comprises U1A signal recognition particle, and the binding partner element comprises U1 hairpin II. In some embodiments, the L^!1A signal recognition particle comprises a sequence of SEQ ID NO: 1827-1828, and the U1 hairpin II is encoded by a sequence comprising SEQ ID NO: 1835. In some embodiments, the NCR protein comprises a QP coat protein, and the binding partner element comprises a QP hairpin. In some embodiments, the Qp coat protein comprises a sequence of SEQ ID NO: 1829, and the Qp hairpin is encoded by a sequence comprising SEQ ID NO: 1836. In some embodiments, the NCR protein comprises a Phage GA coat protein, and the binding partner element comprises a Phage GA hairpin. In some embodiments, the Phage GA coat protein comprises a sequence of SEQ ID NO: 1830, and the Phage GA hairpin is encoded by a sequence comprising SEQ ID NO: 1837. [0165] The RNA binding partner element can be a retroviral psi packaging element inserted into the gRNA variant or is a hairpin stem loop such as MS2 hairpin, PP7 hairpin, Qp hairpin, boxB, phage GA hairpin, phage AN hairpin, iron response element (IRE), transactivation response element (TAR), or U1 hairpin II with affinity to an NCR protein linked to a Mononegavirales structural protein (for example MA), the NCR protein selected from the group consisting of MS2 coat protein, PP7 coat protein, QP coat protein, protein N, protein Tat, phage GA coat protein, iron-responsive binding element (IRE) protein, and U1 A signal recognition particle protein (III A). The interaction of the binding partner element and the NCR protein can facilitate the non-covalent recruitment and incorporation of the gRNA variant (and CRISPR nuclease complexed with gRNA) into the budding PDS particle in the packaging cell.

[0166] As used herein, “binding partner element'’ means a sequence of the gRNA that has binding affinity to non-covalent recruitment (NCR) protein, i.e. a peptide or protein that, when expressed in the packaging cell, facilitates the non-covalent recruitment and incorporation of the gRNA variant and associated CasX protein into the budding PDS particle in the packaging cell. It has been discovered that the incorporation of the binding partner element inserted into the guide RNA and NCR into the fusion protein comprising the Mononegavirales structural protein (e.g,, MA) facilitates the packaging of the PDS particl e due, in part, to the affinity of the CasX for the gRNA, resulting in an RNP, such that both the gRNA variant and CasX variant are associated with Mononegavirales structural protein during the encapsidation process of the PDS particle, increasing the proportion of PDS comprising RNP compared to a construct lacking the binding partner and NCR. In some embodiments, the disclosure provides PDS in which the binding partner element and the NCR component are encoded in their respective plasmids in a 1 : 1 ratio (protein to gRNA). In other embodiments, the disclosure provides PDS in which the binding partner element and the NCR component are encoded in their respective plasmids in a 1:2 ratio (protein to gRNA). In other embodiments, the disclosure provides PDS in which the binding partner element and the NCR component are encoded m their respective plasmids in a 1 :3 ratio (protein to gRNA). In other embodiments, the disclosure provides PDS in which the binding partner element and the NCR component are encoded in their respective plasmids in a 1 :4 ratio (protein to gRNA). In other embodiments, the disclosure provides PDS m which the binding partner element and the NCR component are encoded in their respective plasmids in a 1:5 ratio (protein to gRNA). In some embodiments, the incorporation of the binding partner elements(s) and NCR(s) results in enhanced incorporation of the RNP of the CRISPR nuclease and gRNA (e.g. CasX and a CasX gRNA) into the PDS compared to a system not comprising the binding partner(s) and NCR(s). In some embodiments, the incorporation of the binding partner(s) and NCR(s) results in PDS containing at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 molecules of the RNP of the CRISPR nuclease and gRNA. In a particular embodiment, the incorporation of the binding partner(s) and packaging recruiters ) results in PDS containing at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 molecules of an RNP of a CRISPR nuclease and gRNA (e.g., CasX variant and gRN A variant) of an embodiment described herein. In some embodiments, the incorporation of the binding partner element(s) and NCR(s) results in PDS containing at least about 100 to about 1000 RNP, at least about 200 to about 800 RNP, or at least about 300 to about 600 RNP. In some embodiments, the incorporation of the binding partner elements(s) and NCR(s) results in at least a 2-fold, at a least 3-fold, or at least a 4-fold increase in editing potency of the PDS particles for a target nucleic acid compared to PDS particles without the incorporated binding partner(s) and NCR(s), when assessed in an in vitro assay under comparable conditions. r>. MS2 hairpin variants

[0167] As described, supra, the gRNA can be modified to comprise one or more binding partner elements to facilitate the recruitment of the gRNA and the associated CRISPR nuclease (e.g., CasX) into the budding PDS particle in the packaging host cell. One such binding partner element is MS2 hairpin, incorporated into the extended stem of the gRNA scaffold, which has affinity to its ligand, MS2 coat protein. As described in embodiments herein, PDS have been designed with the MS2 coat protein linked to the MA protein and MS2 hairpins incorporated into the gRNA to facilitate the recruitment and incorporation of a CasXtgRNA complex into the PDS particles. It has been discovered, as described in the Examples, that modifying the sequence of the MS2. hairpin to increase the binding affinity of the MS2 hairpin for its ligand enhances the editing activity of the resulting PDS towards the target nucleic acid when introduced into target cells. In some embodiments, the disclosure provides PDS comprising gRNA comprising one or more MS2 hairpin sequence variants, wherein the variant exhibits a Ko to its ligand of less than 100 nM, less than 50 nM, less than 35 nM, less than 10 nM, less than 3 nM, or less than 2 nM. In some embodiments, the disclosure provides PDS comprising CasX variant complexed with gRNA comprising one or more MS2 hairpin sequence variants, wherein the MS2 variant exhibits a KD to its ligand of less than 100 nM, less than 50 nM, less than 35 nM, less than 10 nM, less than 3 nM, or less than 2 nM and the PDS exhibits improved editing activity towards a target nucleic acid in an in vitro cellular assay, wherein the ECso is less than IO⁸, or less than 10', or less than I0⁶ particles to achieve editing in 50% of the cells. In a particular embodiment, the disclosure provides PDS comprising a gRNA MS2 variant exhibiting a KD to its ligand of less than 10 nM, wherein the PDS exhibits editing activity towards a target nucleic acid in an in vitro cellular assay wherein the ECso is less than 10'' or 10° particles. In some embodiments, the PDS comprises a gRNA MS2 variant wherein the scaffold is selected from the group consisting of gRNA scaffold variants 188, 251, 296-315, corresponding to SEQ ID NOS: 2249, 2309 and 2354-2373. In a particular embodiment, the PDS comprises a gRNA MS2 variant wherein the scaffold is selected from the group consisting of gRNA scaffold variants 188, 251, 296-300, 304, 305, 307 and 313, corresponding to SEQ ID NOS: 2249, 2309, 2354- 2358, 2362-2363, 2365 and 2371.

V. Tropism Factors and Pseudotyping of PDS

[0168] In another aspect, the disclosure relates to the incorporation of tropism factors in the PDS systems and particles to increase tropism and selectivity for target cells, organ or tissues intended for gene editing. Tropism factors of the PDS embodiments include, but are not limited to, envelope glycoproteins derived from viruses, antibody fragments, and receptors or ligands that have binding affinity to target cell markers. The inclusion of such tropism factors on the surface of PDS particles enhances the ability of the PDS particles to selectively bind to and fuse with the cell membrane of a target ceil bearing such target cell markers, increasing the therapeutic index and reducing unintended side effects of the therapeutic payload incorporated into the PDS particles.

[0169] In some embodiments, the PDS comprises one or more gly coproteins (GP) incorporated on the surface of the particle wherein the GP provides for enhanced or selective binding and fusion of the PDS to a cell-surface marker of a target cell to be modified. In other embodiments, the PDS comprises one or more antibody fragments on the surface of the particle wherein the antibody fragments provides for enhanced or selective binding and fusion of the PDS to a cell-surface marker of a target cell. In other embodiments, the PDS comprises one or more cell surface receptors, including G-protem-linked receptors, and enzyme-linked receptors, on the surface of the particle wherein the receptor provides for enhanced or selective binding and fusion of the PDS to a cell-surface marker of a target cell. In some embodiments, the PDS comprises one or more ligands on the surface of the particle wherein the ligand provides for enhanced or selective binding and fusion of the PDS to a target cell bearing a receptor to the ligand on the cell surface. In still other embodiments, the PDS comprises a combination of one or more glycoproteins, antibody fragments, cell receptors, or ligands on the surface of the particle to provide for enhanced or selective binding and fusion of the PDS to a target cell.

[0170] For enveloped viruses, membrane fusion for viral entry is mediated by membrane glycoprotein complexes. Two basic mechanistic principles of membrane fusion have emerged as conserved among enveloped viruses; target membrane engagement and refolding into hairpin-like structures (Plemper, RK. Cell Entry of Enveloped Viruses. Curr Opin Virol. 1:92 (2011)). The envelope glycoproteins are typically observed as characteristic protein “spikes” on the surface of purified virions in electron microscopic images. The underlying mechanism of viral entry by enveloped viruses can be utilized to preferentially direct PDS to target particular cells, tissue, or organs in a process known as pseudotyping. In some embodiments, the PDS of the disclosure are pseudotyped by incorporation of a glycoprotein derived from an enveloped virus that has a demonstrated tropism for a particular organ or cell. Representative glycoproteins within the scope of the instant disclosure are listed in Table 9 as SEQ ID NOS: 601-824, and in the Examples, In some embodiments, the glycoproteins utilized in the PDS of the disclosure include, but are not limited to the cognate glycoproteins of the Mononegavirales virus that is the source of the MA protein incorporated into the PDS. In some embodiments, the glycoproteins utilized in the PDS of the disclosure are selected from the group consisting of the sequences of SEQ ID NOS: 1309-1596 as set forth in Table 22, or a sequence having 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% sequence identity thereto. In other embodiments, the glycoproteins utilized in the PDS of the disclosure include, but are not limited to non-cognate glycoproteins derived from a virus selected from the group consisting of Argentine hemorrhagic fever virus, Australian bat virus, Autographa califormca multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Boma disease virus, Breda vims, Bunyamwera virus, Chandipura vims, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue fever virus, Duvenhage virus. Eastern equine encephalitis virus, Ebola hemorrhagic fever virus, Ebola Zaire virus, enteric adenovirus, Ephemerovirus, Epstein-Bar virus (EBV), European bat virus 1, European bat vims 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 type 6, human herpesvirus type 8, human immunodeficiency virus 1 (HIV-1), human metapneumovirus, human T-lymphotropic virus 1, influenza A, influenza B, influenza C virus, Japanese encephalitis virus, Kaposi's sarcoma- associated herpesvirus (HHV8), Kaysanur Forest disease virus, La Crosse virus, Lagos bat virus, Lassa fever virus, lymphocytic choriomeningitis virus (LCMV), Machupo virus, Marburg hemorrhagic fever virus, measles virus, Middle eastern respiratory syndrome-related coronavirus, Mokola virus, Moloney murine leukemia virus, monkey pox, mouse mammary tumor vinis, mumps virus, murine gammaherpesvirus, Newcastle disease virus, Nipah virus, Nipah virus, Norwalk virus, Omsk hemorrhagic fever virus, papilloma virus, parvovirus, pseudorabies virus, Quaranfil virus, rabies virus, RD114 Endogenous Feline Retrovirus, respiratory syncytial virus (RSV), Rift Valley fever virus, Ross River virus, Rotavirus, Rous sarcoma vims, rubella virus, Sabia-associated hemorrhagic fever virus, SARS-associated coronavirus (SARS-CoV), Sendai virus, Tacaribe vims, Thogotovirus, tick-borne encephalitis causing virus, varicella zoster vims (HHV3), varicella zoster virus (HHV3), variola major virus, variola minor virus, Venezuelan equine encephalitis vims, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus (VSV), Vesiculovirus, West Nile virus, western equine encephalitis virus, and Zika Virus. Non-limiting examples of non-cognate glycoprotein sequences are provided in Table 9 as SEQ ID NOS: 601-824. In one exemplary embodiment, the glycoprotein incorporated into the PDS is glycoprotein G from vesicular stomatitis virus (VSV-G), which has the ability to bind to LDL receptors on a wide variety of mammalian cells (Finkelshtein, D., et al. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. PNAS 110:7306(2013)). In some embodiments, the PDS comprises one or more glycoprotein sequences of Table 9, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto, wherein the glycoproteins are incorporated and exposed on the surface of the PDS, providing tropism and enhanced selectivity for the PDS to the target cell to be edited. In some embodiments, the PDS comprises a glycoprotein selected from the group consisting of the polypeptide sequences of SEQ ID NOS: 601-824 as set forth in Table 9.

Tabie 9: Glycoproteins for PDS

[0171 ] In some embodiments, a PDS particle comprising a glycoprotein derived from an enveloped virus, for example in the capsid, exhibits at least a 2-fold, or at least a 3-fold, or at least a 4-fold, or at least a 5-fold, or at least a 10-fold increase in binding of the PDS particle to a target cell compared to a PDS particle that does not have the glycoprotein, when assayed in an in vitro binding assay under comparable conditions. Representative examples demonstrating enhanced binding and uptake of PDS particles bearing glycoproteins to target cells leading to, in this case, enhance gene editing of target nucleic acid, are provided in the Examples, below.

[0172] In some embodiments, the present disclosure provides PDS particles comprising an antibody fragment linked to the exterior of the particle wherein the antibody fragment has specific binding affinity to a target cell marker or receptor on a target cell, tissue or organ, providing tropism for the PDS for the target cell. In one embodiment, the antibody fragment is selected from the group consisting of an Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, single chain diabody, linear antibody, a single domain antibody, a single domain camelid antibody, and a single-chain variable fragment (scFv) antibody. The target cell marker or ligand can include cell receptors or surface proteins known to be expressed preferentially on a target cell for which nucleic acid editing is desired. In such cases, aPDS particle comprising an antibody fragment in the capsid exhibits at least a 2-fold, or at least a 3-fold, or at least a 4- fold, or at least a 5-fold, or at least a 10-fold increase in binding to a target cell bearing the target cell marker or receptor compared to a PDS particle that does not have the antibody fragment. In the case of antibody fragments with affinity to ceil markers or receptors, the cell markers or receptors can include, but not be limited to cluster of differentiation 19 (CD 19), cluster of differentiation 3 (CD3), CD3d molecule (CD3D), CD3g molecule (CD3G), CD3e molecule (CD3E), CD247 molecule (CD247, or CD3Z), CD8a molecule (CD8), CD7 molecule (CD7), membrane metalloendopeptidase (CD 10), membrane spanning 4-domains Al (CD20), CD22 molecule (CD22), TNF receptor superfamily member 8 (CD30), C-type lectin domain family 12 member A (CLL1), CD33 molecule (CD33), CD34 molecule (CD34), CD38 molecule (CD38), integrin subunit alpha 2b (CD41), CD44 molecule (Indian blood group) (CD44), CD47 molecule (CD47), integrin alpha 6 (CD49f), neural ceil adhesion molecule 1 (CD56), CD70 molecule (CD70), CD74 molecule (CD74), CD99 molecule (Xg blood group) (CD99), interleukin 3 receptor subunit alpha (CD123), prominin 1 (CD133), syndecan 1 (CD138), carbonix anhydrase IX (CAIX), CC chemokine receptor 4 (CCR4), ADAM metallopeptidase domain 12 (ADAM12), adhesion G protein-coupled receptor E2 (ADGRE2), alkaline phosphatase placental-like 2 (ALPPL2), alpha 4 Integrin, angiopoietin-2 (ANG2), B-cell maturation antigen ( BCM A ), CD44V6, carcinoembiyonic antigen (CEA), CEAC, CEA ceil adhesion molecule 5 (CEACAM5), Claudin 6 (CLDN6), CLDN18, C-type lectin domain family 12 member A (CLEC12A), mesenchymal-epithelial transition factor (cMET), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), epidermal growth factor receptor 1 (EGF1R), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP-2), epithelial cell adhesion molecule (EGP-40 or EpCAM), EPH receptor A2 (EphA2), ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), erb-b2 receptor tyrosine kinase 2 (ERBB2), erb-b2 receptor tyrosine kinase 3 (ERBB3), erb-b2 receptor tyrosine kinase 4 (ERBB4), folate binding protein (FBP), fetal nicotinic acetylcholine receptor (AChR), folate receptor alpha (Fralpha or FOLR1), G protein-coupled receptor 143 (GPR143), glutamate metabotropic receptor 8 (GRM8), glypican-3 (GPC3), ganglioside GD2, ganglioside GD3, human epidermal growth factor receptor 1 (HERI), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), , Integrin B7, intercellular cell-adhesion molecule-1 (ICAM-1), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor a2 (IL-13R-a2), K-light chain. Kinase insert domain receptor (KDR), Lewis-Y (LeY), chondromodulin-1 (LECT1), LI cell adhesion molecule (LI CAM). Lysophosphatidic acid receptor 3 (LPAR3), melanoma-associated antigen 1 (MAGE-A1), mesothelin (MSLN), mucin 1 (MUCI), mucin 16, cell surface associated (MUCI 6), melanoma- associated antigen 3 (MAGEA3), tumor protein p53 (p53), Melanoma Antigen Recognized by T cells 1 (MARTI ), glycoprotein 100 (GP100), Proteinase3 (PR1), ephrin-A receptor 2 (EphA2), Natural killer group 2D ligand (NKG2D ligand), New York esophageal squamous cell carcinoma 1 (NY-ESO-1), oncofetal antigen (h5T4), prostate-specific membrane antigen (PSMA), programmed death ligand 1 (PDL-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (TPBG), tumor-associated glycoprotein 72 (TAG-72), tumor-associated calcium signal transducer 2 (TROP-2), tyrosinase, survivin, vascular endothelial growth factor receptor 2 (VEGF- R2), Wilms tumor-1 (WT-1), leukocyte immunoglobulin-like receptor B2 (LILRB2), Preferentially Expressed Antigen In Melanoma (PRAME), T cell receptor beta constant l(TRBCl), TRBC2, and (T-cell immunoglobulin mucin-3) TIM-3, or fragments thereof. In the case of antibody fragments with affinity to neuron receptors, the cell markers or receptors can include, but not be limited to Adrenergic (e.g., al A, alb, al c, aid, a2a, a2b, a2c, a.2d, pl, p2, p3), Dopaminergic (e.g., DI, D2, D3, D4, D5), GABAergic (e.g., GABAA, GABABla, GABAB15, GABAB2, GABAC), Glutaminergic (e.g., NMD A, AMP A, kainate, mGluRl, mGIuR2, mGluR3, mGluR4, mGluR.5, mGluR6, mGluR7), Histaminergic (e.g.. Hl, H2, H3), Cholinergic (e.g., Muscarinic (e.g., Ml, M2, M3, M4, M5; Nicotinic (e.g., muscle, neuronal (a-bungarotoxin-insensitive), neuronal (a-bungarotoxin-sensitive)), Opioid (e.g., p, 51, 52, K), and Serotonergic (e.g., 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F, 5-HT2A, 5- HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5, 5-HT6, 5-HT7).

[0173] In one embodiment, the antibody fragment is conjugated to the PDS particle after its production and isolation from the producing host cell. In another embodiment, the antibody fragment is produced as a part of the PDS capsid expressed by the packaging host cell of the PDS system, for example by transfection of a nucleic acid encoding the antibody fragment into the packaging host cell along with the nucleic acids encoding the remaining components of the PDS. VI. Nucleic Acids Encoding PDS Systems

[0174] In another aspect, the present disclosure relates to nucleic acids encoding components of the PDS system, and the vectors that comprise the nucleic acids, as well as methods to make the nucleic acids and vectors. In some embodiments, the nucleic acids encode the incorporated therapeutic payload(s).

[0175] In some embodiments, the present disclosure provides one or more nucleic acids encoding components including viral-derived PDS structural components, as well as nucleic acids encoding therapeutic payloads and tropism factors. The nucleic acids utilized for the key structural components of the PDS particles of the embodiments can be derived from a variety of viruses from the Mononegavirales order, including but not limited to viruses from the family members Artoviridae, Bomaviridae, Filoviridae, Lispiviridae, Mymonaviridae, Nyamiviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Sunviridae, and Xinmoviridae . In some embodiments, the nucleic acids of the embodiments are incorporated into plasmid vectors that can be transfected into eukaryotic packaging host cells that, when cultured under appropriate conditions, lead to the expression of the PDS structural and processing components, therapeutic payloads, and tropism factors, self-assembly of the PDS particles that encapsidate the therapeutic payloads and incorporate the tropism factor upon budding from the packaging cells. The nucleic acids can be designed to result in PDS in various configurations. Representative, but non-limiting configurations of PDS of the disclosure are presented in Table 10, below, and are described more fully in the Examples. In some embodiments, plasmid 1 can, optionally, encode short linkers of any of the linker embodiments described herein between the MA and NCR or CasX components.

[0176] In some embodiments, the nucleic acid encoding the MA protein is derived from an Artoviridae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Bornavindae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Filoviridae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Lispiviridae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Mymonaviridae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Nyamiviridae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Paramyxoviridae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Pneumoviridae vims. In some embodiments, the nucleic acid encoding the MA protein is derived from a Rhabdoviridae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Sunviridae virus. In some embodiments, the nucleic acid encoding the MA protein is derived from a Xlnmoviri dae virus. In the foregoing embodiments, the disclosure further contemplates incorporation of a nucleic acid encoding a nucleocapsid, which can be from the same Mononegavirales virus as the MA protein, or may be from a different Mononegavirales virus. In some embodiments, the nucleic acid encoding the MA protein is selected from the group of sequences of Table 12 (SEQ ID NOS: 51-67), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0177] In some embodiments, the present disclosure provides nucleic acids encoding sequences for the tropism factors that are incorporated in, and displayed on the surface of the PDS particles upon their release from the eukaryotic packaging cell, wherein the tropism factor confers an increased ability of the PDS to bind and fuse with the membrane of a target cell, organ or tissue. In some embodiments, the tropism factor is a glycoprotein of a Mononegavirales virus, wherein the encoding nucleic acid is selected from the group consisting of the sequences of SEQ ID NOS: 1309-1596 as set forth in Table 22, or a sequence having 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. In other embodiments, the tropism factor is a glycoprotein of a virus other than & Mononegavirales virus, wherein the encoded sequence is selected from the group consisting of the sequences of SEQ ID NOS: 601-824 as set forth in Table 9, or a sequence having 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. In another embodiment, the disclosure provides a nucleic acid encoding a non-viral tropism factor. In some embodiments, the tropism factor is an antibody fragment, wherein the antibody fragment has specific binding affinity for a target cell marker or receptor on a target cell, organ or tissue. In another embodiment, the disclosure provides nucleic acids encoding a cell receptor, wherein the cell receptor has specific binding affinity for a target cell marker on a target cell, organ or tissue. In another embodiment, the disclosure provides nucleic acids encoding a ligand, wherein the ligand has specific binding affinity for a target cell marker or receptor on a target ceil, organ or tissue. By inclusion of the nucleic acids encoding for the tropism factors, it will be understood that the resulting PDS particles will have increased selectivity for the target cell, organ or tissue, resulting in an increased therapeutic index and reduced off-target effects.

[0178] The present disclosure further provides nucleic acids encoding or comprising the therapeutic payloads incorporated into the PDS particles. Exemplary therapeutic payloads have been described herein, supra. In some embodiments, the therapeutic pay load of the PDS is a CRISPR nuclease and one or more guide RNAs. In a particular embodiment of the foregoing, the disclosure provides nucleic acids encoding the CasX nucleases of SEQ ID NOS: 136-176, 180-506 or 1905 as set forth in Table 3, SEQ ID NOS: 7731-7891, SEQ ID NOS: 7978-7980, or a sequence having 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. In some embodiments, the disclosure provides nucleic acids encoding the CasX nucleases of SEQ ID NOS: 190 and 197. Representative examples of such nucleic acids are presented as SEQ ID NOS: 1887-1904. In another particular embodiment of the foregoing, the disclosure provides nucleic acids encoding the gRNA variants consisting of the sequences of SEQ ID NOS: 2238-2258 and 2260-2431 as set forth in Table 8, or a sequence having 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, wherein the gRNA further comprises a targeting sequence complementary' to a target nucleic acid.

[0179] In some embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the PDS system selected from two or more of zMononegavirales MA protein, a Mononegavirales NC protein, a therapeutic payload, an NCR, and a tropism factor, wherein the components are encoded on two, three, or four individual nucleic acids. In some embodiments of the foregoing, a first nucleic acid encodes the MA protein and NCR, a second nucleic acid encodes a therapeutic pay load of any of the embodiments described herein, and a third nucleic acid encodes the tropism factor of any of the embodiments described herein. In some embodiments of the foregoing, a first nucleic acid encodes the MA protein and the CRISPR protein as a therapeutic payload with, optionally, an intervening linker between the two components, a second nucleic acid encodes the tropism factor, and a third nucleic acid encodes the gRN A as a therapeutic payload. In another embodiment of the foregoing, a first nucleic acid encodes the MA protein and an NCR with, optionally, an intervening linker between the two components, a second nucleic acid encodes a CRISPR protein as a therapeutic payload, a third nucleic acid encodes a gRNA as a therapeutic pay load, and a fourth nucleic acid encodes a tropism factor. In another embodiment of the foregoing, a first nucleic acid encodes the MA protein, the NC protein, and an NCR with, optionally, an intervening linker between the components, a second nucleic acid encodes a CRISPR as a therapeutic payload, a third nucleic acid encodes a gRNA as a therapeutic payload, and a fourth nucleic acid encodes a tropism factor. In another embodiment of the foregoing, a first nucleic acid encodes the MA protein, the NC protein, and the CRISPR protein, with, optionally, an intervening linker between the components, a second nucleic acid encodes a gRNA tropism factor, and a third nucleic acid encodes the tropism factor. In another embodiment of the foregoing, a first nucleic acid encodes the MA protein and an NCR with, optionally, an intervening linker between the two components, a second nucleic acid encodes a dXR fusion protein comprising a dCasX and one or more repressor domains selected from the group consisting of Kruppel associated box (KRAB) domain selected from the group consisting of the sequences of SEQ ID NOS: 7720-7728, DNMT3A catalytic domain, DNMT3L interaction domain, and DNMT3A ADD domain as a therapeutic payload, a third nucleic acid encodes a gRNA as a therapeutic payload, and a fourth nucleic acid encodes a tropism factor. In particular embodiments of the foregoing, the CRISPR protein, dXR and gRNA are from the CasX system, including the sequences of Tables 3, 6, and 8.

Table 10: Representative PDS plasmid configurations

*5’ to 3' orientation

[0130] In some embodiments, the nucleic acids encoding the PDS system of any of the embodiments described herein further comprises a donor template nucleic acid wherein the donor template comprises a sequence to be inserted into a target nucleic acid to either correct a mutation or to knock-down or knock-out a gene. In some embodiments, the donor template sequence comprises a non-homologous sequence flanked by two regions of homology 5’ and 3’ to the break sites of the target nucleic acid (i.e., homologous arms), facilitating insertion of the non-homologous sequence at the target region which can be mediated by HDR or HITT. The exogenous donor template inserted by HITI can be any length, for example, a relatively short sequence of between 1 and 50 nucleotides in length, or a longer sequence of about 50- 1000 nucleotides in length. The lack of homology can be, for example, having no more than 20-50% sequence identity and/or lacking in specific hybridization at low stringency. In other cases, the lack of homology can further include a criterion of having no more than 5, 6, 7, 8, or 9 bp identity. In such cases, the use of homologous arms facilitates the insertion of the non-homologous sequence at the break site(s) introduced by the nuclease. In some embodiments, the donor template polynucleotide comprises at least about 10, at least about 50, at least about 100, or at least about 200, or at least about 300, or at least about 400, or at least about 500, or at least about 600, or at least about 700, or at least about 800, or at least about 900, or at least about 1000, or at least about 10,000, or at least about 15,000 nucleotides. In other embodiments, the donor template comprises at least about 10 to about 15,000 nucleotides, or at least about 100 to about 10,000 nucleotides, or at least about 400 to about 8,000 nucleotides, or at least about 600 to about 5000 nucleotides, or at least about 1000 to about 2000 nucleotides. The donor template sequence may comprise certain sequence differences as compared to the genomic sequence; e.g., restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor nucleic acid at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus). Alternatively, these sequence differences may include flanking recombination sequences such as FLPs, loxP sequences, Cre sequences, or the like, that can be activated at a later time for removal of the marker sequence. In another embodiment, the donor template comprises a nucleic acid encoding at least a portion of a target gene wherein the donor template nucleic acid comprises all or a portion of the wildtype sequence compared to the target gene comprising a mutation, wherein the donor template is inserted into the target nucleic acid of the cell by HDR during the gene editing process. In such cases, upon insertion into the target nucleic acid, the target gene is corrected such that the functional gene product can be expressed. In some embodiments, each of the individual nucleic acids are incorporated into plasmid vectors appropriate for transfection into a eukaryotic packaging host cell, examples of which are detailed more fully, below, such that the PDS system will involve two, three, or four plasmids. In each case, the nucleotide sequence encoding the components of the PDS system are operably linked to (under the control of) promoters and accessory elements operable in a eukaryotic cell and appropriate for the component to be expressed.

[0181 ] Non-limiting examples of Pol 11 promoters include, but are not limited to EFlalpha, EF-1 alpha core promoter, Jens Tornoe (JeT), promoters from cytomegalovirus (CMV), CMV immediate early (CMVTE), CMV enhancer, herpes simplex virus (HSV) thymidine kinase, early and late simian virus 40 (SV40), the SV40 enhancer, long terminal repeats (LTRs) from retrovirus, mouse metallothionein-I, adenovirus major late promoter (Ad MLP), CMV promoter full-length promoter, the minimal CMV promoter, the chicken P-actin promoter (CBA), CBA hybrid (CBh), chicken P-actin promoter with cytomegalovirus enhancer (CB7), chicken beta- Actin promoter and rabbit beta-Globin splice acceptor site fusion (CAG), the rous sarcoma virus (RSV) promoter, the HIV-Ltr promoter, the hPGK promoter, the HSV TK promoter, a 7SK promoter, the Mini-TK promoter, the human synapsin I (SYN) promoter which confers neuron-specific expression, beta-actin promoter, super core promoter I (SCP1), the Mecp2 promoter for selective expression in neurons, the minimal IL-2 promoter, the Rous sarcoma virus enhancer/promoter (single), the spleen focusforming virus long terminal repeat (LTR) promoter, the TBG promoter, promoter from the human thyroxine-binding globulin gene (Liver specific), the PGK promoter, the human ubiquitin C promoter (UBC), the UCOE promoter (Promoter of HNRPA2B1-CBX3), the synthetic CAG promoter, the Histone H2 promoter, the Histone H3 promoter, the Ulal small nuclear RNA promoter (226 nt), the Ulal small nuclear RNA promoter (226 nt), the Ulb2 small nuclear RNA promoter (246 nt) 26, the GUSB promoter, the CBh promoter, rhodopsin (Rho) promoter, silencing-prone spleen focus forming virus (SFFV) promoter, a human Hl promoter (Hl), a POLI promoter, the TTR minimal enhancer/promoter, the b-kinesin promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, the human eukaryotic initiation factor 4A (EIF4A1) promoter, the ROSA26 promoter, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, tRNA promoters, and truncated versions and sequence variants of the foregoing. In a particular embodiment, the Pol II promoter is EF-1 alpha, wherein the promoter enhances transfection efficiency, the transgene transcription or expression of the CRISPR nuclease, the proportion of expression- positive clones and the copy number of the episomal vector in long-term culture. Nonlimiting examples of Pol III promoters include, but are not limited to U6, mini IJ6, U6 truncated promoters, BiHl (Bidrectional Hl promoter), BiU6, Bi7SK, BiHl (Bidirectional U6, 7SK, and Hl promoters), gorilla U6, rhesus U6, human 7SK, human Hl promoter, and truncated versions and sequence variants thereof. In the foregoing embodiment, the Pol III promoter enhances the transcription of the gRNA.

[0182] Recombinant expression vectors of the disclosure can also comprise accessory elements that facilitate robust expression of the CasX proteins and the gRNA of the disclosure. For example, recombinant expression vectors can include one or more of a poly adenylation signal (Poly ( A), an intronic sequence or a post-transcriptional regulatory element such as a woodchuck hepatitis post-transcriptional regulatory element (WPTRE). Exemplary poly(A) sequences include hGH poly(A) signal (short), HSV TK poly(A) signal, synthetic polyadenylation signals, SV 40 poly(A) signal, p-globin poly(A) signal and the like. A person of ordinary' skill in the art will be able to select suitable elements to include in the recombinant expression vectors described herein.

[0183] In some embodiments, the PDS system of the disclosure comprises two nucleic acids. In some embodiments, the PDS system of the disclosure comprises three nucleic acids. In some embodiments, the PDS sy stem of the disclos ure comprises four nucleic acids. In some embodiments, the PDS system of the disclosure comprises five nucleic acids.

Exemplary embodiments of the nucleic acids (and plasmids) and the configuration of the components encoded by each the nucleic acids are presented in Table 10, as well as in the Examples, below. It will be understood that in each case, the CRISPR protein, the gRNA, the NCR, and the tropism factor of the table can comprise any of the embodiments described herein.

VII. PDS Packaging Ceils

[0184] In another aspect, the present disclosure relates to packaging host cells utilized in the production of PDS. It has been discovered that components derived, in part, from Mononegavirales viruses can be utilized to create PDS particles within packaging cells for delivery' of a therapeutic payload to the target cells. In some embodiments, the packaging cell transformed with the PDS system plasmids produce PDS that facilitate delivery' of the encapsidated RN P of a CRI SPR Class 2 nuclease and a gRNA (e.g., CasX:gRNA) system to cells to effect editing or modifi cation of a target nucleic acid in a cell. In a particular embodiment, the packaging cell transformed with the PDS system plasmids produce PDS particles that facilitate delivery of the encapsidated RNP of a CasX:gRNA system to cells to effect editing or modification of a target nucleic acid in a cell.

[0185] As used herein, the term “packaging cell” or “packaging host cell” is used in reference to cell lines that do not contain a packaging signal, but do stably or transiently express viral structural proteins and replication enzymes which are necessary or useful for the correct packaging of PDS particles. In some embodiments, the cell line can be any cell line suitable for the production of PDS, including primary ex vivo cultured cells (from an individual organism) as well as established cell lines. Cell types may include bacterial cells, yeast cells, and mammalian cells. Exemplary bacterial cell types may include E. colt. Exemplary yeast cell types may include Saccharomyces cerevisiae. Exemplary mammalian cell types may include mouse, hamster, and human primary cells, as well as cell lines such as human embryonic kidney 293 (HEK293) cells, Lenti-X 293T cells, baby hamster kidney (BHK) cells, HepG2 cells, Saos-2 cells, HuH7 cells, NSO cells, SP2/0 cells, ¥0 myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS cells, WI38 cells, MRC5 cells, A549 cells, HeLa cells, Chinese hamster ovary (CHO) cells, or HT1080 cells. The choice of the appropriate vector for the cell type will be readily apparent to the person of ordinary skill in the art.

[0186] In some embodiments, the packaging host cell can be modified to reduce or eliminate cell surface markers or receptors that would otherwise be incorporated into the PDS, thereby reducing an immune response to the cell surface markers or receptors by the subject receiving an administration of the PDS. Such markers can include receptors or proteins capable of being bound by major histocompatibility complex (MHC) receptors or that would otherwise trigger an immune response in a subject. In some embodiments, the packaging host cell is modified to reduce or eliminate the expression of a cell surface marker selected from the group consisting of B2M, CUT A, PD1, and HLA-E, wherein the incorporation of the marker is reduced on the surface of the PDS. In some embodiments, the packaging host cell is modified to express one or more cell surface markers selected from the group consisting of CD46, CD47, CD55, CD59, CD24, CD58, SLAMF4, and SLAMF3 (serving as "don't eat me" signals), wherein the cell surface marker is incorporated onto the surface of the PDS, wherein said incorporation disables PDS engulfment and phagocytosis by host surveillance cells such as macrophages and monocytes,

[0187] In some embodiments of the PDS system, vectors are introduced into the packaging host cell that encode the particular therapeutic payload (e.g., a CasX:gRNA designed for editing target nucleic acid), as well as the other viral-derived structural components, detailed above, (e.g., the MA protein, the tropism factor, and, optionally, the donor template nucleic acid sequence). The vectors can remain as extra-chromosomal elements or some or all can be integrated into the host cell chromosomal DNA to create a stably-transformed packaging host cell.

[0188] In some embodiments, the vectors comprising the nucleic acids of the PDS system are introduced into the cell via transfection, transduction, lipofection or electroporation to generate a packaging host cell line. The introduction of the vectors can use one or more of the commercially available TransMessenger® reagents from Qiagen, Stemfect RNA Transfection Kit from Stemgent, and TransIT-mRNA Transfection Kit from Minis Bio LLC, Lonza nucleofection, Maxagen electroporation and the like. Methods for transfection, transduction or infection are well known to those of skill in the art.

[0189] In some cases, the packaging vectors are introduced into the cells together with a dominant selectable marker, such as neo, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be linked physically to genes encoding by the packaging vector.

[0190} Assembly and release of PDS with the encapsidated therapeutic payload from the transfected host cell can be mediated by the viral structural protein, MA. Utilizing an Mononegavirales system, it is sufficient to express the MA protein to allow the efficient, production of PDSs from cells. Upon expression, the MA protein is targeted to the cell membrane and incorporated in the PDS during membrane budding. In those cases where the therapeutic protein (e.g., CasX or a CasX variant) is fused to the MA protein, the therapeutic protein is encapsidated within the PDS particle upon assembly. In those cases where an NCR is fused to the MA protein, the gRNA and the complexed CasX are recruited in anon- covalent fashion into the PDS particle during its assembly. The tropism factor is incorporated onto the surface of the PDS particle during the budding of the PDS from the host packaging cell ,

VIII. PDS Expression Systems and Methods of Producing PDS

[0191] In another aspect, the present disclosure provides a recombinant expression system for use in the production of PDS particles in a selected host packaging host cell, comprising an expression cassette comprising the nucleic acids of the PDS system described herein operably linked to promoters and accessory elements compatible with expression in the selected host cell. The expression cassettes may be included on one or more vectors as described herein and in the Examples, and may use the same or different promoters. Exemplary accessory elements include a transcription promoter, MMLV-ltr trans-activator, internal ribosome entry' site (IRES) or p2A peptide to permit translation of mul tiple genes from a single transcript, metallothionein, a transcription enhancer element, a transcription termination signal, poly adenylation sequences, sequences for optimization of initiation of translation, and translation termination sequences. It will be understood that the choice of the appropriate control element will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0192] In some embodiments, a nucleotide sequence encoding each therapeutic payload (e.g., a gRNA, a gRNA variant, a CasX or a CasX variant protein) is operably linked to an inducible promoter, a constitutively active promoter, a spatially restricted promoter (i.e., transcriptional control element, enhancer, tissue specific promoter, cell type specific promoter, etc.), or a temporally restricted promoter. In certain embodiments, suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. In other embodiments, individual nucleotide sequences encoding the gRNA or the CasX are linked to one of the foregoing categories of promoters, which are then introduced into the cells to be modified by conventional methods, described below.

[0193] Non-limiting examples of Pol II promoters include, but are not limited to UBC, CMV, SV40, CAG, CB7, PGK, JeT, GUSB, CBh, EF-lalpha, beta-actin, RSV, SFFV, CMVdl promoter, truncated human CMV (tCMVd2), minimal CMV promoter, chicken p- actin promoter, HSV TK promoter, Mini-TK promoter, minimal IL-2 promoter, GRP94 promoter, Super Core Promoter I, Super Core Promoter 2, MLC, MCK, GRKI protein promoter, Rlio promoter, and CAR protein promoter, hSyn Promoter, U1A promoter, Ribsomal Rpl and Rps promoters (for example, hRp!30 and hRpsl 8), CMV53 promoter, minimal SV40 promoter, CMV53 promoter, SFCp promoter, pJB42CAT5 promoter, MLP promoter, EFS promoter, MeP426 promoter, MecP2 promoter, MHCK7 promoter, CK7 promoter, and CK8e promoter. In a particular embodiment, the Pol II promoter is EF-lalpha, wherein the promoter enhances transfection efficiency, the transgene transcription or expression of the CRISPR nuclease, the proportion of expression- positive clones and the copy number of the episomal vector in long-termculture. pH 941 Non-limiting examples of Pol III promoters include, but are notlimited to U6, mini U6, 7SK, and Hl variants, BiHl (Bidrectional Hl promoter), BiU6, Bi7SK, BiHl (Bidirectional U6, 7SK, and Hl promoters), gorilla U6, rhesus U6, human 7SK, and human Hl promoters. In the foregoing embodiment, the Pol III promoter enhances the transcription of the gRNA. Selection of the appropriate promoter is well within the level of ordinary skill in the art, as it relates to controlling expression, e.g., for modifying a gene or other target nucleic acid.

JOI 95 | In some embodiments, the present disclosure provides methods of making an PDS particle comprising a therapeutic payload (e.g., an RNP of a CasX protein and a gRNA), the method comprising propagating the packaging host cell of the embodiments described herein comprising the expression cassettes or the integrated nucleic acids encoding the PDS of anyone of the embodiments described herein under conditions such that PDS particles are produced with the encapsi dated therapeutic payload, followed by harvesting the PDS particles produced by the packaging host cell, as described below or in the Examples. In some embodiments, the packaging host cell produces PDS particles comprising RNP of a CasX and gRNA and, optionally, a donor template for the editing of the target nucleic acid by HDR.

[0196] The packaging host cell can be, for example, a mammalian cell (e.g., HEK293 cells, Lenti-X 293T cells, BHK cells, HepG2 cells, Saos-2 cells, HuH7 cells, NSO cells, SP2/0 cells, YO myeloma cells, A549 cells, P3.X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS ceils, WI38 cells, MRC5 cells, A549 ceils, HeLa cells, CHO cells, and HT1080 cells), an insect cell (e.g., Trichoplusia ni (Tn5) or SIP), a bacterial cell, a plant cell, a yeast cell, an antigen presenting cell (e.g., primary, immortalized or tumor-derived lymphoid ceils such as macrophages, monocytes, dendritic cells, B-celis, T-cells, stem cells, and progenitor cells thereof). Packaging ceils can be transfected by conventional methods, including electroporation, use of cationic polymers, calcium phosphate, virus-mediated transfection, transduction, or lipofection. In some embodiments, the packaging host cell can be modified to reduce or eliminate cell surface markers or receptors that would otherwise be incorporated into the PDS, thereby reducing an immune response to the cell surface markers or receptors by the subject receiving an administration of the PDS. In some embodiments, the packaging host cell is modified to reduce or eliminate the expression of a cell surface marker selected from the group consisting of B2M, CD47 and HLA-E, wherein the incorporation of the marker is reduced at least about 2-fold, at least about 5-fold, or at least about 10-fold on the surface of the PDS released from the packaging host cell compared to PDS released from a packaging host cell that has not been so modified. In the foregoing embodiment, the reduction or elimination of the cell surface marker reduces a host immune response to the PDS administered to a subject. In some embodiments, the packaging host cell is modified to express one or more cell surface markers selected from CD46, CD47, CD55, CD59, CD24, CD58, SLAMF4, and SL.AMF3 (serving as "don't eat me" signals), wherein the incorporation of the marker is increased at least about 2-fold, at least about 5-fold, or at least about 10-fold on the surface of the PDS released from the packaging host cell compared to PDS released from a packaging host cell that has not been so modified. In the foregoing embodiment, the increased expression of the cell surface marker disables PDS engulfment and phagocytosis by host surveillance cells such as macrophages and monocytes.

[0197] The introduction of the vectors into the packaging host cell can use one or more of the commercially available TransMessenger reagents from Qiagen, Stemfect RNA Transfection Kit from Stemgent, and TransIT-mRNA Transfection Kit from Minis Bio LLC, Lonza nucleofection, Maxagen electroporation and the tike. Methods for transfection, transduction or infection are well known to those of skill in the art.

[0198] In one embodiment, PDS particles are produced by the incubation of the transfected packaging host cells in appropriate growth medium for 48 to 96 hours and are collected by filtration of the grow th medium. In some cases, the PDS can be further concentrated by centrifugation in a 10% or a 10-30% density-’ gradient sucrose buffer. In other cases, the PDS can be concentrated by column chromatography, such as by use of an ion-exchange resin or a size exclusion resin.

IX. Applications

[0199} The PDS particles and systems provided herein are useful in methods for delivery' of the therapeutic payload to a cell and in methods of modifying of a target nucleic acid in a gene. In some embodiments, the disclosure provides methods of delivery' and use of the PDS particles systems comprising RNP of a CRISPR Class 2 nuclease and gRNA provided herein for modifying or editing target nucleic acids in cells. In some embodiments, the disclosure provides methods of modifying of a target nucleic acid in a gene using PDS particles comprising RNPs of a CasX variant and gRNA variant of any of the embodiments provided herein. In some embodiments of the method, the method utilizes any of the PDS particles mbodiments comprising RNPs of the CasX:gRNA systems described herein, and optionally includes a donor template embodiment described herein. In some cases, the method knocks- down the expression of a mutant protein in cells comprising the target nucleic acid. In other cases, the method knocks-out the expression of the mutant protein. In still other cases, the method results in the correction of one or more mutations in the target nucleic acid, resulting in the expression of a functional gene product.

[0001 j In other embodiments, the disclosure provides methods of repression of a target nucleic acid in a gene using PDS particles comprising RNPs of a dXR and gRNA variant of any of the embodiments provided herein. In some cases, the method results in repression of transcription of the target nucleic acid. In other cases, the method results in the epigenetic modification of the target nucleic acid such that repression of transcription is heritable and is stable through one or more cell divisions. In some embodiments of the method, repression of transcription is stable through 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 cell divisions, or more.

[0200 [ In some embodiments, the method comprises contacting the cells comprising the target nucleic acid with an effective dose of PDS particles comprising RN Ps of a CasX protein or dXR and a guide ribonucleic acid (gRNA) comprising a targeting sequence complementary' to the target nucleic acid, wherein said contacting results in modification of the target nucleic acid by the CasX protein or dXR. In another embodiment, the PDS further comprises a donor template wherein contacting the cell with the PDS results in insertion of the donor template into the target nucleic acid sequence. In some cases, the donor template is used in conjunction with the RNP to correct a mutation a target gene, while in other cases the donor template is used to insert a mutation to knock-down or knock-out expression of the expression product of the target gene.

[0201] In some embodiments, the method of modifying a target nucleic acid in a cell comprises contacting the cells comprising the target nucleic acid with an effective dose of PDSs wherein the cell is modified in vitro or ex vivo.

[0202] In other embodiments of the method of modifying a target nucleic acid in a cell, the cells are modified in vivo, wherein a therapeutically-effective dose of the PDS is administered to a subject. The method has the advantage over viral delivery' systems in that the RNP of the PDS particles are comparatively short-lived relative to the nucleic acids delivered in viral systems such as AAV. A further advantage of the PDS system is the ability to match the particles produced by system to specific cell types by manipulating the tropism of the PDS. In some embodiments, the half-life of the delivered RNP is about 24 hours (h), or about 48h, or about 72h, or about 96h. or about 120h, or about 1 week. By the methods of treatment, the administration of the PDS results in the improvement of one, two, or more symptoms, clinical parameters or endpoints associated with the disease in the subject,

[0203] In some embodiments, the subject administered the PDS particles is selected from the group consisting of mouse, rat, pig, non-human primate, and human. In a particular embodiment, the subject is a human. In some embodiments, the PDS particles are administered to the subject in a therapeutically effective dose. In one embodiment, the PDS is administered by a route of administration selected from the group consisting of subcutaneous, intradermal, intraneural, intranodal, intramedullary, intramuscular, intravenous, intra-arterial, intracerebroventricular, intraci sternal, intrathecal, intracranial, intralumbar, intratracheal, intraosseous, mhalatory, intracontralateral striatum, intraocular, mtravitreal, intralymphatical, intraperitoneal routes and sub-retinal routes, wherein the administering method is injection, transfusion, or implantation.

[0204] In another embodiment, the disclosure provides a method of treatment of a subject having a disease according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose of a PDS particle of any of the embodiments described herein. In one embodiment of the treatment regimen, the therapeutically effective dose is administered as a single dose. In another embodiment of the treatment regimen, the therapeutically effective dose is administered to the subject as two or more doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months, or once a year, or every 2 or 3 years.

X. Kits and Articles of Manufacture

[0205] In another aspect, provided herein are kits comprising the compositions of the embodiments described herein. In some embodiments, the kit comprises a plurality' of PDS particles comprising a therapeutic pay load of any of the embodiment described herein, an excipient and a suitable container (for example a tube, vial or plate). In a particular embodiment, the therapeutic payload is an RNP of a CasX protein and a gRNA. In some embodiments, the kit comprises nucleic acids encoding the PDS particle structural components (e.g., MA and NC), the therapeutic payload(s) of any of the embodiments described herein, and the tropism factor of any of the embodiments described herein, together with an excipient and a suitable container. [0206] In some embodiments, the kit further comprises a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing. In some embodiments, the kit further comprises a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the kit further comprises instructions for use.

[0207] The present description sets forth numerous exemplary' configurations, methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments. Embodiments of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments and is not limited to combinations of embodiments explicitly provided below.

ILLU STRATIVE EMBODIMENTS

[0208] The invention can be understood with reference to the following illustrative, enumerated embodiments:

[0209] 1. A particle delivery system (PDS) comprising components selected from: (a) a fusion protein comprising one or more Mononegavirales structural proteins and at least one heterologous protein; (b) one or more therapeutic payloads; and (c) a tropism factor.

[0210] 2. The PDS of embodiment 1, wherein the Mononegavirales structural protein is a matrix protein (MA), a nucleocapsid protein (NC), or is both MA and NC.

[0211] 3. The PDS of embodiment 1 or embodiment 2, wherein the at least one heterologous protein is selected from the group consisting of one or more non-covalent recruitment (NCR) proteins and a therapeutic protein.

[0212] 4. The PDS of any one of embodiments 1-3, wherein the at least one heterologous protein is linked to the Mononegavirales structural protein with a linker selected from the group consisting of SEQ ID NOS: 1253-1308.

[0213] 5. The PDS of embodiment 3 or embodiment 4, wherein the one or more non- covalent recruitment (NCR) proteins are selected from the group consisting of an MS2 coat protein, a PP7 coat protein, a QD coat protein, a U1 A signal recognition particle, a protein N, a protein Tat, a phage GA coat protein, an iron-responsive binding element (IRE) protein, and an HIV Rev protein.

[0214] 6. The PDS of any one of embodiments I -5, wherein the one or more therapeutic payloads comprise a protein, a nucleic acid, or comprise both a protein and a nucleic acid. [0215] 7. The PDS of embodiment 6, wherein the protein is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, ribonuclease (RNAse), deoxyribonuclease (DNAse), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, a transcription factor, granulocyte-macrophage colony -stimulating factor (GMCSF), transposon, reverse transcriptase, viral interferon antagonists, a tick protein, and an anti-cancer modality.

[0216] 8. The PDS of embodiment 7, wherein the CRISPR protein is a Class 2 CRISPR protein.

[0217] 9. The PDS of embodiment 8, wherein the Class 2 CRISPR protein is selected from the group consisting of a Type II, a Type V, and a Type VI protein.

[0218] 10. The PDS of embodiment 9, wherein the Type V protein is selected from the group consisting of Cast 2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f, Casl2g, Casl 2h, Casl2i, Cast 2j, Cast 2k, Casl4, and Cas<R

[0219] 11. The PDS of embodiment 10, wherein the CasX comprises a sequence selected from the group consisting of SEQ ID NOS: 136-176, and 180-506, 1905, 7731-7891, and 7978-7980, or a sequence having 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% sequence identity thereto.

[0220] 12. The PDS of embodiment 10, wherein the CasX comprises a sequence selected from the group consisting of SEQ ID NOS: 136-176, and 180-506, 1905, 7731 -7891, and 7978-7980, or a sequence having 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% sequence identity thereto. [0221] 13. ’The PDS of embodiment 10, wherein the CasX comprises a sequence selected from the group consisting of SEQ ID NOS: 136-176, and 180-506 and 1905. [0222. ] 14. The PDS of embodiment 10, wherein the CasX comprises a chimeric CasX comprising domains derived from two different CasX proteins.

[0223] 15. 'The PDS of embodiment 10, wherein the CasX compri ses a sequence selected from the group consisting of SEQ ID NOS: 190, 197, 273, 348, 351, 355 and 478.

[0224] 16. The PDS of embodiment 10, wherein the CasX is cataly tically -dead.

[0225] 17. The PDS of embodiment 16, wherein the catalytically-dead CasX (dCasX) comprises a sequence selected from the group consisting of SEQ ID NOS: 7716 and 7937- 7959, or a sequence having 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% sequence identity thereto.

[0226] 18. The PDS of any one of embodiments 11-17, wherein the CasX or dCasX is a fusion protein that comprises one or more heterologous proteins,

[0227] 19. The PDS of embodiment 18, wherein the one or more heterologous proteins comprises an NTS selected from the group of sequences consisting of SEQ ID NOS: 507-600 and SEQ ID NOS: 7589-7639, wherein the NTS are located at or near the N-terminus and/or the C -terminus of the CasX and, optionally, the one or more NLS are linked to the CasX or to adjacent NLS with a linker peptide.

[0228] 20. The PDS of embodiment 19, wherein the linker peptide is selected from the group consisting of SR, RS, (G)n (SEQ ID NO: 7640), (GS)n (SEQ ID NO: 7641), (GSGGS)n (SEQ ID NO: 7642), (GGSGGS)n (SEQ ID NO: 7643), (GGGS)n (SEQ ID NO: 7644), GGSG (SEQ ID NO: 7645), GGSGG (SEQ ID NO: 7646), GSGSG (SEQ ID NO: 7647), GSGGG (SEQ ID NO: 7648), GGGSG (SEQ ID NO: 7649), GSSSG (SEQ ID NO: 7650), GPGP (SEQ ID NO: 7651), GGP, PPP, PPAPPA (SEQ ID NO: 7652), PPPG (SEQ ID NO: 7653), PPPGPPP (SEQ ID NO: 7654), PPP(GGGS)n (SEQ ID NO: 7655), (GGGS)nPPP (SEQ ID NO: 7656), AEAAAKEAAAKEAAAKA (SEQ ID NO: 7657), TPPKTKRKVEFE (SEQ ID NO: 7658), and GP AEAAAKEAAAKEAAAKA (SEQ ID NO: 1932), where n is 1 to 5.

[0229] 21. The PDS of embodiment 19 or 20, wherein the CasX comprises anuclear export sequence (NES) linked to the C-terminus of the NLS linked to the C-terminus of the CasX. [0230] 22. The PDS of embodiment 21, wherein the NES is selected from the group consisting of the sequences of SEQ ID NOS: 1838-1886, or a sequence having 1, 2, 3 amino acid insertions, deletions or substitutions relative thereto.

I I I [0231 | 23. The PDS of embodiment 19 or embodiment 21, wherein the NES is linked to the NTS by a sequence cleavable by a protease.

[0232] 24. The PDS of embodiment 23, wherein the sequence cleavable by a protease is cleavable by an HIV- 1 protease or TEV protease.

[0233] 25. The PDS of embodiment 23 or embodiment 24, wherein the sequence cleavable by a protease is SQNYPIVQ (SEQ ID NO: 100) or ENLYFQS (SEQ ID NO: 98).

[0234] 26. The PDS of embodiment 18, wherein the fusion protein comprises a dCasX and one or more repressor domains selected from the group consisting of a Kruppel associated box (KRAB) domain, a DNMT3A catalytic domain, a DNMT3L interaction domain, and a DNMT3A ADD domain.

[0235] 27. The PDS of embodiment 26, wherein the fusion protein comprises a KRAB domain selected from the group consisting of the sequences of SEQ ID NOS: 7720-7728 and one or more linker peptides.

[1)236] 28. The PDS of embodiment 27, wherein the fusion protein comprises, from N~ terminus to C -terminus, DNMT3A ADD domain-DNMT3A cataly tic domain -linker- DNMT3L interaction domain-linker- KRAB-linker-dCasX (dXR).

[0237] 29. The PDS of any one of embodiments 6-28, wherein the therapeutic payload comprises a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASO), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, an RNA aptamer, a CRISPR guide ribonucleic acid (gRNA), a donor template, or any combination thereof.

[0238] 30. The PDS of embodiment 29, wherein the nucleic acid is a gRNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 15 and 20 nucleotides and is complementary' to a target nucleic acid sequence of a cell.

[0239] 31. The PDS of embodiment 30, wherein the gRNA is a single-molecule guide RNA.

[0240 ] 32. The PDS of embodiment 31, wherein the gRN A scaffold comprises a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1907), or a sequence with at least at least 1, 2, 3, 4, or 5 mismatches thereto.

[0241] 33. The PDS of any one of embodiments 30-32, wherein the gRNA scaffold sequence comprises a sequence selected from the group consisting SEQ ID NOS: 2101-2258, 2260-2431, or a sequence having 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% sequence identity thereto. [0242] 34. The PDS of any one of embodiments 30-32, wherein the gRNA scaffold sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 2101- 2258, 2260-2431.

[0243] 35. The PDS of any one of embodiments 30-32, wherein the gRNA scaffold sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 2238, 2275, 2281, 2293, and 2309.

[0244] 36. The PDS of any one of embodiments 30-35, wherein the gRNA and the CRISPR protein are capable of forming a ribonucleoprotein (RNP) complex.

[0245] 37. The PDS of any one of embodiments 30-36, wherein the scaffold of the gRNA variant further comprises one or more RNA binding partner elements selected from the group consisting of: (i) a stem IIB of Rev response element (RRE), (ii) a stem II-V of RRE; (hi) a stem II of RRE; (iv) a Rev-binding element (RBE) of Stem IIB; and (v) a full-length RRE, wherein the one or more components are capable of binding Rev.

[0246] 38. The PDS of any one of embodiments 30-37, wherein the scaffold of the gRNA variant comprises one or more non-covalent recruitment components selected from the group consisting of: (i) an MS2 hairpin; (ii) a PP7 hairpin; (iii) a Qp hairpin; (iv) a boxB; (v) a phage GA hairpin; (vi) a phage AN hairpin; (vii) an iron responsive element (IRE); (viii) a transactivation response element (TAR); and (ix) a L^!1A hairpin II, wherein the non-covalent recruitment components have binding affinity to NCR selected from the group consisting of MS2 coat protein, PP7 coat protein, Q coat protein, U1 A signal recognition particle, protein N, protein Tat, phage GA coat protein, and iron-responsive binding element (IRE) protein, facilitating the non-covalent recruitment of the therapeutic protein into the PDS.

[0247] 39. The PDS of any one of embodiments 2-38, wherein the MA protein comprises a sequence selected from the group consisting of SEQ ID NOS: 1039-1252, or a sequence having 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% sequence identity thereto.

[0248] 40. The PDS of embodiment 2-38, wherein the MA protein comprises a sequence selected from the group consisting of SEQ ID NOS: 1039-1252.

[0249] 41. The PDS of any one of embodiments 2-40, wherein the NC protein comprises a sequence derived from the

Mononega.viral.es virus as the MA protein. [0250 | 42. The PDS of any one of embodiments 2-40, wherein the NC protein comprises a sequence derived from a different Mononegavirales virus as the MA protein.

[0251] 43. The PDS of embodiment 41 or embodiment 42, wherein the NC protein comprises a sequence selected from the group consisting of SEQ ID NOS: 1597-1810, or a sequence having 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% sequence identity thereto.

[0252] 44. Tire PDS of embodiment 41 or embodiment 42, wherein the NC protein comprises, wherein the NC protein comprises a sequence selected from the group consisting of SEQ ID NOS: 1597-1810.

[0253] 45. The PDS of any one of embodiments 1-40, wherein the tropism factor is selected from the group consisting of a glycoprotein, an antibody fragment, a receptor, and a ligand to a target cell marker.

[0254 ] 46. The PDS of embodiment 45, wherein the tropism factor is a cognate gly coprotein derived from the Mononegavirales virus.

[0255] 47. The PDS of embodiment 46, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 1309-1596, or a sequence having 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% sequence identity thereto.

[0256 [ 48. The PDS of embodiment 46, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 1309-1596.

[0257] 49. The PDS of embodiment 41 , wherein the tropism factor is a glycoprotein derived from a virus other than a Mononegavirales virus,

[0258] 50. The PDS of embodiment 49, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 601-824, or a sequence having 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% sequence identity thereto.

[0259] 51. The PDS of embodiment 49, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 601-824.

[0260] 52. A nucleic acid encoding the fusion protein of any one of embodiments 1-51. H12611 53. The nucleic acid of embodiment 52. wherein the encoded components are configured, 5‘ to 3': (a) MA-NCR; (b) MA-NC-NCR; (c) MA-CasX; (d) MA-dXR; (e) MA- NCR-CasX; or (f) M A-NCR-dXR.

[0262] 54. A nucleic acid encoding the therapeutic payload of any one of embodiments 1- 51.

[0263] 55. The nucleic acid of embodiment 54, wherein the therapeutic payload comprises a CasX variant.

10264] 56. The nucleic acid of embodiment 55, wherein the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 190, 197, 273, 348, 351, 355, and 478.

[0265] 57. The nucleic acid of embodiment 54, wherein the therapeutic payload comprises a fusion protein comprising a dCasX and one or more repressor domains selected from the group consisting of Kruppel associated box (KRAB) domain, DNMT3A catalytic domain, DNMT3L interaction domain, and DNMT3A ADD domain.

[0266] 58. The nucleic acid of embodiment 54, wherein the therapeutic payload comprises a gRNA.

[0267] 59. The nucleic acid of embodiment 57, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 2238, 2275, 2281, 2293, and 2309. [0268] 60. A nucleic acid encoding the tropism factor of any one of embodiments 1-51. [0269] 61. A plasmid comprising the nucleic acid of any one of embodiments 54-60, wherein the nucleic acid is operably linked to a promoter.

[0270] 62. The plasmid of embodiment 61, wherein the promoter is selected from the group consisting of EF-1 alpha, EF-lalpha core promoter, Jens Tomoe (JeT), cytomegalovirus promoter (CMV), CMV immediate early (CMVIE), minimal CMV promoter, herpes simplex virus (HS V) promoter, simian virus 40 (SV40), adenovirus major late promoter (Ad MLP), chicken P-actin promoter (CB A), CBA hybrid (CBh), chicken p-actm promoter with cytomegalovirus enhancer (CB7), chicken beta- Actin promoter and rabbit beta-Globin splice acceptor site fusion (CAG), rous sarcoma vims (RSV) promoter, HIV-Ltr promoter, hPGK promoter, HSV TK promoter, Mini-TK promoter, human synapsin I (SYN) promoter, betaactin promoter, super core promoter 1 (SCP1), Mecp2 promoter, minimal IL-2 promoter, TBG promoter, PGK promoter, human ubiquitin C promoter (UBC), UCOE promoter, Histone H2 promoter, the Histone H3 promoter, II lai small nuclear RNA promoter, GUSB promoter, CBh promoter, rhodopsin (Rho) promoter, silencing-prone spleen focus forming virus (SFFV) promoter, POLI promoter, b-kinesin 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, U6, mini U6, U6 truncated promoters, BiU6, Bi7SK, BiFIl, bidirectional U6, bidirectional 7SK, bidirectional Hl, gorilla U6, rhesus U6, human 7SK, and human Hl.

[0271] 63. A eukaryotic cell comprising the plasmid(s) of embodiment 61 or embodiment 62.

[0272] 64. The eukaryotic cell of embodiment 63, wherein the components of the PDS are encoded on three or four plasmids.

[0273] 65. The eukaryotic cell of embodiment 64, wherein a first plasmid encodes the fusion protein of embodiment 52 or embodiment 53.

[0274] 66. The eukaryotic cell of embodiment 64 or embodiment 65, wherein a second plasmid encodes the CasX or the fusion protein of any one of embodiments 55-57.

[0275] 67. The eukaryotic cell of any one of embodiments 64-66, wherein a third plasmid encodes the gRNA of embodiment 58 or embodiment 59.

[0276] 68. The eukaryotic cell of any one of embodiments 64-67, wherein a fourth plasmid encodes the tropism factor of embodiment 60.

[0277] 69. The eukaryotic cell of any one of embodiments 64, wherein the eukaryotic cell is selected from the group consisting of human embryonic kidney 293 (HEK293) cells, Lenti- X 293T cells, HEK293T cells, Lenti-X 293T cells, baby hamster kidney (BHK) cells, HepG2, Saos-2 cells, HuH7 cells, NS0 cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS cells, WI38 cells, MRC5 cells, A549, HeLa cells, CHO cells, and HT 1080 cells.

[0278] 70. The eukaryotic cell of embodiment 69, wherein the encoded components are capable of self-assembling into a PDS when the plasmids are introduced into the eukaryotic cell and the components are expressed.

[0279] 71. The eukaryotic cell of any one of embodiments 63-70, wherein the eukaryotic cell is modified to reduce or eliminate expression of a cell surface marker.

[0280] 72. The eukaryotic cell of embodiment 71, wherein the cell surface marker is one or more of B2M, CUT A, or PD-1. [0281 | 73. The eukaryotic cell of any one of embodiments 62-72. wherein the eukaryotic cell is modified to express one or more cell surface markers selected from the group consisting of CD46, CD47, CD55, CD59, CD24, CD58, SLAMF4, and SL.AMF3.

[0282] 74. A PDS particle produced by the eukaryotic cell of any one of embodiments 63- 73.

[0283] 75. The PDS particle of embodiment 74. having a diameter of less than about 50, about 60, about 70, about 80, about 90 nm, or less than about 100 nm.

[0284] 76. The PDS particle of embodiment 74 or embodiment 75, wherein the therapeutic pay load is encapsidated within the PDS particle upon self-assembly of the PDS particle in the eukaryotic cell.

[0285] 77. The PDS particle of embodiment 76, wherein the therapeutic payload is a ribonucleoprotein (RNP) of the CasX variant or the fusion protein comprising a dCasX and one or more repressor domains selected from the group consisting of Kruppel associated box (KRAB) domain, DNMT3A catalytic domain, DNMT3L interaction domain, and DNMT3A ADD domain and the gRNA.

[0286] 78. The PDS particle of any one of embodiments 74-77, wherein the one or more binding partner elements incorporated into the gRNA are capable of binding the one or more NCR, wherein the binding facilitates incorporation of increased numbers of RNP into the PDS particles during self-assembly compared to a PDS particle not comprising the one or more binding partner elements and the one or more NCR.

[0287] 79. The PDS particle of any one of embodiments 76-78, wherein the particle contains at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 molecules of the therapeutic pay load.

[0288] 80. The PDS particle of embodiment 78 or embodiment 79, wherein the increased numbers of RNP incorporated into the PDS particle results in at least a 2-fold, at a least 3- fold, at least a 4-fold, at least a 5-fold increase in editing potency of the PDS particles for the target nucleic acid compared to PDS particles without the one or more binding partner elements and the non-covalent recruitment proteins, when assayed in vitro under comparable conditions.

[0289] 81. The PDS particle of any one of embodiments 74-80, wherein the tropism factor is incorporated on the PDS particle surface upon self-assembly of the PDS particle in the eukaryotic cell. [0290] 82. The PDS particle of embodiment 81, wherein the tropism factor has binding affinity for a cell surface marker of a target cell and facilitates entry of the PDS particle into the target cell ,

[0291 [ 83. The PDS particle of embodiment 81 or embodiment 82, wherein incorporation of the tropism factor results in at least a 2-fold, at a least 3-fold, at least a 4-fold, at least a 5- fold, at least a 6-fold, at least a 7-fold, or at least an 8-fold increase in editing potency of the PDS particle for the target nucleic acid compared to PDS particle without the incorporated tropism factor, when assayed in vitro under comparable conditions.

[0292] 84. The PDS particle of any one of embodiments 74-83, wherein the cell surface marker of any one of B2M, CUT A, or PD-1 is eliminated or is reduced at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold compared to PDS produced from a eukaryotic cell that has not be modified.

[0293] 85. The PDS particle of any one of embodiments 74-84, wherein the cell surface marker of any one of CD46, CD47, CD55, CD59, CD24, CD58, SLAMF4, or SLAMF3 is increased at least 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5 -fold, or at least about 10-fold compared to PDS produced from a eukaryotic cell that has not be modified.

[0294] 86. A method of making a PDS particle comprising a therapeutic pay load, the method comprising propagating the eukaryotic cell of any one of embodiments 63-73 under conditions such that the components are expressed and seif-assemble into PDS particles that release from the eukary otic cell.

[0295] 87. The method of embodiment 86, wherein the eukaryotic cell is an HEK293T cell.

[0296] 88. The method of embodiment 86 or embodiment 87, wherein the therapeutic payload is a ribonucleoprotein (RNP) of the CasX variant or a dXR and the gRNA,

[0297] 89. The method of embodiment 88, wherein the incorporation of the binding partner element(s) and the one or more NCR results in incorporation of increased numbers of RNP into the PDS particle during self-assembly compared to a PDS particle not comprising the one or more binding partner elements and the one or more NCR.

[0298] 90. The method of embodiment 89, wherein the particle contains at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 molecules of the therapeutic payload. [0299] 91. The method of embodiment 89 or embodiment 90. wherein the PDS particle exhibits at least a 2-fold, at a least 3-fold, or at least a 4-fold increase in editing potency compared to a PDS particle without the incorporated binding partner element(s) and NCR, when assayed in vitro under comparable conditions.

[0300] 92. A method of modifying a target nucleic acid sequence of a gene in a population of cells, the method comprising contacting the cells with a plurality of the PDS particles of any one of embodiments 74-85, wherein said contacting comprises introducing the into the cell the RNP, wherein the target nucleic acid targeted by the guide RNA is modified by the CRISPR protein.

[0301] 93. The method of embodiment 92, wherein the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence.

[0302] 94. The method of embodiment 92, wherein the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence.

[0303] 95. The method of any one of embodiments 92-94, wherein the modification comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the target nucleic acid sequence of the cells.

[0304] 96. The method of any one of embodiments 92-95, wherein the gene is knocked- down or knocked out.

[0305] 97. The method of embodiment 92, wherein transcription of the gene is repressed.

[0306] 98. The method of embodiment 97, wherein the repression of transcription is heritable through one or more cell divisions.

[03071 99. The method of any one of embodiments 92-98, wherein the cells are modified in vitro or ex vivo.

[0308] 100, The method of any one of embodiments 92-98, wherein the cells are modified in vivo.

[0309] 101 . The method of embodiment 100, wherein the PDS particles are administered to a subject.

[0310] 102. The method of embodiment 101, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, and non-human primate,

[0311] 103. The method of embodiment 101, wherein the subject is human.

[0312] 104, The method of any one of embodiments 101-103, wherein the PDS particles are administered by a route of administration selected from the group consisting of subcutaneous, intradermal, intraneural, intranodal, intramedullary, intramuscular, intravenous, intracerebroventricular, intracisternal, intrathecal, intracranial, intralumbar, intratracheal, intraosseous, inhalatory, intracontralateral striatum, intraocular, intravitreal, intralymphatical, intraperitoneal and sub-retinal routes.

[0313] 105. The method of any one of embodiments 101-104, wherein the PDS particles are administered to the subject using a therapeutically effective dose.

[0314] 106. The method of any one of embodiments 101-105, wherein the PDS particles are administered to the subject according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose of the PDS particles.

[0315] 107. The method of embodiment 106, wherein the therapeutically effective dose is administered to the subject as two or more doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months, or once a year, or every 2 or 3 years.

[0316] 108. A packaging cell, comprising: (a) a first plasmid encoding the fusion protein of embodiment 52 or embodiment 53; (b) a second plasmid encoding a CasX variant comprising a sequence selected from the group consisting of SEQ ID NOS: 190, 197, 273, 348, 351, 355, and 478; (c) a third plasmid encoding a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 2238, 2275, 2281, 2293, and 2309; (d) a fourth plasmid encodes the tropism factor of any one of embodiments 45-51.

[0317] 109. The packaging cell of embodiment 108, wherein the packaging cell is selected from the group consisting of human embryonic kidney 293 (HEK293) cells, Lenti-X 293T cells, HEK293T cells, Lenti-X 293T cells, baby hamster kidney (BHK) cells, HepG2, Saos-2 cells, HuH7 cells, NS0 cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS cells, WI38 cells, MRC5 cells, A549, HeLa cells, CHO cells, and HT1080 cells.

286526972 EXAMPLES

Example 1: Generation and assessment of potency of Mononegavirales-based PDS particles with CasX fused to the viral matrix protein

[0318] Viruses of the Momnegavirales order are unique in that their viral envelope is comparatively simpler than those harbored by the Retroviridae family, given that the envelope is composed of a single protein. Harnessing the simplicity' of this architectural design potentially confers a distinct advantage in packaging and production of virus-like particles. Here, experiments were conducted to engineer novel CasX particle delivery' system (PDS) particles derived from the Mononegavirales order and assess their editing potency. The possibility of using matrix proteins from different viral species within the Mononegavirales order was assessed in two different architectural variations. In one variation, described in this example, the encoding sequence for the CasX protein was specifically fused to the sequence encoding a. viral matrix protein derived from various species of the Vesiculovirus genus wi thin the Rhabdovirus family of the Mononegavirales order. Upon expression of the components, PDS particles were generated in which the CasX RNPs were recruited and incorporated into the PDS because of direct covalent fusion of the CasX protein to the Mononegavirales matrix protein.

Materials and Methods:

PDS construct cloning:

[0319] In this example, all plasmids encoding CasX utilized the CasX 491 variant (SEQ ID NO: 190), RNA fold structures were generated with RNAfold web server and VARNA Javabased software.

[0320] Briefly, amplified and puri fled fragments encoding CasX 491 and matrix proteins from various species within the Mononegavirales order were cloned into plasmid backbones according to standard methods. These PDS plasmid constructs contained sequences coding for CasX protein 491, guide scaffold variant 174, a spacer targeting the tdTomato STOP cassette (spacer 12.7; CUGCAUUCUAGUUGUGGUUU; SEQ ID NO. 1906), and the VSV- G glycoprotein, and were generated using standard molecular cloning techniques. Cloned and sequence-validated plasmid constructs were maxi-prepped and assessed for quality' prior to use for PDS production. Sequences of the various PDS constructs are shown in Tables 11-13. Table 11. DNA sequences of PDS constructs*

Table 12. Sequences of Mononegavirales matrix proteins

Table 13. Sequences encoding guide scaffold gRNA and VSV-G glycoprotein constructs

Production of PDS particles:

[0321] PDS particles containing ribonucleoproteins (RNPs) of CasX variant 491, and a single guide RNA with scaffold 174 and a spacer targeting the tdTomato locus were produced using adherent HEK293T Lenti-X™ cells. Briefly, HEK293T Lenti-X™ ceils were maintained in 10% fetal bovine serum (FBS) supplemented Dulbecco’s Modified Eagle Medium (DMEM) with N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) and GlutaMAX™ (Thermo Fisher). Cells were seeded in 15 cm dishes at 20 x 10E6 cells per dish in 20 mL of media 24 hours before transfection. Once the cells reached 70-90% confluence', the cells were transfected with the following plasmids using PEI MAX® (Polypus): PDS structural plasmids, a plasmid encoding the scaffold gRNA with a spacer targeting tdTomato (pSG005), and a plasmid encoding the VSV-G glycoprotein (pGP2) (sequences in Tables I I- 13). Media was aspirated from the plates 24 hours post-transfection and replaced with Opti- MEM™ (ThermoFisher). PDS-containing media was collected 72 hours post-transfection and filtered through a 0.45 pm PES filter. The supernatant was concentrated and purified via centrifugation. PDS particles were resuspended in freezing buffer or NPC media.

PDS particle transduction of mouse tdTomato neural progenitor cells (NPCs):

[0322] tdTomato NPCs were grown in DMEM/F12 supplemented with Glutamax, HEPES, Non-Essential Amino Acid (NEAA), Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested and seeded on PLF-coated 96-well plates. 48 hours later, cells were transduced with PDS particles containing the tdTomato targeting spacer, starting with 50 pL of concentrated PDS particles and proceeding through 5 half-log serial dilutions. Cells were then centrifuged for 15 minutes at 1000 x g. Transduced NPCs were grown for 96 hours before analyzing tdTomato fluorescence by flow' cytometry as a marker of editing at the tdTomato locus. Assays were run 2-3 times for each sample, with similar results. XDP version 168 (VI 68), which served as a positive control, was generated using methods described in International Publication No. WO2021113772A1, served as an experimental control. Briefly, .XDP VI 68 refers to a virus-like particle derived from lent! viral -based HIV harboring a Gag-CasX fusion configuration. Version 168 XDPs are described in detail in International Publication No. WO202226I I50A2.

Results:

[0323] Mononegavirales-bas&i PDS particles with CasX fused to the viral matrix protein were generated and assessed for their editing efficiency at the tdTomato locus in mouse NPCs. Percent editing at the tdTomato locus was measured by quantifying tdTomato fluorescence using flow cytometry when 50 pL of concentrated PDS preps were used to transduce mouse NPCs. The results demonstrate that PDS particles constructed using the matrix protein from different viral species of the Mononegavirales order used to deliver CasX RNPs into NPCs exhibited editing at the tdTomato locus ranging from 3 to 57% (FIG. 3). The data support that protein components derived from different Mononegavirales strains can be used to engineer PDS particles to deliver CasX RNPs effectively to cells to edit the target nucleic acid. Example 2: Generation and assessment of potency of Mononegavirales-based PDS particles using an MS2-based recruitment of CasX RNP

[0324] In Example 1, CasX was fused to the viral matrix protein derived from various species of the Vesiculovirus genus within the Rhabdovirus family of the Mononegavirales order. In this example, the MS2 bacteriophage packaging system was utilized as a mechanism for non-covalent recruitment (NCR) of CasX RNPs into PDS particles. Briefly, the packaging system to produce PDS particles used two major components to recruit the CasX ribonucleoprotein (RNP) into the particle: the phage coat protein and its cognate binding partner, which is a short hairpin RNA stem loop structure. In this orthogonal phage RNA- based recruitment system, the short hairpin stem loop structure was engineered into the gRNA, and the sequence encoding the phage coat protein was fused to the sequence encoding the Mononegavirales matrix protein. This design enables the recruitment of CasX RNP into the PDS particle by the non-covalent interaction between the short hairpin stem loop structure engineered into the gRNA of the CasX RNP and the phage coat protein fused to the matrix protein. Here, Mononegavirales-baseti PDS particles were generated where the CasX RNP was recruited into the PDS by fusing the high affinity variant MS2353 coat protein to the Mononegavirales matrix protein in the PDS construct.

Materials and Methods:

PDS construct cloning:

[0325 ] All plasmids encoding CasX proteins incorporated sequences encoding either the CasX 491 or CasX 676 variant protein. RNA fold structures were generated as described in Example 1

[0326] Briefly, to generate the PDS structural plasmids, the gag-pol sequence was removed from a plasmid. Amplified and purified fragments encoding CasX 491 or CasX 676, matrix proteins from various species within the Mononegavirales order, and MS2 coat protein components (MS353 having sequence MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQK RKYT1KVEVPKVATQ1VGGVELPVAAWRSYLNMELTIP1FATNSDCEL1VKAMQGLL KDGNPIPSA1AANSGIY, SEQ ID NO: 1929) were cloned into plasmid backbones using standard techniques. These plasmid backbones also included a sequence encoding an HIV-1 Rev protein. The PDS plasmid constructs comprising sequences coding for CasX protein 491 (SEQ ID NO: 190) or 676 (SEQ ID NO: 355), guide scaffold variant 251 (SEQ ID NO: 2309), and a spacer targeting the tdTomato STOP cassette (spacer 12.7, SEQ ID NO: 1906) were generated using standard molecular cloning techniques. The guide scaffold variant 251 was composed of scaffold 174 with two MS2 hairpin elements incorporated into the extended stem, as well as a portion of the HIV-1 Rev response element, termed “RBE” Cloned and sequence- validated constructs were maxi-prepped and subjected to quality assessment prior to use for PDS particle production. Table 14 summarizes the plasmids used to encode the components of the MA-MS2 PDS configuration tested in this example. Sequences for the Mononegavirales-based PDS constructs used in this example are listed in Table 15 and Table 16.

Table 14. Summary of PDS configuration tested in this example

Table 15. Sequences of PDS constructs*

Components are listed in a 5’ to 3’ order within the constructs. The backbone of the plasmid expresses Rev.

Table 16. DNA and amino add sequences of Mononegavirales matrix proteins

Table 17. Sequences of guide scaffold gRNA, VSV-G glycoprotein, and CasX variant constructs

[0327] Production of PDS particles was performed as described in Example 1, using the plasmids with sequences listed in Tables 15-17. In this example, the scaffold gRNA and VSV-G glycoprotein were encoded by pSG73 and pGP2 respectively.

PDS particle titering and morphology and. size assessment:

[0328] PDS particles were diluted in IX PBS at 1:200 and 1:400 and were subsequently assessed on the NanoSight NS300 to determine the mean titer and mode size (nm). The morphology' of version 372 PDS particles was visualized using transmission electron microscopy (TEM).

[0329] Transduction of murine tdTomato NPCs were performed as described in Example 1. XDP version 206 (V206), which was generated using methods described in International Publication No. WO2021113772A1, served as an experimental control. Briefly, XDP V206 was derived from lenti viral -based HIV harboring a configuration in which the MS2 353 coat protein was fused to the Gag polyprotein and the cognate MS2 hairpin was incorporated into the gRN A. Version 206 XDPs are described in detail in International Publication No.

WO2022261150A2.

[0330] Results:

[0331] Mononegavirales-basod PDS particles incorporating the MS2 phage RNA system for CasX RNP recruitment were generated by fusing the MS2 high affinity coat protein variant MS2353 to the Mononegavirales matrix protein and assessed for the ability to edit the tdTomato locus in murine NPCs. The matrix proteins used to generate PDS versions 322- 326 (V322-V326) were derived from viral species of the Bornaviridae family; for PDS V327-V333, the matrix proteins were from species of the Filoviridae family; for PDS V334, the matrix proteins were derived from species of the Nyamivmdae family; for PDS V335- V363, the matrix proteins were derived from species of the Paramyxoviridae family; for PDS V364-V366, the matrix proteins were derived from species of the Pneumoviridae family; and for PDS V367-V415, the matrix proteins were derived from species of the Rhabdoviridae family. Specific species and matric protein sequences are provided in Table 16.

[0332] Titers were quantified for each version of the PDS particle produced using the NanoSight NS300, an indication that

-based PDS particles could be produced using the MS2353 coat protein for CasX RNP recruitment. Comparable titers across various PDS versions were observed, with -99% of PDS particles having titers between 1E9 and 1E10 PDSs/mL. These titers were comparable with the titer obtained for V206, the experimental control.

[0333] The diameter size was also assessed for the generated PDS particles using the NanoSight NS300, and the mode size was determined. The findings revealed that a vast majority (-96%) of versions of Mononegavirales-boseA PDS particles generated were smaller in diameter compared to the diameter measured for the experimental control V206, which had a. mode of - 157 nm. Versions of PDS particles with a mode diameter that measured <100 nm in size are listed in Table 18. Notably, most of the smaller PDS particles were generated using matrix proteins derived from viral species that fall within the Rhabdoviridae and Paramyxoviridae families.

[0334] The editing efficiency of the generated PDS particles at the tdTomato locus was assessed in murine NPCs. Percent editing at the tdTomato locus was measured by quantifying tdTomato fluorescence using flow' cytometry when 5.5 pL of concentrated PDS preparations were used to transduce mouse NPCs, and the data are shown in Table 19. The results demonstrate that the PDS particles exhibited percent editing at the tdTomato locus ranging from <1% to -99%, with certain families exhibiting high editing efficiencies (Table 19). For instance, PDS particles V327-V333, which utilized the matrix protein derived from the Filoviridae family, were able to edit at -60-96% efficiency (Table 19), Use of the matrix proteins derived from the Avian metaavulavirus species, which fall within the Paramyxoviridae family, also demonstrated high editing (>90%). Matrix proteins derived from other viral species within the Paramyxoviridae family that notably resulted in high editing levels include those derived from the Narmo virus ( V352 ), Jeilongvirus (V346), and Orthorubulavirus (V360) (Table 19). Among the Rhabdoviridae family of viral species, use of the matrix protein derived from the Ephernerovirus (V372, V376), Hapavirus (V379), Vesiculovirus (V404, V408), and Zahedan zarhavirus (V414) resulted in the production of PDS particles with high editing efficiencies (Table 19).

[0335] The morphology of version 372 PDS particles with CasX RNPs packaged w as examined using TEM. While Rhabdoviral particles typically have a cone-shaped structure, the version 372 PDS particles with CasX RNPs instead appeared spherical (data not shown). [0336] Altogether, these data support that structural proteins derived from various Mononegavirales strains could be used to engineer PDS particles to deliver CasX RNPs to the target cells to induce editing of the target nucleic acid. These PDS particles could be further explored in different types of cells and tissues, where they could potentially offer unique advantages over viral -like particles generated based on HIV viruses.

Table 18. Mnnnnegnv/rafes-based PDS versions with mode diameter <100 nm

Table 19. Representative measurements of percent editing at the tdTomato locus

control. Example 3: Generation and assessment of Tfonon^nvfrnfes-based PDS particles using additional MS2 variants for CasX RNP recruitment

[0337} In Example 2, MS2 353, the high affinity coat protein variant, was used in the generation of Mononegavirales-baseti PDS particies for the recruitment of CasX RNP, resulting in functional PDSs that were able to edit the tdTomato locus in vitro. In this example, the ability of an MS2-based recruitment system using the MS2 wild-type (WT) coat protein, a non-dimerizing MS2 340 coat protein variant, or a dual MS2 (either with MS2 WT or MS2340), each of which has a different affinity to the MS2 hairpin compared to that of MS2 353, is evaluated for activity enhancements inMononegavirales-based PDSs.

Materials and Methods:

[0333] Plasmids containing constructs for expression of CasX variants 491 (SEQ ID NO: 190), 515 (SEQ ID NO: 197), 668 (SEQ ID NO: 348), 672 (SEQ ID NO: 351), or 676 (SEQ ID NO: 355) are used. The amino acid sequences of the CasX variants are provided in Table 3. A separate plasmid encodes for guide scaffold variant 251 and spacer 12.7 targeting the tdTomato STOP cassette.

[0339] PDS structural plasmids are generated as described in Example 2, Here, the MS2 coat protein used to fuse to an individual Mononegavirales matrix protein is MS2 WT or MS2 340 (sequences listed in Table 20), or a dual MS2 (either with dual MS2 WT or dual MS2 340). The Mononegavirales matrix proteins (Table 16) that resulted in PDS particles demonstrating the highest editing potency (as determined in Example 2) is tested in this example. PDS particles are pseudotyped with the VSV-G glycoprotein.

Table 20. Sequences of an MS2 WT or MS2340 coat protein

[0340] PDS particle production, titering of PDS particles, and transduction of murine tdTomato NPCs are performed as described in Example 1. XDP V206 serves as an experimental control.

[0341] The results of this experiment are expected to show that Mononegavirales -based PDS particles produced with leading matrix protein candidates identified from Example 2 exhibit varying editing efficiencies at the tdTomato locus in transduced mouse NPCs. The findings are expected to show that, in addition to the MS2 353 coat protein variant, it is functionally feasible to fuse the MS2 WT, MS2 340, dual MS2 WT, or dual MS2340 coat protein to the Mononegavirales matrix protein to recruit CasX RNPs into the PDS particles to transduce target cells and edit the target gene. EC50 analysis also is expected to reveal the editing potency of these Mononegavirales -based PDS versions relative to one another and to the experimental control XDP V206 (in which the MS2 coat protein is fused to the HIV Gag polyprotein).

Example 4: Optimization of linker sequences between the viral matrix protein and either the MS2 coat protein or CasX in the generation of Mononeguvfrafes-based PBS particles

[0342] Experiments are performed to assess whether the packaging efficiency of CasX RNPs within the Mononegavirales -based PDS can be improved by optimizing the linker sequence between the viral matrix protein and either CasX for covalent fusion or the MS2 coat protein for non-covalent recruitment of CasX RNP. In the preceding examples, the linker sequences used in the fusion of either CasX or MS2 to the Mononegavirales matrix protein were either a GGS linker or an 8x GGS linker. It is believed that the linker can be optimized by altering its length, tuning its flexibility by changing the glycine content, or substituting an entirely different flexible or rigid linker. Optimizing the linker sequence is believed to have the potential to increase the number of CasX RNPs packaged within & Mononegavirales ~ based PDS particle, and therefore could result in the production of a more potent PDS particle.

Materials and Methods:

[0343] Plasmids containing constructs for expression of CasX variants 491, 515, 593, 668, 672, 676, or 812 (SEQ ID NOS: 190, 197, 273, 348, 351, 355, and 478, respectively) are used. A separate plasmid encodes guide scaffold variant 251 and spacer 12.7 targeting the tdTomato STOP cassette is also used. [0344] PDS structural plasmids are generated following similar methods described in Example 2. Sequences of linker peptides to be used in this example are listed in Table 21. 'The leading Mononegavirales matrix protein that resulted in the generation of PDS particles exhibiting the highest editing potency, as determined in Examples 2 and 3, is used in the generation of PDS particles to assess the effects of linker optimization on CasX RNP packaging efficiency in this example. PDS particles are pseudotyped with the VSV-G glycoprotein.

Table 21. Linker peptides for fusing CasX or MS2 protein to the Mononegavirales matrix protein

[0345] PDS particle production, titering and size assessment of PDS particles is determined using the NanoSight NS300. Transduction of murine tdTomato NPCs is performed as described in Example 1. XDP V206 serves as an experimental control.

Quantification of CasX RNPs via Western blot analysis:

[0346] To determine the number of CasX molecules per PDS particle produced, a semi- quantitative Western blot analysis is performed. The protein amount in PDS particles is measured using a Pierce 660 assay. PDS particles are lysed in Laemmli sample buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using a polyclonal antibody against the CasX protein. A standard curve of known CasX amounts is included on the gel. The resulting immunoblot is imaged using a ChemiDoc Touch, and CasX protein level is quantified by densitometry using the Image Lab software from BioRad. Quantification of CasX molecules in each PDS particle sample is determined using a standard curve.

[03471 The results of these experiments are expected to show that altering the length of the existing GGS linker or using an alternative flexible or rigid linker affects the packaging of CasX RNPs within each Mononegavirales -based PDS particle. It is possible that increasing the length of the linker or swapping in a more flexible linker peptide could improve the packaging efficiency, such that substantially more CasX RNPs could be packaged within each PDS particle. Semi -quantitative Western blot analysis is expected to reveal the estimated number of CasX molecules packaged within an individual PDS particle for each experimental condition assessing a particular linker peptide. Furthermore, the findings resulting from the assessment of tdTomato editing using Mononegavirales-baseA PDS particles are expected to provide insight into the level of editing potency achieved when using a particular linker peptide for fusion of either CasX or MS2 to the viral matrix protein.

Example 5: Assessment of tropism and editing potency of PDS particles pseudotyped with glycoproteins from diverse viruses

[0348] In the preceding examples, PDS particles were pseudotyped with the vesicular stomatitis vims envelope (VSV-G) glycoprotein. However, VSV-G pseudoty ped viral vectors have been shown to be susceptible to human complement inactivation; as a result, alternative envelope glycoproteins that can protect viral particles against complement inactivation are expiored. Furthermore, incorporating alternative glycoproteins into engineered PDS is expected to improve specific cellular tropism and, therefore, enhance their potency in the target cell with minimal editing in off-target cell types. In this example, experiments were performed to generate PDS particles pseudotyped with envelope glycoproteins derived from different viral species and determine their ability to 1) confer increased tropism for a particular’ cell or tissue type and 2) induce improved editing at the target genomic locus of target cells after successful delivery of the incorporated CasX RNPs in vitro or in vivo. Specifically, PDS particles were generated with cognate glycoproteins, i.e., glycoproteins derived from the same Mononegavirales as the PDS structural proteins.

[0349] Materials and Methods:

[0350] To produce PDS particles for in vitro experiments in mouse NPCs, plasmids containing constructs encoding for CasX variant 491, 515, 668, 672, or 676, along with a separate plasmid encoding for guide scaffold variant 251 and spacer 12.7 targeting the tdTomato STOP cassette are used. For in vitro experiments in various human cell lines, plasmids containing constructs encoding for CasX 491, 515, 668, 672, or 676, along with a separate plasmid encoding for guide scaffold 251 and spacer 7.37 (GGCCGAGAUGUCUCGCUCCG, SEQ ID NO: 1930) targeting the endogenous bela-2- microglobulin (B2M) gene are used for PDS production. For in vivo mouse model experiments, plasmids containing constructs encoding for CasX 491, 515, 668, 672, or 676, along with a plasmid encoding for guide scaffold 251 and spacer 35.2 (AGAAGAUGGGCGGGAGUCUU, SEQ ID NO: 1931) targeting the safe harbor ROSA26 locus are used for PDS production.

[0351] Specifically, for the experiment described in this example, plasmids containing constructs encoding CasX 491 along with a separate plasmid encoding guide scaffold 251 with spacer 7.37 (GGCCGAGAUGUCUCGCUCCG, SEQ ID NO: 1930) targeting the endogenous B2M gene were used for PDS production.

[0352} PDS structural plasmids were generated as described in Example 2. Cognate Mononegavirales glycoproteins to be tested for pseudotyping are listed in Table 22 below. Non-cognate glycoproteins to be tested for pseudotyping are listed in Table 9. PDS particles using the top-performing Mononegavirales matrix protein fused with an MS2 coat protein demonstrated in Examples 2 and 3 are pseudotyped with envelope glycoproteins listed in Tables 9 or 22 to assess the ability to enhance tropism and improve editing potency.

Table 22. Amino acid sequences of cognate Mononegavirales glycoproteins for pseudotyping

[0353] Specifically, for the experiment described in this example, PDS version number 327, 328, 329, 330, 331, 332, 333, 336, 350, 352, 360, 362, 367, 372, 379, 384, 393, 408, 414, 404 particles were pseudotyped with either VSV-G or their cognate glycoproteins. The sequences of the Mononegavirales matrix proteins for each PDS version number are listed in Table 16, and the sequences of the cognate glycoprotein for each Mononegavirales species are provided in Table 22, above.

[03541 PDS particles were produced as described in Example 1, and titered using the NanoSight NS300. Transduction of murine tdTomato NPCs is performed as described in Example 1. XDP V206 serves as an experimental control.

PDS particle transduction of various human cell, lines, followed by HLA immunostaining and flow cytometry:

[0355] To further characterize their tropism and editing potency, PDS particles pseudotyped with different glycoproteins (listed in Table 22) are used to transduce various human cell lines, for example human NPCs, Jurkat cells (T lymphocytes), K-562 cells (lymphoblasts), ARPE-19, WERI-RB1, human astrocytes, human induced neurons, human skeletal muscle cells, and HepG2 cells (hepatocyte carcinoma cells). Specifically, for the experiment described in this example, ARPE-19 and Jurkat cells were transduced with PDS particles,

[0356] -20,000 cells were seeded on 96-well plates and transduced with PDS particles 24- 48 hours after seeding, starting with 50 pL of concentrated PDS particles followed by five half-log dilutions thereof. Five days post-transduction, PDS-treated cells were harvested B2M expression analysis viaHLA immunostaining followed by How cytometry. B2M protein expression was determined by using an antibody (BioLegend) to detect the B2M-dependent HL A protein expressed on the cell surface. The number of HLA+ cells was measured using an Attune™ NxT flow cytometer. Decreased or lack of HLA protein expression indicated successful editing at the B2M locus in these treated cells.

[0357] For editing analysis by NGS, cells are seeded and transduced as described above, and genomic DNA (gDNA) from harvested cells is extracted using the Zymo Quick-DN A™ Miniprep Plus kit following the manufacturer’s instructions. Target amplicons are formed by amplifying regions of interest from 200 ng of extracted gDNA with a set of primers specific to the human B2M locus and further processed for NGS as described below'.

In vivo administration of PDS particles and tissue processing to assess editing:

[0358] To assess PDS-mediated genome editing in vivo, PDS particles containing CasX and gRNA with a spacer targeting the ROSA26 locus, which are pseudotyped with various glycoproteins listed in Table 22, are produced as described in Example 1. These PDS particles are administered intravenously via the facial vein of C57BL/6J neonate mice. Naive, untreated mice serve as experimental controls. Four weeks post-injection, mice are euthanized and various tissues (e.g., liver, heart, skeletal muscles, brain, and spinal cord) are harvested for gDNA extraction using the Zymo Quick-DNA/RNA™ miniprep Kit following the manufacturer's instructions. For editing analysis by NGS, target amplicons are amplified from 200 ng of extracted gDNA with a set of primers targeting the mouse ROSA26 locus and further processed for NGS as described below. In addi tion, o ther routes of administration for in vivo delivery of PDS particles are explored (e.g., mtrastriatal, subretinal, intravitreal, intracerebroventricular, or intramuscular administration).

NGS processing and analysis:

[0359] Gene-specific primers amplifying the locus of interest to form target amplicons contain an additional sequence at the 5' end to introduce an Illumina adapter and a 16- nucleotide unique molecular identifier. Amplified DNA products are purified with the Ampure XP DNA cleanup kit. Quality and quantification of the amplicon is assessed using a Fragment Analyzer DNA Analysis kit (Agilent, dsDNA 35-1500bp). Amplicons are sequenced on the Illumina Miseq according to the manufacturer's instructions. Raw fastq files from sequencing are quality-controlled and processed using cutadapt v2.1, flash! v2.2.00, and CRISPResso2 v 2.0.29. Each sequence is quantified for containing an insertion or deletion (indel) relative to the reference sequence, in a window' around the 3’ end of the spacer (30 bp window' centered at -3 bp from 3’ end of spacer). CasX editing activity is quantified as the total percent of reads that contain insertions, substitutions, and/or deletions any where within this window for each sample.

[0360] Results:

[0361] Mononegavirales PDS version number 327, 328, 329, 330, 331, 332, 333, 336, 350, 352, 360, 362, 367, 372, 379, 384, 393, 404, 408, and 414 particles pseudotyped with VSV-G did not result in substantial levels of editing of the 52Mlocus in ARPE-19 and Jurkat cells (data not shown). Specifically, while 85.4%-87.1% of the ARPE-19 cells in the sample transduced with XDP V206 exhibited editing of the B2M locus, the most potent Mononegavirales PDS (version 328) produced only 5.7%-7.1% edited ARPE-19 cells when transduced at the same dilution. All other tested Mononegavirales PDS particles produced even lower levels of editing, with approximately 1-2% of ARPE-19 cells exhibiting editing of the B2M locus. The results were similar in Jurkat cells.

[0362] Similarly, Mononegavirales PDS version number 327, 328, 329, 330, 331, 332, 333, 336, 350, 352, 360, 367, 372, 379, 384, 393, and 414 particles pseudotyped with cognate glycoproteins also did not result in substantial levels of editing of the B2M locus in ARPE-19 and Jurkat ceils (data not show'n). In Jurkat cells, version 352 PDS particles with the cognate Tupai a n anno virus glycoprotein produced 43% edited cells at the highest dilution of PDS particles transduced, but less than 1% edited cells at all other dilutions. All other tested Mononegavirales PDS particles produced lower levels of editing, with no more than 3% of Jurkat cells exhibiting editing of the B2M locus. In comparison, XDP V206 produced 69.8% edited Jurkat cells at the highest dilution transduced, and 70.6%-15.9% edited cells at all other dilutions transduced.

[0363] In ARPE-19 cells, Mononegavirales PDS particles again produced little to no editing of the B2Mlocus.

[0364] There were not differences in titer between the Mononegavirales PDS particles pseudotyped with VSV-G or with cognate glycoproteins, which suggests that the experimental conditions were comparable and that differences in editing w'ere not due to differences in the number of particles in the various experiments. [0365] PDS version number 362 (Sosuga pararubulavirus), 404 (Indiana vesiculovirus), 408 (Maraba vesiculovirus) particles were not tested with their cognate glycoproteins.

Example 6: Use of the Mononegavirales nucleocapsid protein in combination with the matrix protein to generate potent PDS particles for CasX RNP delivery

[0366] In earlier examples, the Mononegavirales matrix protein was the primary' structural protein used to engineer PDS particles to package and deliver CasX RNPs. Experiments were performed to evaluate whether incorporation of other viral structural proteins, such as the nucleocapsid, could strengthen the structural integrity of the PDS particle and/or improve recruitment of CasX RNPs, thereby enhancing overall editing potency. This example investigates how the use of the Mononegavirales nucleocapsid protein, alone or in combmation with its cognate matrix protein, affects the assembly and production of PDS particles, as well as their ability to successfully deliver CasX RNPs to cells and edit the target genomic locus. Different configurations incorporating one or both structural proteins derived from the same viral species were designed and tested to generate Mononegavirales-basQd PDS particles using an MS2-based recruitment system of CasX RNP.

Materials and Methods:

[0367] Various architectural configurations of the PDS particles are designed to encompass RNPs of CasX variant proteins 491, 515, 668, 672, or 676, complexed with a single gRNA with scaffold 251 and either spacer 12,7 targeting the tdTomato locus or spacers 7.37 and 7.9 (GUGUAGUACAAGAGAUAGAA; SEQ ID NO: 177) targeting the B2M locus, for in vitro experiments. For in vivo mouse model experiments, these alternative configurations of the PDS particles are designed to contain CasX RNPs with gRNA containing either spacer 12,7 targeting the tdTomato locus or spacer 35.2 targeting the ROSA26 locus.

[0368] PDS structural plasmids are generated following similar methods described in Example 2. The resulting structural plasmids contain constructs that encode for one of the following three alternative architectural configurations, where the MS2 coat protein is fused to either the matrix (MA) protein or a cognate nucleocapsid (NC) protein or both: 1) MA- MS2 + NC; 2) MA-MS2 + NC-MS2; or 3) MA + NC-MS2. PDS particles without either the NC or MA protein are used as controls. Sequences of Mononegavirales nucleocapsid proteins are listed in Table 23. PDS particles are pseudotyped with VSV-G or with a Mononegavirales-derived glycoprotein as described in Example 5.

[0369] Specifically, for the experiments producing the results described in this example, CasX 491 or 676 were used along with gRNA with scaffold 251 and spacer 12, 7 targeting the tdTomato locus (see Tables 24-26). Each PDS version was tested with either CasX 491 or 676. The results provided in Tables 24-26 were generated using the same CasX nuclease (either 491 or 676) within each row, but not necessarily between rows. PDS particles were generated in which the MS2 coat protein was fused to the MA protein and the NC was encoded on a separate structural plasmid (MA-MS2 +NC). PDS particles were pseudotyped with VSV-G. The experiment was also repeated with spacers targeting the B2M locus (data not shown), with similar results.

[0370] In this example, the PDS particle “version numbers" refer to the assignment of different species ofMononegavirales matrix proteins to version numbers as described for the MA-MS2 fusion proteins listed in Example 2, above.

Table 23. Amino arid sequences of Mononegavirales imdeocapsid proteins

[0371] PDS particle production, as well as titering and size assessment of PDS particles using a NanoSight NS300, were performed as described in Example 1.

[0372} The morphology' of the PDS particles with different architectural configurations is visualized using transmission electron microscopy (TEM).

[0373] To assess the editing potency of the engineered PDS particles, murine tdTomato NPCs were transduced as described in Example 1 , and editing of the tdTomato locus was measured. ’The experiment was repeated for each PDS version in each configuration with similar results.

[0374] PDS particles are further evaluated in their ability to assess editing at the endogenous B2M locus in various human cell lines as described in Example 5, Furthermore, in vivo genome editing mediated by these PDS particles with alternative configurations is evaluated using the methods described in Example 5. To evaluate tdTomato editing in vivo, the Ai9 mouse model is used. Ai9 is a Cre reporter tool strain designed to have a loxP flanked STOP cassete preventing the transcription of a CAG promoter-driven tdTomato marker. These mice express tdTomato following Cre-mediated recombination to remove the STOP cassete.

[0375] For the various in vitro and in vivo experiments assessing editing potency, XDP V206 and PDS particle harboring the MA-MS2 configuration assessed in Example 2 or a NC- MS2 configuration served as experimental controls. Quantification ofCasX RNPs:

[0376] The number of CasX molecules recruited and packaged within each PDS particle is determined via a semi-quantitative Western blot analysis as described in Example 4.

Furthermore, absolute quantification of gRNAs within each PDS particle is determined using droplet digital PCR (ddPCR) using the Bio-Rad QX200 Droplet Digital PCR instrument. Briefly, purified PDS particles are treated with DNase and RNase to eliminate unpackaged nucleic acids, followed by lysis of PDS particles to extract packaged gRNAs, which is serially diluted and subjected to droplet formation using a droplet generator. Within each droplet, a PCR amplification reaction is performed using a primer-probe set specific to the target gRNA. Subsequently, the total number of positive (gRNA-containing) droplets and negative droplets was determined for the sample using the Bio-Rad QuantaSoft software to calculate the absolute number of gRNAs within each PDS particle.

Results:

[0377] Editing of the tdTomato locus was measured in mouse NPCs transduced with serial dilutions of PDS particles in MA-MS2, MA-MS2 + NC, or NC-MS2 architectural configurations. Die results are provided in Tables 24-26, below'.

Table 24. Percentage of mouse NPCs with edited tdTomato locus following transduction

* PDS versions 337, 384, 404, 408, 413, 414, and 415 were not tested in the NC-MS2 configuration.

Table 25. Percentage of mouse NPCs with edited td'Tomato locus following transduction with MA-MS2 + NC PDS particles

Table 26. Percentage of mouse NPCs with edited tdTomato locus following transduction with MA-MS2 PDS particles

[0378] As shown in Table 24, the NC-MS2 configuration produced low levels of editing across the PDS particle versions tested, with the possible exception of version 379 (derived from Manitoba hapavirus). Specifically, version 379 produced 63,5% editing when 16.67 pl of PDS particles were transduced, while all other PDS versions produced no more than 23.1% editing at that dilution (Table 24). Therefore, it is believed that the nucleocapsid protein was not sufficient to generate a PDS particle capable of recruiting and encapsulating a CasX RNP, [0379] Results for the MA-MS2 + NC configuration were inconsistent between PDS versions. Some PDS versions tested produced relatively high levels of editing in the MA- MS2 + NC configuration; for example, PDS versions 327, 329, 335, 360, 372, 376, 379, 380, and 414 all produced greater than 70% editing when 5.56 pl of PDS particles were transduced (Table 25). Other PDS versions tested produced relatively low levels of editing in the MA-MS2 + NC configuration. Specifically, PDS versions 328, 333, 337, 352, 364, 366, 370, 384, 387, 393, 398, and 402 all produced less than 10% editing when 5.56 ul of PDS particles were transduced (Table 25).

[0380] Overall, for most PDS versions tested, the MA-MS2 architectural configuration was the most potent editor of the tdTomato locus in mouse NPCs (Table 26). For example, 20 of the 34 PDS versions tested (versions 327, 328, 329, 330, 333, 335, 336, 337, 352, 356, 360, 367, 370, 372, 376, 379, 402, 404, 408) produced greater than 80% editing when 5.56 ul of PDS particles were transduced (Table 26). The least potent PDS particle in the MA-MS2 configuration was version 366, which produced only 21.5% editing; all other PDS versions tested were more potent than version 366. Notably, most of the PDS versions tested were more potent in the MA-MS2 configuration than the MA-MS2 + NC configuration (compare Tables 25 and 26). Specifically, at the 5.56 pl dilution, only four of the tested PDS versions (327, 329, 380, and 414) produced lower levels of editing in the MA-MS2 architectural configuration than they did in the MA-MS2 + NC configuration.

[0381] Accordingly, it is believed that for most Mononegavirales-denvod PDS particles, the nucleocapsid protein is not necessary, compared to the MA-MS2 configurations, to generate a PDS particle capable of recruiting and encapsulating CasX RNP. In most cases, the MA-MS2 architectural configuration produced PDS particles that were the most potent for gene editing.

Example 7: Use of orthogonal CasX RNP recruitment systems with non-covaient recruitment RNA-binding proteins linked to the Mononegavirales matrix protein [0382] In the preceding examples, the MS2 bacteriophage packaging system was explored as a means for recruiting CasX RNPs into PDS particles, where the MS2 coat protein as the non-covalent recruitment (NCR) protein was fused to the matrix protein, and its cognate binding partner element, the MS2 hairpin, was incorporated into the gRNA. In this example, experiments are performed to evaluate the use of alternative NCR proteins linked to the Mononegavirales matrix protein and their cognate RNA hairpin partners incorporated into the gRNA scaffolds to improve incorporation of CasX RNP and PDS particle generation.

Materials and Methods:

[0383] Plasmids containing constructs encoding CasX variant proteins 491, 515, 668, 672, or 676 are used. A separate plasmid encoding for a gRNA containing spacer 12.7 targeting the tdTomato STOP cassette is used. Generated PDS particles are pseudotyped with the VSV- G glycoprotein.

[0384] PDS structural plasmids are generated following similar methods described in Example 2. In this example, the resulting structural plasmids encode for an individual NCR protein (listed in Table 27) linked to the leading Mononegavirales matrix protein identified from earlier examples (Table 37).

[0385] The extended stem region of the accompanying gRNA scaffold is highly modifiable. Novel guide scaffold variants are engineered such that the various RNA hairpins (listed in Table 27) are integrated with either guide scaffold 174 or 235 (Table 36). Use of each scaffold variant is expected to result in a different binding affinity between CasX RNP to an individual alternative NCR protein. These guide plasmids, which include spacer 12.7 targeting the tdTomato locus, are generated following standard molecular cloning techniques. Table 27. Sequences of NCR proteins and their cognate RNA hairpin partners

[0336] PDS particles are produced and titered using the NanoSight NS300, as described in Example 1. The morphology of these PDS particles with alternative NCR-based recruitment systems is visualized using transmission electron microscopy (TEM).

[0387] To assess the editing potency of the engineered PDS particles, transduction of murine tdTomato NPCs is performed as described in Example 1 to assess editing at the tdTomato locus. XDP V206 and PDS particles harboring the MA-MS2 configuration assessed in Example 2 serve as experimental controls.

[0388] The number of CasX molecules recruited and packaged within each PDS particle is determined via a semi-quantitative Western blot analysis as described in Example 4. The absolute quantification of gRNAs within each PDS particle is determined using ddPCR as described in Example 6,

[0389] The results of these experiments are expected to demonstrate that incorporation of alternative NCR proteins into the PDS architecture, especially those with higher binding affinities for the RNA hairpin partner, may produce more potent PDS particles. Furthermore, the guide RN A scaffolds are modified to incorporate multiple hairpin elements that are expected to result in enhanced binding affinity and recruitment of CasX RNPs and, therefore, improve the potency of the PDS particle. While this example focuses on the use of alternative NCR proteins linked to the Mononegavirales matrix protein (sequences in Table 37), these alternative NCR proteins may also be linked with the Mcmonegavirales nucleocapsid protein (sequences in Table 38).

Example 8: Assessment of PDS particle size, morphology, and level of CasX RNP packaging efficiency on tropism and editing potency

[0390] Experiments are performed to assess whether use of the nucleocapsid protein to produce different architectural designs, alternative linkers, different glycoproteins for pseudotyping, other RNA binding proteins (RBPs) for use as NCR proteins and their cognate RNA hairpin partners would affect the size, morphology, and CasX RNP packaging efficiency of the PDS particle. It is conceivable that varying the particle size and morphology, as well as increasing the particle packaging efficiency, can impact cellular and tissue tropism, editing potency, and in vivo biodistribution.

Materials and Methods:

[0391] Methods of altering and assessing the linker peptides fusing either CasX or an RBP to the matrix protein in the generation of PDS particles are described in Example 4. Methods of pseudotyping and assessing PDS particles with different glycoproteins are described in Example 5. Methods of modifying and assessing the architectural designs by including the nucleocapsid structural protein in the generation of PDS particles are described in Example 6, Methods of producing and investigating the use of alternative RBPs and hairpin partners are described in Example 7. [0392] The size of PDS particles is assessed using the NanoSight NS300, as described in Example 1. The morphology of these PDS particles with alternative RBP-based recruitment systems is visualized using transmission electron microscopy (TEM), The number of CasX molecules recruited and packaged within each PDS particle is determined via a semi- quantitative Western blot analysis as described in Example 4. The absolute quantification of gRNAs within each PDS particle is determined using ddPCR as described in Example 6. Editing potency assessment is informed by transducing mouse NPCs with PDS particles and evaluating the editing rate at the tdTomato locus, as described in Example 1. Assessment of in vivo tropism and editing is performed as described in Examples 5 and 6.

Results:

[0393J Results from NanoSight and TEM analyses are expected to provide data on the size of the PDS particles generated, which are expected to vary widely. It is possible that use of a specific matrix and/or nucleocapsid protein combination derived from certain viruses could generate PDS particles with an average size of < lOOnm. The ability to control particle size confers different capabilities; for instance, larger particles would offer the benefit of packaging more cargo (e.g., RNPs), while smaller particles would likely be able to cross tissue barriers more easily and have higher cellular uptake to deliver the therapeutic cargo in vivo. Furthermore, understanding the particle size of these PDS particles is expected to provide insight into their biodistribution and editing potency in vivo. Similarly, TEM analyses are anticipated to unveil details of the resulting geometry of these nanostructures. Understanding PDS morphology may also inform shape-dependent cellular uptake and biodistribution. Lastly, the packaging efficiency of the generated PDS particles is anticipated to be informed by the quantification of CasX molecules and gRNAs within an individual PDS, and results are expected to show that the number of CasX RNPs per PDS particle varies widely depending on particle size, shape, and structural composition.

Example 9: Assessment of interferon antagonism by the Mononegavirales matrix protein used in the generation of PDS particles

[0394] To ensure delivery of PDS particles containing CasX RNPs to the target cell or tissue, avoidance by the innate immune system is desirable. Experiments were performed to test whether the Mononegavirales matrix protein used in the generation of PDS particles antagonizes interferon (IFN) signaling and the response to this signaling, which would result in innate immune evasion and enhanced delivery of CasX RNPs to induce editing at the target locus. Materials and Methods:

[0395] Plasmids containing constructs for CasX expression encoding a CasX variant, and a plasmid encoding a gRNA containing spacer 12,7 targeting the tdTomato STOP cassette were used. As described in Example 2, the resulting Afozzozzegav/raZes '-based PDS particles utilized the MS2 packaging system as the mechanism for non-covalently recruiting CasX RNPs.

[0396] PDS structural plasmids were generated as described in Example 2, following standard molecular cloning techniques. PDS version 329 (derived from Bombali ebolavirus) was used, as well as version 329 PSD particle with a cognate Bombali ebolavirus VP35 protein, as described in Example 13. PDS particles were pseudotyped with the VSV-G glycoprotein.

[0397] PDS particle production, titering of PDS particles using the NanoSight NS300, and transductions of Jurkat cells, K562 cells, or ARPE-19 cells were performed as described in Example 1. XDP V206, replication-competent recombinant lentivirus (LV), and recombinant AAV particles served as experimental controls.

Quantifying the expression of interferon-stimulated genes by qPCR:

[0398] To determine whether the IFN response was induced or suppressed by the Mononegavirales matrix protein used in generating PDS particles, qPCR was performed to assess the expression of a panel of interferons genes, including the type 1 interferons alpha and beta, the type 2 interferon gamma, and the type three interferon lambda. RNA was extracted from harvested ARPE-19, Jurkat, and K562 cell lines treated with PDS particles using the Zymo Quick-RNA™ 96 kit and used as input for reverse transcription. The resulting cDNA served as input for qPCR reactions to measure the expression of the interferons using SYBR™ Green-based detection. Expression of a housekeeping gene was used for normalization, and expression data of the interferons was analyzed according to the double delta Ct method and compared relative to an untreated control.

[0399] In parallel, an enzyme-linked immunosorbent assay (ELISA) testing for the presence of effector cytokines activated in response to IFN stimulation is performed to corroborate qPCR analyses.

[0400] Results:

[0401] Innate immune response profiles for ARPE-19, Jurkat, and K562 cells treated with version 329 PDS particles or various other particles are provided in FIG. 4, FIG. 5, and FIG, 6, respectively.

[0402] In ARPE-19 cells, all of the tested particles induced interferon alpha and all of the tested particles except for the replication-competent recombinant lentiviral particle induced interferon beta relative to the untreated control (FIG. 4). XDP V206 produced the highest levels of interferon alpha and beta, and the version 329 PDS particles with or without VP35 induced about 2-4 fold lower interferon alpha and beta than did XDP V206. Interferon gamma was not induced in ARPE-19 cells, and very low levels of interferon lambda were induced (FIG. 4).

[0403] In Jurkat cells, V206 XDPs and version 329 PDS particles with VP35 produced the highest levels of interferon beta and interferon lambda expression (FIG. 5). Very little interferon alpha or interferon gamma was induced by any of the particles tested (FIG. 5). [0404] In K562 cells, none of the tested particles induced interferon alpha relative to the untreated control, and only recombinant AAV8 particles induced interferon beta relative to the untreated control (FIG. 6). Most of the tested particles induced interferon gamma, with the exceptions of version 329 PDS particles with VP35, and recombinant AAV8 and AAV9 particles (FIG. 6). Finally, replication-competent recombinant lentivirus particles, recombinant AAV9 particles, and version 32.S* PDS particles with VP35 induced interferon lambda (FIG. 6).

Example 10: Packaging of CasX RNPs within PDS particles by appending an NES sequence and/or mutationally inactivating the NLS sequence on the matrix protein [0405] NLS (nuclear localization signal) sequences are commonly found in viral proteins; for instance, native NLS or NLS-like sequences are likely present in the Mononega.virales matrix proteins being utilized to generate PDS particles. NLS presence could potentially hamper the packaging of CasX RNPs in the cytoplasm by sequestering CasX RNPs in the nucleus of packaging cells. Therefore, to improve packaging of the RNP in the PDS, thereby enabling enhanced PDS particle potency, the following strategies are employed: 1) mutational inactivation of the NLS and/or 2) appending an NES (nuclear export signal) sequence to the matrix protein, and/or 3) appending an NES sequence to the C-terminus of CasX to facilitate CasX export from the nucleus and promote packaging of CasX RNPs into PDS particles. Experiments are performed to demonstrate that CasX RNP packaging may be improved by employing these strategies, which would therefore enhance tire editing potency of PDS particles.

Materials and Methods:

[0406] Plasmids encoding CasX variant proteins 491, 515, 668, 672, or 676, and a plasmid encoding for guide scaffold variant 251 and spacer 12,7 targeting the tdTomato STOP cassette are used. [0407] PDS structural plasmids are generated following similar methods described in Example 2. NES sequences that can be appended to the C-terminus of CasX are listed in Table 28. Additionally, the C-terminus of the CasX sequence contains one or two HIV protease cleavage sites (SQNYPIVQ; SEQ ID NO: 100) followed by a rigid linker (GPAEAAAKEAAAKEAAAKA; SEQ ID NO: 1932), both of which precede the appended NES sequence. Alternatively, the Tobacco Etch Virus (TEV) protease cleavage (ENLYFQS; SEQ ID NO: 98) is used in place of the aforementioned HIV protease cleavage site.

Furthermore, a separate plasmid encoding for a polyprotein composed of zMononegavirales matrix protein linked with an HIV1 protease or a temperature sensitive TEV protease is incorporated into the construct to aid in the cleavage of the appended NES, The sequences for the polyprotein containing HIV1 protease or TEV protease are listed in Tables 34 and 35, respectively. The leading Mononegavirales matrix protein that resulted in the generation of PDS particles exhibiting the highest editing potency, as determined in Examples 2 and 3, is used in these experiments. The NLS or NLS-like sequence of the leading matrix protein is mutationally inactivated via site-directed mutagenesis. The resulting generated PDS particles are pseudotyped with the VSV-G glycoprotein.

Table 28. NES sequences for CasX C-terminal tagging

[0408] PDS particle production, titering and size assessment of generated PDS particles using the NanoSight NS300, and transduction of murine tdTomato NPCs is performed as described in Example 1. XDP V206 serves as an experimental control.

[ 04091 The number of CasX molecules recruited and packaged within each PDS particle is determined via a semi-quantitative Western blot analysis as described in Example 4. The absolute quantification of gRNAs within each PDS particle is determined using ddPCR as described in Example 6.

[0410] The results of these experiments are expected to show that tagging an NES sequence to either the CasX, matrix protein, or both CasX and matrix protein helps prevent nuclear sequestration of both the matrix protein and CasX RNPs and improve packaging efficiency, potentially yielding more potent PDS particles. Appending different NES sequences to CasX is expected to result in varying numbers of CasX molecules and gRNAs being packaged into PDS particles as RNPs, and therefore the resulting PDS particles are anticipated to exhibit a range of editing efficiencies at the tdTomato locus in transduced mouse NPCs. The findings are also anticipated to show that mutating the native NLS sequence in the Mononegaviral.es matrix protein helps impede nuclear accumulation of CasX RNPs during PDS particle production. It is possible that the combination of inactivating the NLS and incorporating the NES substantially enhances packaging, thereby producing more potent PDS particles containing CasX RNPs.

Example 11: Generation and assessment of potency of Afnmwe^owrafes-based PDS partides using alternative configurations of PDS particle constructs

[0411] In the preceding examples, Mononegavirales-\)3&eA PDS particles were generated by transfecting the packaging cells with four individual plasmids: 1) a structural plasmid encoding the matrix-MS2 fusion protein, 2) a plasmid encoding the CasX variant, 3) a plasmid encoding a single gRNA and a targeting spacer, and 4) a plasmid encoding a glycoprotein (e.g., VSV-G) for pseudotyping. Reducing the number of plasmids necessary for PDS particle generation without compromising particle delivery and efficacy is expected to be ultimately beneficial for the manufacturing process. In this example, experiments are performed to generate and assess the edi ting potency of Mononegavirales-\wsQ& PDS particles produced by using alternative configurations of PDS constructs encoding for the components to identify the most efficient plasmid system for packaging and production of PDS particles containing CasX RNPs.

Materials and Methods:

[0412] PDS structural plasmids are generated using similar methods described in Example

2. Sequences encoding CasX variant proteins 491 , 515, 668, 672, or 676 and guide scaffold variant 251 and spacer 12,7 targeting the tdTomato STOP cassette are used. The configurations of the resulting plasmids used for transfection to generate PDS particles are listed in Table 29, including the four-plasmid system (configuration version 1) utilized for production in the preceding examples. Here, the leadingAfononegnvzra/es matrix protein that resulted in the generation of PDS particles exhibiting the highest editing potency as determined in Examples 2 and 3 is used. The resulting plasmids are Sanger-sequenced to ensure correct assembly prior to midi-prepping for use for PDS particle production.

Table 29. Alternative configurations of PDS particle constructs. "MA" denotes matrix protein

[0413] PDS particle production, titering of generated PDS particles using the NanoSight

NS300, and transduction of murine tdTomato NPCs is performed as described in Example 1.

Varying amounts of PDS particle plasmids are tested to determine the optimal ratios of plasmids for transfection into Lenti-X packaging cells, which are assessed via titering. [0414] The number of CasX molecules recruited and packaged within each PDS particle is determined via a semi-quantitative Western blot analysis as described in Example 4. The absolute quantification of gRNAs within each PDS particle is determined using ddPCR as described in Example 6.

[0415] These experiments are expected to show that alternative configurations of plasmids encoding the PDS particle constructs are able to generate PDS particles effectively in transfected packaging ceils, which are expected to be abie to successfully’ deliver CasX RNPs to cells to edit the target genomic locus (here, the tdTomato locus). The results are expected to determine whether these alternative configurations are able to achieve at least a similar level of editing potency as the four-plasmid system used for PDS particle production as demonstrated in Examples 2 and 3. In addition, an alternative strategy of PDS particle production to be tested includes engineering a packaging cell line to express VSV-G or other pseudotyping factors, and subsequently transfect these cells with the structural plasmids listed in columns 1-3 of Table 29 to produce PDS particles containing CasX RNPs.

Example 12: Generation of hypo-immunogenic packaging ceil lines derived from HEK293T Lenti~X cells for use in generating Mtmonegavirales-bsised PDS particles with reduced immunogenicity

[0416] The preceding examples have demonstrated the practicality of generating Mononegavirales-based PDS particles to deliver therapeutic payloads such as CasX RNPs to the target cells for editing. It is possible that specific components or contaminants of the PDS particle, possibly derived from the packaging cells and incorporated into the PDS surface, would trigger undesired immune responses in vivo. Experiments are performed to build hypo-immunogenic packaging cell lines for use in the generation of PDS particles, which is anticipated to be more effective or have improved safety’ profiles for in vivo applications. In this example, a packaging cell line with a knock-out of the beta-2 microglobulin (B2A4) gene with a concurrent CD47 knock-in was generated, which is used for the production of Mononegavirales-based PDS particles.

[0417] Materials and Methods:

[0418] Plasmids encoding CasX variant proteins 491, 515, 668, 672, or 676, and a plasmid encoding for guide scaffold variant 251 and spacer 12.7 targeting the tdTomato STOP cassette are utilized. The leading Mononegavirales matrix protein that resulted in the generation of PDS particles exhibiting the highest editing potency as determined in Examples 2 and 3 is used. PDS particles are pseudotyped with the VSV-G glycoprotein. [0419] PDS structural plasmids are generated using methods described in Example 2, and the resulting sequence-verified plasmids is used for PDS production using the engineered hypo-immunogenic packaging lines as described below.

Generation of HEK293T cells with B2M knock-out and CD47 knock-in (B2MfCD47^'f: [0420] 293T Lenti-X cells were nucleofected with purified RNP targeting the B2M locus and a CD47 single-stranded oligo DNA nucleotide (ssODN) to knockout the B2M gene and concurrently knock-in the CD47 gene at the B2.M locus to overexpress CD47. Nucleofected cells were then seeded in a well of a 6-weil plate and subsequently expanded prior to trypsinization to sort for B2M'^/~;CD47^+/i cells using an Attune™ NxT flow cytometer. Sorted B2M'^/';CD47¹ ' ' cells were expanded and frozen for subsequent use in the packaging of Monone gavirales -based PDS particles described herein.

[0421] In addition, other genetic knockouts and/or knock-ins are made using similar methods to engineer a variety of hypo-immunogenic packaging lines derived from HEK293T Lenti-X cells. Genetic modifications for generating hypo-immunogenic lines are listed in Table 30.

Table 30. Genetic modifications to use to engineer hypo-immunogenic packaging ceils

[0422] PDS particle production, titering of PDS particles using the NanoSight NS300, and transduction of murine tdTomato NPCs are performed as described in Example 1. XDP V206 serves as an experimental control. In addition. Mononegavirales-based PDS particles generated using unmodified Lenti-X cells for packaging serves as an experimental control. Surface characterization of PDS particles:

[0423] To identify expression of proteins encoded by the genes listed in Table 30 on the surface of PDS particles, samples of isolated PDS particles are subjected to immunolabeling using the appropriate antibodies to detect the target surface protein. Immunolabeled PDS particles are subsequently subjected to nanoparticle tracking analysis (NT A) using the NanoSight NS300 to characterize surface immunophenotype of the PDS particles. In parallel, PDS particles are harvested for Western blotting analysis to detect for the presence or absence of proteins encoded by the genes listed in Table 30.

[0424] PDS particles produced using various hypo-immunogenic packaging cell lines are utilized in assays for additional characterization, including assessing complement inactivation and macrophage phagocytic activity in vitro.

[0425] Briefly, to assess whether PDS particles produced from packaging cells with CD59, CD55, or CD46 knock-in bypass complement activation, human plasma containing intact complement proteins is incubated with PDS particles produced from unmodified or CD59^+/+ (or CD55^1/+ or CD46 '^{/ l}) -modified packaging cells. Following incubation, the plasma-PDS particle mixture is used to transduce tdTomato NPCs to evaluate editing efficiency. As a comparator, human plasma, that has been heat-inactivated in order to inactivate the complement proteins isincubated with PDS particles and subsequently used to transduce tdTomato NPCs.

[0426] Briefly, for evaluating macrophage phagocytic activity' in vitro, U937 cells, a human lymphoblast line grown in RPM1-1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (PenStrep), are seeded on wells coated with poly-L-lysine and cultured for three days to induce differentiation into macrophages. The first 24 hours of culturing are supplemented with phorbol myristate acetate, which induces differentiation into macrophages. Subsequently, the differentiated cells are treated with GFP -labeled PDS particles for six hours before being harvested for immunostaining with the CDllb antibody to label macrophages for imaging. PDS particles used for treatment are produced from unmodified or CD47^+/+ (or CD24^_/‘ or CD58‘^/_) -modified packaging cells.

[0427] Assessment of whether the in vivo editing potency is improved using PDS particles produced from hypo-immunogenic packaging cells is performed using similar methods described in Examples 5 and 6.

[0428] The experiments discussed in this example are expected to demonstrate that PDS particles produced from using hypo-immunogenic packaging lines exhibit improved editing efficiency compared to that exhibited by PDS particles produced from unmodified packaging hues, in vitro and in vivo. The reason for this expected outcome is that PDS particles produced using hypo-immunogenic cells are anticipated to have substantially reduced immunogenicity. Therefore, it is expected that PDS particles produced from CD59^+/+ (or CD55^+/+ or ('1)46 ' 1 -modified packaging cells are able to bypass complement activation when incubated with human plasma to induce editing. Furthermore, GFP-Iabeled PDS particles produced from CD47'^,/+ (or CD24"⁷" or CD58’^A) -modified packaging cells are expected to reduce macrophage phagocytosis. Producing PDS particles with more resistance to complement activation or macrophage phagocytosis is anticipated to result in improved delivery and editing efficiency of CasX RNPs.

Example 13: Use of the Filoviridae VP35 protein to generate PDS partides for CasX RNP delivery

[0429] As described in Example 2, PDS particles derived from members of the Filoviridae family of Mononegavirales were effective for delivering CasX RNPs that edited the genome. The filovirus structural protein VP35 can improve production of virus-like partides derived from filoviruses, as shown, for example, by Johnson et al, (Johnson RF, Bell P, Harty RN. Effect of Ebola virus proteins GP, NP and VP35 on VP40 VLP morphology. Virol J. 2006 May 23;3:31 ). Experiments were performed to test whether adding filovirus VP35 proteins to PDS particles would improve the structure and function of the PDS particles.

Materials and Methods:

[0430] CasX 491 was used along with gRNA with scaffold 251 and spacer 12.7 targeting the tdTomato locus. PDS versions 327, 328, 329, 330, 331, 332, 333, 428, 429, and 430, as described in Example 2, were used with structural plasmids encoding architectural configurations in which the MS2 coat protein was fused to the matrix (MA) protein, and cognate nucleocapsid (NC) protein and/or cognate VP35 proteins were encoded on separate plasmids, PDS structural plasmids were generated as described in Example 2. The plasmids used to encode the components of these PDS configurations are summarized in Table 31, below. DNA and amino acid sequences of the VP35 protein and the VP35 protein fused to MS2 are provided in Table 32. The PDS particles were pseudotyped with VSV-G.

Table 31. Summary of PDS configurations tested in this example

Table 32. DNA and amino acid sequences of VP35 proteins and VP35 proteins fused to

MS2

[0431] PDS particles were produced and titered as described in Example 1. The morphology of PDS particles was visualized using transmission electron microscopy (TEM). To assess the editing potency of the PDS particles, murine tdTomato NPCs were transduced as described in Example 1, and editing of the tdTomato locus was measured.

Results:

[0432] Table 33, below, provides the percentage of mouse NPCs with an edited tdTomato locus following transduction with serial dilutions of filovirus-derived PDS particles in various architectural configurations with or without VP35 or VP35 fused to MS2.

Table 33. Percentage of mouse NPCs with edited tdTomato locus

[0433] Overall, the results provided in Table 33 show that PDS particles can be produced with the nucleocapsid protein anchor VP35 in addition to MA-MS2. However, inclusion of nucleocapsid protein (or nucleocapsid protein fused to MS2) or VP35 (or VP35 fused to MS2) did not increase transduction efficiency in TdT NPCs compared to the PDS particles with the MA-MS2 architectural configuration.

[0434] For PDS version 428 (Bundibugyo ebolavirus) the MA-MS2 + VP35, MA-MS2 + VP35-MS2, and MA-MS2 + NC-MS2 + VP35 configurations produced higher levels of editing than the MA-MS2 and MA-MS2 + NC-MS2. + VP35-MS2 configurations.

[0435] PDS version 327 (Lloviu cuevavirus) produced high levels of editing activity that was similar across the tested configurations.

[0436] Transmission electron microscopy (TEM) was performed to examine the morphology version 330 (derived from Reston ebolavirus) PDS particles generated with MA-MS2 or MA-MS2 + VP35 + NC architectures, with or without CasX (data not shown). The MA-MS2 particles appeared spherical, whether or not CasX was packaged in the particles. While the MA-MS2 + VP35 + NC particles without CasX produced the expected filamentous morphology that is characteristic of the Filoviridae family, MA-MS2 + VP35 + NC particles with CasX were spherical. These results suggest that the recruitment of CasX affected the PDS packaging process and prevented the formation of a filamentous structure.

Table 34: Sequences of polyprotein comprising a Mononegavirales matrix protein linked with an HIV protease

Table 35: Sequences of polyprotein comprising a Mononegavirales matrix protein linked

Table 36: Sequences of guide scaffold variants with incorporated RNA hairpins for alternative non-covalent recruitment systems

Table 37: Sequences of ma trix (MA) protein linked with NCR proteins

Table 3§: Sequences of nuckocapsid (NC) proieiu linked with MS2 WT and alternative

NCR proteins

Example 14: CasX:gRNA In Vitro Cleavage Assays

[0437] Experiments were performed to assess in vitro DNA cleavage by CasXtgRNA ribonucleoproteins (RNPs).

Materials and Methods:

Assembly of UNP:

[0438] RNPs of either CasX 119 (SEQ ID NO: 136), CasX 491 (SEQ ID NO: 190), CasX 515 (SEQ ID NO: 197), or CasX 812 (SEQ ID NO: 478) were assembled with single guide RNAs (sgRNA) with scaffold 316

(ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGU AGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG; SEQ ID NO: 7976) and one of two spacers, as described in detail below. Separately, RNPs of CasX 515 were assembled with sgRNA with either scaffold 2 (SEQ ID NO: 5), 174 (SEQ ID NO: 2238), 235 (SEQ ID NO: 2293), or 316 and one of two spacers.

[0439] Purified RNP of CasX and sgRNA were prepared same-day prior to experiments. For experiments where protein variants were being compared, the CasX protein was incubated with sgRNA at 1:1.2 molar ratio. When scaffolds were compared, the protein was added in 1.2:1 ratio to guide. Briefly, sgRNA was added to Buffer #1 (20 niM Tris HC1 pH 7.5, 150 mM NaCI, 1 mM TCEP, 5% glycerol, 10 mM MgCb) on ice, then CasX was added to the sgRNA solution, slowly with swirling, and immediately incubated at 37 °C for 20 minutes to form RNP complexes. RNP complexes were centrifuged at 4 °C for 5 minutes at 16,000 x g to remove any precipitate. Formation of competent (active) RNP w^zas assessed as described below.

In vitro cleavage assays:

104401 The ability of CasX variants to form active RNP compared to reference CasX was determined using an in vitro cleavage assay. The beta-2 microglobulin (B2M) 7.9 and 7.37 target for the cleavage assay was created as follows. DNA oligos (sequences in Table 39) were generated with 5" terminal amino modification for conjugation to Cy-dyes with an amino-reactive handle (N-hydroxy succinimide). Oligo-dye conjugation reactions of 100 uM oligo and 1 mM dye were performed in 100 mM sodium borate pH 8.3 at 4 °C for 16 h. Target strands (TS) were labeled with Cy5.5 and non-targeting strands (NTS) were labeled with Cy7.5. After quenching the reactions with 1 mM Tris pH 7.5, the conjugated oligos w⁷ere purified via ethanol precipitation. Double-stranded DNA (ds DNA) targets were formed by mixing the oligos in a 1: 1 ratio in lx hybridization buffer (20 mM Tris HC1 pH 7.5, 100 mM KCL 5 mM MgCb.), heating to 95 °C for 10 minutes, and allowing the solution to cool to room temperature.

Table 39: DNA sequences and descriptions of target DNAs

*5AmMC6 indicates the 5' Amino Modifier C6. The target sequences are underlined.

The Kcleave assay using the mismatched position 5 dsDNA target was run at 37 °C.

Determining cleavage-competent fractions for RNPs:

[0441] Cleavage reactions were prepared with final RNP concentrations of 100 nM and final target concentration of 100 nM. Reactions were carried out at 37 °C and initiated by the addition of the dye-labeled dsDNA target. Aliquots were taken at 5, 30, and 60 minutes and quenched by adding to 95% formamide, 25 mM EDTA. Samples were denatured by heating at 95 °C for 10 minutes and run on a 10% urea-PAGE gel. The gels were imaged with a

Cytiva Typhoon and quantified using the Cytiva IQTL software. Kcleave assay:

[0442] Cleavage reactions were set up with a final RNP concentration of 200 nM and a final target concentration of 10 nM. Reactions were carried out at 16 °C, except where otherwise noted, and initiated by the addition of the target DNA. Aliquots were taken at 15, 30, 60, 120, 180, 240, and 480 seconds, and quenched by adding to 95% formamide, 25 mM EDTA. Samples were denatured by heating at 95 °C for 10 minutes and run on a 10% urea-PAGE gel. lire gels were imaged with a Cytiva Typhoon and quantified using the Cytiva IQTL software. The apparent first-order rate constant of non-target strand cleavage (kdeave) was determined for each CasX:sgRNA combination replicate individually.

[0443] To test the relative specificities of engineered proteins in vitro, apparent cleavage rate constants were compared for targets with mismatched bases at various positions (5, 10, and 15 nt downstream of PAM, Table 39). Cleavage assays were performed in large excess of RNP (200 nM RNP and 1 nM target dsDNA) at 16 °C, with the exception of assays measuring cleavage of the target with a mismatch at 5 nt, which were conducted at 37 °C in order to observe measurable cleavage rates. Aliquots were taken at 15, 30, 60, 120, 180, 240, and 480 seconds, and quenched by adding to 95% formamide, 25 mM EDTA. Samples were denatured by heating at 95 °C for 10 minutes and run on a 10% urea-PAGE gel. The gels were imaged with a Cytiva Typhoon and quantified using the Cytiva IQTL software. The apparent first-order rate constant of non-target strand cleavage (kcleave) v/as determined for each CasX:sgRNA combination replicate individually.

Results:

Determining cleavage-competent fractions for protein variants compared to reference CasX 119

[0444] To determine the cleavage-competent fraction for the tested CasX proteins, it was assumed that CasX acts essentially as a single-turnover enzyme under the assayed conditions, as indicated by the observation that sub-stoichiometric amounts of enzyme fail to cleave a greater- than-stoi chi ometric amount of target even under extended time-scales and instead approach a plateau that scales with the amount of enzyme present. Thus, the fraction of target cleaved over long time-scales by an equimolar amount of RNP is indicative of what fraction of the RNP is properly formed and active for cleavage. Thus, the active (competent) fraction for each RNP was derived from the cleaved fraction over the total signal al the 60-minute timepoint, upon confirming an increase in cleaved fraction from the 5-minute timepoint, and relative plateau in cleaved fraction from the 30-minute timepoint. [0445] Apparent competent fractions were determined for the RNPs with various CasX proteins, and are provided in Table 40.

Table 40: Protein variant RNP comparison of fraction competence and Kcleave rates

* Active fraction was calculated by averaging three experimental replicates.

** The Kcleave assay using the mismatched position 5 dsDNA target was run at 37 °C.

[0446] For protein variant comparison, the following CasX proteins were used with guide scaffold 316 and spacer 7.9 or guide 316 and spacer 7.37: CasX 119, CasX 491, CasX 515, and CasX 812, CasX 119 had the lowest active fraction for both spacers, indicating that CasX 491, CasX 515, and CasX 812 form more active and stable RNP with the identical guides under the tested conditions as compared to CasX 119. CasX proteins 491, 515, and 812 did not show consistent trends in their competent fractions across the two spacers, consistent with the expectation that the additional engineering following CasX 491 primarily affects target engagement and cleavage, rather than guide binding or stability’.

Kcleave assay to understand specificity of RNPs formed from protein variants:

[0447] Assays were performed to measure the apparent first-order rate constant of non-target strand cleavage (kcleave), and the results are presented in Table 40, above. A drastic effect on the kinetics of CasX 812 RNP cleavage was observed for on-target versus the mismatched dsDNA target for both spacers. CasX 812 had comparable on-target cleavage rates to CasX 491 and CasX 515 for both spacers, with a slightly higher cleavage rate than 515 on spacer 7.9, which might be explained by the lower competent fraction observed for the 515 RNP with that spacer, and a lower cleavage rate on 7.37.

[0448] The off-target rates for CasX 812 were much more substantially reduced for most of the mismatched substrates. The difference in kcleave rates was readily apparent for the target with a mismatch at position 10, with 812 having a roughly 6-fold (7.9) and 2-fold (7.37) reduction in cleavage rate, as compared to its on-target rate. CasX 515, by comparison, exhibited a 2.4-fold and a 25% reduction on the same targets. A substantial difference was also observed for the position 5 mismatch targets. Even though the assay was run at 37 °C to enable measurable cleavage rates, as the position 5 mismatch targets were essentially uncleaved by the CasX RNPs at the lower temperature used for the other targets, CasX 812 against spacer 7.9 exhibited a 9-fold reduction in cleavage rate from on-target rate run at 16 °C and a 2-fold reduction for the 7.37 spacer with a position 5 mismatch. CasX 515 showed a 2-fold reduction for mismatched 7.9 and a nearly equivalent cleavage rate for 7.37 with the position 5 mismatch (note that the ‘’equivalent’' cleavage rate is due to the increased temperature).

[0449] For the position 15 mismatch substrate, CasX 812 exhibited modest reductions in cleavage rates relative to on-target rates, comparable to the reduction observed for 515. This suggests that the increased sensitivity of CasX 812 to mismatches declines by the PAM distal region, at least for the specific mismatches and spacers tested here. The increased sensitivity at positions 5 and 10 in particular correlates with the position of the G329K mutation present in CasX 812. This mutation introduces a positive charge near the RNA spacer around position 8 and may help CasX to better read out distortions caused by mismatches. Mismatches closer to this new site of contact would be more likely to significantly disrupt either R-loop propagation or allosteric activation of the RuvC (depending on the precise mechanism of increased specificity’), while mismatches farther away’ (as in the position 15 mismatch) might have more variable effects depending on the nature of the mismatch and its effects on the broader heteroduplex structure. Taken together, these data confirm that CasX 812 is inherently’ more sensitive to mismatches between the RNA spacer and the DNA target and is not simply a less active enzyme, as the decrease in cleavage rate at mismatched targets is in excess of the decrease in cleavage rate at properly matched targets. This is consistent with results described in Examples 15 and 16 that indicate that CasX 812 is a highly specific enzyme, with lower off-target editing compared to the other nucleases tested.

Determining cleavage-competent fractions for single guide variants relative to reference single guide 2: [0450] RNPs were complexed using the aforementioned methods. To isolate the effect of sgRNA identity on RNP formation, guide-limiting conditions were employed. sgRNAs with scaffolds 2, 174, 235, or 316 with spacers 7.9 or 7.37 were mixed with CasX 515 at final concentrations of 1 pM for the guide and 1.2 pM for the protein. Fraction competence was calculated as described above, and the results are provided in Table 41 .

Table 41. Guide variant RNP comparison of fraction competence and Kcleave assay

* active fraction was calculated by averaging two experimental replicates

[0451 ] Given the complex folding structure of the CasX guide, fraction competence is expected to largely be determined by how much of the guide is properly folded for interaction with the protein. AH guides with engineered scaffolds show-ed improvements over scaffold 2, but guides with scaffold 235 or 316 showed improvements relative to 174 for spacer 7.37. ’This is consistent with the introduction of mutations in the pseudoknot and triplex that are expected to stabilize the properly folded form.

[0452] Higher competent fractions of all guides were observed for spacer 7.9. For this spacer, scaffold 174 had the highest competent fraction, followed by scaffolds 316, 235, and 2.

Proper guide folding is expected to be highly dependent on the potential for undesired interactions betw-een the scaffold and spacer sequences, so the observed differences may be attributable to differential sequence-specific interactions, variations in prep quality, or noise in the assay.

Determining kcleave for single guide variants compared to reference scaffold 2: [0453] Cleavage assays were performed with CasX 515 and guides with reference scaffold 2 compared to guides with scaffolds 174, 235, or 316 with spacer 7.9 or 7.37 to determine relati ve cleavage rates. The mean and standard deviation of three repli cates with independent fits are presented in Table 41, above.

[0454] To reduce cleavage kinetics to a range measurable with the assay, the cleavage reactions were incubated at 16 °C. Under these conditions, all guides supported faster cleavage rates as compared to scaffold 2. For spacer 7.37, the cleavage kinetics aligned with those guides that contributed to the highest fraction competence, with the highest cleavage rate being sg!74 (0.1723 s’¹), followed by scaffold 235 (0.1696 s'¹) and scaffold 316 (0.1413 s’¹), versus scaffold 2 (0.1346 s"¹). For spacer 7,9, scaffold 316 yielded the highest cleavage rate (0.0851 s'¹), followed by scaffold 235 (0.0647 s'¹) and sg!74 (0.0534 s'¹), versus scaffold 2 (0.0204 s’¹). The fraction competence and kcieave data did not demonstrate differences across the engineered variants that were consistent across both spacers, although all are consistently better than scaffold 2. This suggests that the improvements seen for scaffold 235 and 316 over 174 are primarily due to behavior in the cell, whether it be stability in the cytoplasm, folding in the cytoplasm, transcription when delivered via plasmid or AAV, or refolding ability when delivered via L.NP, that are not captured by guides that have been in vitro transcribed, refolded, and tested for cleavage biochemically.

Example 15: Identification of CasX proteins with enhanced activity or specificity relative to CasX 515

[0455] An experiment was performed to identify CasX proteins with single mutations and increased editing activity or improved specificity relative to CasX 515.

Materials and Methods:

[0456] A multiplexed pooled approach was taken to assay clonal proteins derived from CasX 515 using a pooled activity and specificity (PASS) assay. A pooled HEK cell line, which was adapted to suspension culture from adherent cells, was generated and termed PASS VI.03. Methods to complete the production of the PASS_V1 .03 line were previously described in International Publication No. WO2022120095 A 1. incorporated herein by reference.

[0457] CasX proteins were expressed using a relatively weakly-expressing promoter to reduce CasX protein expression and thereby improve the sensitivity of the assay. Samples were tested in quadruplicate. The list of CasX proteins tested and their mutations relative to CasX 515 is provided in Tables 42 and 43, below. All of the tested CasX proteins had single mutations (i.e., a single amino acid substitution, deletion, or insertion) relative to CasX 515, except for CasX 676. which has three mutations relative to CasX 515. Streptococcus pyogenes Cas9 without a guide RNA served as a negative control.

[0458] To assess the editing activity and specificity of the tested CasX proteins at human target sites, two sets of target sites were quantified. First, editing was quantified at TTC PAM on-target sites in which the twenty' nucleotides of each gRNA spacer targeting these on-target sites were perfectly complementary to the target site. For each sample and spacer-target pair, data based on < 500 reads were removed. Fraction indel values for each sample and spacertarget pair were subtracted by the average fraction indel value across Cas9-treated samples with the same spacer-target pair; Cas9 served as a negative control due to the absence of a compatible guide RNA. Second, editing was quantified at TTC PAM off-target sites, in which one of the twenty nucleotides of the spacer was mismatched with the target site. As above, for each sample and spacer-target pair, data based on < 500 reads were removed, and fraction indel values for each sample and spacer-target pair were subtracted by the average fraction indel value across Cas9-treated samples with the same spacer-target pair. Finally, for those TTC PAM spacer-target pairs that had both an on-target and an off-target version, the average editing activity and standard error of the mean (SEM) were calculated.

Results:

[0459] Table 42 provides the level of on-target editing produced by various CasX proteins with mutations relative to CasX 515, ranked from highest to lowest activity.

Table 42. Average on-targeting editing activity, ranked from highest to lowest

*Positions of mutations are shown relative to a CasX 515 sequence with an N-terminal methionine residue (i.e., SEQ ID NO: 197, with the addition of an N-terminal methionine). [0460] As shown in Table 42, CasX proteins 607, 532, 676, 592, 788, 583, and 555 produced higher levels of on-target editing than did CasX 515. CasX proteins 569, 787, 561, 577, 585, and 572 also produced relatively high levels of on-target editing, with at least 90% of the activity' of CasX 515 (i.e., greater than 1.88E-01 on-target editing).

[0461] Table 43 provides the level of off-target editing produced by various CasX proteins with mutations relative to CasX 515, ranked from lowest to highest activity'.

Table 43. Average off-targeting editing activity, ranked from lowest to highest

’^Positions of mutations are shown relative to a CasX 515 sequence with an N -terminal methionine residue (i.e., SEQ ID NO: 197, with the addition of an N-tenninal methionine). [0462] As shown in Table 43, many of the tested CasX proteins showed lower levels of off- target editing than did CasX 515. For example, consistent with previous results, CasX 812 produced relatively low' levels of off-target editing. Further, some of the tested CasX proteins showed even lower levels of off-target editing than did CasX 812 (specifically, CasX 528, 535, 573, 824, 631, 587, 538, and 702).

[0463] Based on these results, a set of mutation conferring a high degree of editing activity and/or specificity was chosen for introducing in pairs into CasX 515. First, high activity mutations were defined as those that showed a level of on-target editing equal to at least 87.3% of the level of on-target editing by CasX 515. CasX 607, 532, 676, 592, 788, 583, 555, 569, 787, 561, 577, 585, 572, 536, 656, 559, 777, and 584 met this threshold, and were therefore selected as potential activity-enhancing mutations (see Table 44). Second, high specificity mutations were defined as those producing 80% or lower of the level of off-target editing produced by CasX 515, while maintaining at least 79.95% of the on-target editing activity of CasX 515. This 80% on-target editing activity requirement was implemented to avoid selecting mutations that were simply loss-of-function mutations and would therefore not be expected to be useful as gene editors. CasX 593, 572, 818, 638, 584, 562, and 784 met these criteria, and were therefore selected as potential specificity-enhancing mutations (see Table 44).

[ 0464 ] In total, 22 individual mutations were chosen as candidates for introducing in pairs into CasX 515 and testing for improved properties, as described in Example 16, below. The positions of the individual mutations relative to full-length CasX 515 protein, as well as amino acid sequences of full-length CasX proteins with the individual mutations, are provided in Table 44. Table 45, below, shows the ammo acid sequences and coordinates of the domains of CasX 515, and Table 46 shows the positions of the 22 individual mutations within the domains of CasX 515, as well as the amino acid sequences of domains with each individual mutation.

Table 44. Summary of positions of single mutations within the CasX 515 protein

^Positions of mutations are shown relative to a CasX 515 sequence with an N-terminal methionine residue (i.e., SEQ ID NO: 197, with the addition of an N-terminal methionine).

Table 45: CasX 515 domain sequences and coordinates

Table 46. Summary of positions of single mutations within CasX 515 protein domains

’^Positions of mutations within domains are shown relative to the CasX 515 domain sequences provided in Table 45, above. fMutated residues are bolded and underlined.

Example 16: Engineered CasX proteins with pairs of mutations relative to CasX 515 [0465] Engineered CasX proteins were generated with pairs of mutations relative to CasX 515, and assessed for their on and off-target gene editing activity.

Materials and Methods:

[0466] Pairs of mutations listed in Tables 44 and 46, above, were introduced into the CasX 515 amino acid sequence to generate 161 ammo acid sequences of engineered CasX proteins. The pairs of mutations and full-length amino acid sequences of the 161 engineered CasX proteins tested are listed in Tables 47, and Table 48 provides the ammo acid sequences of each of the domains of the 161 engineered CasX proteins.

Table 47. Pairs of mutations and amino acid sequences of engineered CasX proteins

*Positions of mutations are shown relative to a CasX 515 sequence with an N-terminal methionine residue (i,e., SEQ ID NO: 197, with the addition of an N-terminal methionine).

Table 48. Amino add sequences of domains of engineered CasX proteins, N- to C- terminus

*Positions of mutations are shown relative to a CasX 515 sequence with an N-terminal methionine residue (i.e., SEQ ID NO: 197, with the addition of an N-terminal methionine). [0467] A subset of these 161 engineered CasX proteins were cloned using methods standard in the art, and are listed in Tables 50, 52, and 53, below; In addition, an engineered CasX protein termed CasX 1001 was generated by combining mutations from engineered CasX protein 812. and CasX variant 676 (27. -.R, 169. L.K, and 329.G.K mutations relative to CasX 515), which have been previously validated as a highly specific and highly active CasX proteins, respectively (the PAM-altering 224. G.S mutation also present in CasX 676 was not included). Engineered CasX protein 969 was generated by combining 27. - R, 171.A.D, and 224.G.T mutations relative to CasX 515. Finally, engineered CasX protein 973 was generated by combining 35.R.P, 171. A.Y, and 304. M.T mutations relative to CasX 515. The amino acid sequences of engineered CasX proteins 969, 973, and 1001 are provided in Table 49, below.

Table 49. Amino acid sequences of engineered CasX proteins 969, 973, and 1003

[0463] A multiplexed pooled PASS assay was performed and analyzed as described in Example 15. As noted in Example 15, CasX proteins were expressed using a relatively weakly-expressing promoter to reduce CasX protein expression and thereby improve the sensitivity of the assay. Samples were tested in duplicate, except for engineered CasX protein 1006, which was tested in quadruplicate. In Tables 50, 52, and 53, below, the results for the CasX 1006 samples are reported in two separate rows, each the average of two samples. Streptococcus pyogenes Cas9 without a guide RNA served as a negative control. CasX 515, CasX 676, and engineered CasX protein 812 were also included as controls.

Results:

[0469] Table 50 provides the level of on-target editing produced by various CasX proteins with mutations relative to CasX 515, ranked from highest to lowest activity. Table 50. Average on-targeting editing activity of engineered CasX proteins, ranked from highest to lowest

*Positions of mutations are shown relative to a CasX 515 sequence with an N-terminal methionine residue (i.e., SEQ ID NO: 197, with the addition of an N-terminal methionine). [0470] As shown in Table 50, 41 of the tested engineered CasX proteins produced higher levels of on-target editing than did CasX 515; the 41 CasX proteins are bolded in Table 50, Engineered CasX protein 1018 had 9.K.G and 891. S.Q amino acid substitutions and produced the highest level of on-target editing in the assay. The CasX 676 control was more active than CasX 515, and CasX 812 was less active than CasX 515, which is consistent with previous results.

[0471 ] A large number of the tested CasX proteins produced lower levels of on-target editing than CasX 515. This suggests that not all combinations of mutations, including combinations of mutations that were relatively active for on-target editing when introduced into CasX 515 as single mutations (see Example 15), are compatible for producing highly active CasX proteins.

[0472] To understand the amino acid residues that may be causal for improving CasX activity, the identity' of the mutations in the engineered CasX proteins with two or three mutations resulting in improved on-target editing activity relative to CasX 515 was examined (Table 51).

Table 51. Summary of mutations in engineered CasX proteins with greater on-target editing activity than CasX 515

*ExcIuding CasX 676,

[0473] As shown in Table 51, certain positions were mutated in several members of the set of engineered CasX proteins with higher on-target editing activity than CasX 515. For example, the serine to glutamine substitution at position 891 (891. S.Q), in the TSL domain, was found in 13 members of the engineered CasX proteins with improved on-target editing activity relative to CasX 515. The TSL domain is a dynamic domain involved in coordinating the introduction of the target strand to the RuvC active site, and the substitution of serine for the longer glutamine may allow for additional hydrogen bonding interactions with the target strand and more efficient transfer to the nuclease domain.

[0474] One of two substitutions at position 169 (169. L.K or 169. L.Q), in the NTSB domain, were found in 12 members of the engineered CasX proteins with higher on-target editing activity than CasX 515. This position is proximal to the second and third nucleotides of the unwound non-target strand in structures of the non-target strand loading state, and the introduction of either a charged residue or one capable of multiple hydrogen-bonding interactions likely allows for the stabilization of the unwound state and thus more efficient unwinding. It should be noted that 169. L.K was more enriched than 169.L.Q among the engineered CasX proteins with improved on-target editing activity, which suggests that while a polar interaction increases enzymatic activity, a charge-charge interaction is more suitable for this position.

[0475] One of three substitutions at position 171 (171. A.S, 171. A.D, or 171. A. Y), also in the NTSB domain, were found in 11 members of the engineered CasX proteins with improved on-target editing activity. Residue 171 is solvent-exposed, so a polar residue is likely more favorable at this position. While the residue is not in a position that interacts with the nontarget strand in published structures, the dynamic nature of the NTSB domain may allow these residues to make hydrogen- bonding interactions with the target DNA at some point in the unwinding process. A serine is present at this position in the wild-type CasX 2 (SEQ ID NO: 2) sequence and is an alanine in CasX variants containing the chimeric NTSB from CasXl, meaning that the 171.A.S mutation in particular represents a reversion to a wild-type sequence. Notably, 171. A. Y was also found in several of the variants performing worse than CasX 515, which suggests that a tyrosine at position 171 might create too much steric hindrance for proper hydrogen-bonding interactions with the target DNA.

[0476] While the 169.L.K and 27. -.R mutations found in CasX 676 were well-represented among the high activity variants, there were a number of orthogonal mutations with distinct mechanisms that may allow for increased activity without the loss of specificity seen in CasX 676. 891. S.Q in particular was found in a number of top-performing activity variants that also have a higher specificity ratio than CasX 515 (see below).

[0477] Table 52, below, provides the level of off-target editing produced by various CasX proteins with two or three mutations relative to CasX 515, ranked from lowest to highest activity.

Table 52. Average off-targeting editing activity of engineered CasX proteins, ranked from lowest to highest

104781 As shown in Table 52, the majority of the tested CasX proteins with pairs of mutations relative to CasX 515 produced lower levels of off-target editing than did CasX

515; these samples are bolded in Table 52. [0479] Table 53. below, provides the specificity ratio (i.e., the average level of on-targeting editing divided by the average level of off-target editing) of the tested CasX proteins with two or three mutations relative to CasX 515, ranked from the highest to lowest ratio. CasX proteins with higher specificity ratios than CasX 515 are bolded in Table 53.

Table 53. Specificity ratios of engineered CasX proteins, ranked from highest to lowest*

[0480] As shown in Table 53, the majority' of the tested engineered CasX proteins had higher on-target to off-target editing ratios than CasX 515. While the previously validated high- specificity variant CasX 812 had the highest specificity ratio, many engineered CasX proteins demonstrated high specificity ratios without as significant a loss in on-target activity as was observed for CasX 812.

[0481] The 35.R.P mutation was commonly observed in variants with very high specificity ratios. This residue is in the OBD and believed to be involved in binding the guide RNA. Mutation to a proline at this position may have complex effects on allosteric regulation. Notably, these variants also tended to have low' activity', suggesting that apparent specificity' may be in part the result of less efficient RNP formation due to the disruption of this guidebinding interaction. Overall, an inverse correlation was observed betw-een specificity ratio and activity. This suggests that it is difficult to fully avoid trade-offs between activity and specificity. However, it is also evident that the strategy of combining activity and specificity mutants can compensate for this trade-off and result in variants with both characteristics improved.

[0482] Notably, some engineered CasX variants produced both higher levels of on-target editing and lower levels of off-target editing than did CasX 515, namely engineered CasX proteins 977, 978, 980, 982, 983, 985, 989, 992, 993, 994, 1001, 1005, 1009, 1016, 1018, 1026, 1028, 1029, 1031, 1040, and 1041. An even greater number had higher on-target activity and a higher specificity ratio, specifically, engineered CasX proteins 977, 978, 980, 982, 983, 985, 989, 992, 993, 994, 996, 999, 1000, 1001, 1005, 1006, 1009, 1014, 1016, 1018, 1026, 1028, 1029, 1031, 1040, and 1041 . Such engineered CasX proteins are therefore interpreted to be highly active and highly specific.

[0483] Taken together, the results described herein demonstrate that mutations to CasX 515 can be introduced into tire sequence that result in engineered CasX with improved gene editing activity and/or specificity.

285526993

Claims

CLAIMS What is claimed is:

1. A particle delivery system (PDS) comprising components selected from:

(a) a fusion protein comprising one or more Mononegavirales structural proteins and at least one heterologous protein;

(b) one or more therapeutic payloads; and

(c) a tropism factor.

2. The PDS of claim 1, wherein the Mononegavirales structural protein is a matrix protein (MA), a nucleocapsid protein (NC), or is both MA and NC.

3. The PDS of claim 1 or claim 2, wherein the at least one heterologous protein is selected from the group consisting of one or more non-covalent recruitment (NCR) proteins and a therapeutic protein.

4. The PDS of any one of claims 1-3, wherein the at least one heterologous protein is linked to the Mononegavirales structural protein with a linker selected from the group consisting of SEQ ID NOS: 1253-1308.

5. Tire PDS of claim 3 or claim 4, wherein the one or more non-covalent recruitment (NCR) proteins are selected from the group consisting of an MS2 coat protein, a PP7 coat protein, a QP coat protein, a U1A signal recognition particle, a protein N, a protein Tat, a phage GA coat protein, an iron-responsive binding element (IRE) protein, and an HIV Rev protein.

6. The PDS of any one of claims 1-5, wherein the one or more therapeutic payloads comprise a protein, a nucleic acid, or comprise both a protein and a nucleic acid.

7. The PDS of claim 6, wherein the protein is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, ery thropoietin, ribonuclease (RNAse), deoxyribonuclease (DNAse), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, a transcription factor, granulocyte-macrophage colony-stimulating factor (GMCSF), transposon, reverse transcriptase, viral interferon antagonists, a tick protein, and an anti-cancer modality.

8. The PDS of claim 7, wherein the CRISPR protein is a Class 2 CRISPR protein.

9. The PDS of claim 8, wherein the Class 2 CRISPR protein is selected from the group consisting of a Type II, a Type V, and a Type VI protein,

10. The PDS of claim 9, wherein the Type V protein is selected from the group consisting of Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), C as 12d (CasY), Casl2e (CasX), Casl2f, Casl 2g, Casl2h, Casl 2i, Casl2j, Casl2k, Casl 4, and CasCD.

11. 'The PDS of claim 10, wherein the CasX comprises a sequence selected from the group consisting of SEQ ID NOS: 136-176, and 180-506, 1905, 7731-7891, and 7978-7980 or a sequence having at least about 70%, at ieast 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% sequence identity thereto.

12. The PDS of claim 10, wherein the CasX comprises a chimeric CasX comprising domains derived from two different CasX proteins.

13. The PDS of claim 10, wherein the CasX comprises a sequence selected from the group consisting of SEQ ID NOS: 190, 197, 273, 348, 351, 355 and 478.

14. The PDS of claim 10, wherein the CasX comprising a sequence selected from the group consisting of SEQ ID NOS: 7731-7891 and 7978-7980, 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.

15. The PDS of claim 14, wherein the CasX comprises two or more modifications relative to a CasX protein of SEQ ID NO: 197 (CasX variant 515), and wherein the two or more modifications act to increase editing activity, editing specificity, specificity ratio, editing activity and editing specificity, or editing activity and specificity ratio of the engineered CasX protein.

16. Die PDS of claim 10, wherein the CasX is catalytically-dead.

17. 'The PDS of claim 16, wherein the catalytically-dead CasX (dCasX) comprises a sequence selected from the group consisting of SEQ ID NOS: 7716 and 7937-7959, or a sequence having at least about 70%, at least about 80%, at least about 85%, at ieast about 90%, at least about 91%, at least about 92%, at ieast 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.

18. The PDS of any one of claims 11 -17, wherein the CasX or dCasX is a fusion protein that comprises one or more heterologous proteins.

19. The PDS of claim 18, wherein the one or more heterologous proteins comprises an NLS selected from the group of sequences consisting of SEQ ID NOS: 507-600 and SEQ ID NOS: 7589-7639, wherein the NLS are located at or near the N-terminus and/or the C- terminus of the CasX and, optionally, the one or more NLS are linked to the CasX or to adjacent NLS with a linker peptide.

20. The PDS of claim 19, wherein the CasX comprises a nuclear export sequence (NES) linked to the C -terminus of the NLS linked to the C-terminus of the CasX.

21. The PDS of claim 19 or claim 20, wherein the NES is linked to the NLS by a sequence cleavable by a protease.

22. The PDS of claim 18, wherein the fusion protein comprises a dCasX and one or more repressor domains selected from the group consisting of a Kruppel associated box (KRAB) domain, a DNMT3A catalytic domain, a. DNMT3L interaction domain, and aDNMT3A ADD domain.

23. The PDS of claim 22, wherein the fusion protein comprises, from N-terminus to C- terminus, DNMT3A ADD domain-DNMT3A catalytic domain-linker-DNMT3L interaction domain-linker- KRAB-linker-dCasX (dXR).

24. The PDS of any one of claims 6-23, wherein the therapeutic payload compri ses a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASO), a double-stranded RNA interference (RNAi) molecule, aDNA aptamer, an RNA aptamer, a CRISPR guide ribonucleic acid (gRNA), a donor template, or any combination thereof.

25. The PDS of claim 24, wherein the nucleic acid is a single molecule gRNA comprising a. scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 15 and 20 nucleotides and is complementary' to a target nucleic acid sequence of a cell.

26. The PDS of claim 25, wherein the gRNA scaffold sequence comprises a sequence selected from the group consisting SEQ ID NOS: 2101-2258, 2260-2431, or a sequence having 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% sequence identity' thereto.

27. The PDS of claim 25, wherein the gRN A scaffold sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 2238, 2275, 2281, 2293, and 2309.

28. The PDS of any one of claims 25, wherein the scaffold of the gRNA variant further comprises one or more RNA binding partner elements selected from the group consisting of: i) a stem IIB of Rev response element (RRE), ii) a stem II- V of RRE; iii) a stem II of RRE; iv) a Rev-binding element (RBE) of Stem IIB; and v) a full-length RRE, wherein the one or more components are capable of binding Rev.

29. The PDS of any one of claims 25-28, wherein the scaffold of the gRNA variant comprises one or more non-covalent recruitment components selected from the group consisting of: i) an MS2 hairpin; it) a PP7 hairpin; iii) a QP hairpin; iv) a boxB; v) a phage GA hairpin; vi) a phage .AN hairpin; vii ) an iron responsive element (IRE); viii) a transactivation response element (TAR); and ix) a U1 A hairpin II, wherein the non-covalent recruitment components have binding affinity to NCR selected from the group consisting of MS2 coat protein, PP7 coat protein, QP coat protein, U1A signal recognition particle, protein N, protein Tat, phage GA coat protein, and ironresponsive binding element (IRE) protein, facilitating the non-covalent recruitment of the therapeutic protein into the PDS.

30. The PDS of any one of claims 2-29, wherein the MA protein comprises a sequence selected from the group consisting of SEQ ID NOS: 1039-1252, or a sequence having 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% sequence identity thereto.

31. The PDS of any one of claims 2-30, wherein the NC protein comprises a sequence selected from the group consisting of SEQ ID NOS: 1597-1810, or a sequence having 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% sequence identity thereto.

32. The PDS of any one of claims 1-31, wherein the tropism factor is selected from the group consisting of a glycoprotein, an antibody fragment, a receptor, and a ligand to a target cell marker.

33. Tire PDS of claim 32, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 601 -824, or a sequence having 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% sequence identity thereto.

34. A nucleic acid encoding the fusion protein of any one of claims 1 -33.

35. The nucleic acid of claim 34, wherein the encoded components are configured, 5' to 3’:

(a) MA-NCR;

(h) MA-NC-NCR;

(c) MA-CasX;

(d) MA-dXR;

(e) MA-NCR-CasX; or

(f) MA-NCR-dXR,

36. A nucleic acid encoding the therapeutic payload of any one of claims 1-33.

37. A nucleic acid encoding the tropism factor of any one of claims 1 -33.

38. A plasmid comprising the nucleic acid of any one of claims 34-37, wherein the nucleic acid is operably linked to a promoter.

39. A eukaryotic cell comprising the plasmid(s) of claim 38.

40. The eukaryotic cell of claim 39, wherem the components of the PDS are encoded on three or four plasmids.

41. The eukaryotic cell of claim 39 or 40, wherein the encoded components are capable of self-assembling into a PDS when the plasmids are introduced into the eukary otic cell and the components are expressed.

42. A PDS particle produced by the eukaryotic cell of any one of claims 39-41.

43. The PDS particle of claim 42, having a diameter of less than about 50, about 60, about 70, about 80, about 90 nm, or less than about 100 nm.

44. The PDS particle of claim 42 or claim 43, wherein the therapeutic payload is encapsidated within the PDS particle upon self-assembly of the PDS particle in the eukary otic ceil.

45. A method of making a PDS particle comprising a therapeutic payload, the method comprising propagating the eukaryotic cell of any one of claims 39-41 under conditions such that the components are expressed and self-assemble into PDS particles that release from the eukaryotic cell.

46. The method of claim 45, wherein the therapeutic payload is a ribonucleoprotein (RNP) of the CasX variant or a dXR and the gRNA.

47. The method of claim 46, wherein the incorporation of the binding partner element(s) and the one or more NCR results in incorporation of increased numbers of RNP into the PDS particle during self-assembly compared to a PDS particle not comprising the one or more binding partner elements and the one or more NCR.

48. The method of claim 47, wherein the particle contains at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 molecules of the therapeutic payload.

49. A method of modifying a target nucleic acid sequence of a gene in a population of cells, the method comprising contacting the cells with a plurality of the PDS particles of any one of claims 42-44, wherein said contacting comprises introducing the into the cell the RNP, wherein the target nucleic acid targeted by the guide RNA is modified by the CRISPR protein.

50. A packaging cell, comprising:

(a) a first plasmid encoding the fusion protein of claim 34 or claim 35;

(b) a second plasmid encoding a CasX variant comprising a sequence selected from the group consisting of SEQ ID NOS: 190, 197, 273, 348, 351, 355, and 478;

(c) a third plasmid encoding a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 2238, 2275, 2281, 2293, and 2309;

(d) a fourth plasmid encodes the tropism factor of claim 32.