EP4069716A1 - Particle delivery systems - Google Patents

Abstract

Provided herein are delivery particle systems (XDP) useful for the delivery of payloads of any type. In some embodiments, a XDP particle system with tropism for target cells of interest is used to deliver CRISPR/Cas polypeptides (e.g. CasX proteins) and guide nucleic acids (gNA), for the modification of nucleic acids in target cells. Also provided are methods of making and using such XDP to modify the nucleic acids in such cells.

Description

PARTICLE DELIVERY SYSTEMS

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. provisional patent application numbers 62/944,982, filed on December 6, 2019, 62/968,915, filed on January 31, 2020, 62/983,460, filed on February 28, 2020, 63/035,576, filed on June 5, 2020 and 63/120,864, filed on December 3, 2020, the contents of each of which are incorporated herein by reference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] This application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 4, 2020 is named SCRB_024_05WO_SeqList_ST25.txt and is 3.14 MB in size.

BACKGROUND

[0003] 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. Even with highly selective targeting modalities, such as monoclonal antibodies, the selectivity of the system for the target cells or organs is not absolute, and off- target toxicity can be a consequence.

[0004] The Retroviridae family of viruses encompass several genera of viruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The Retroviridae family includes Othoretrovirinae (Lentivirus, Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus), and Spumaretrovirinae . The best known lentivirus is the Human Immunodeficiency Virus (HIV), which causes acquired immune deficiency syndrome (AIDS). As with all retroviruses, lentiviruses have gag, pol and env genes, coding for viral proteins in the order: 5'-gag-pol-env- 3'. The lentivirus system has been adapted to introduce gene editing systems into human or animal cells by the creation of virus-like particles (VLP) containing the gene editing systems. Retroviral systems have advantages over other gene-therapy methods, including high-efficiency infection of dividing and non-dividing cells, long-term stable expression of a transgene, and low immunogenicity. Lentiviruses have been successfully used for transduction of diabetic mice with the gene encoding PDGF (platelet-derived growth factor), a therapy being considered for use in humans (Lee JA, et al. Lentiviral transfection with the PDGF-B gene improves diabetic wound healing. Plast. Reconstr. Surg. 116 (2): 532 (2005)). However, one major difficulty with use of certain therapeutics, like CRISPR nucleases, in VLP is off-target effects, particularly with long term expression of the nuclease when traditional expression methods such as via plasmid/viral vectors are used. Accordingly, there remains a need for improved systems for delivery of gene editing systems using particles derived from viral vectors.

SUMMARY

[0005] The present disclosure provides delivery particle (XDP) systems for the delivery of therapeutic payloads, including proteins, nucleic acids, small molecules and the like to target cells and tissues.

[0006] In some embodiments, the XDP system comprises nucleic acids encoding components selected from all or a portion of a retroviral gag polyprotein, a therapeutic payload, and a tropism factor, 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. In one embodiment of the foregoing, the tropism factor is a glycoprotein having a sequence selected from the group of sequences consisting of SEQ ID NOS: 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496,

498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534,

536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572,

574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594 and 596 as set forth in Table 4, 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 a particular embodiment, the glycoprotein is VSV-G. In a particular embodiment, the glycoprotein comprises a sequence of SEQ ID NO: 438.

[0007] The therapeutic payload can be a protein, a nucleic acid, or both a protein and a nucleic acid. In some embodiments of the XDP system, 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, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality. In one embodiment, the therapeutic payload is a Class 1 or Class 2 CRISPR protein, wherein the Class 2 CRISPR protein selected from the group consisting of a Type II, Type V, or Type VI protein. In one embodiment, the Class 2 CRISPR Type V protein is selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX, wherein the CasX comprises a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397 as set forth in Tables 1, 7, 8, 9, or 11, 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 some embodiments, the CasX comprises a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388- 397. In some embodiments, the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid, wherein the CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 14 and 30 nucleotides and is complementary to a target nucleic acid sequence, and wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781 as set forth in Table 3, 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 some embodiments, the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781.

[0008] In some embodiments, the XDP system further comprises nucleic acids encoding one or more components selected from one or more protease cleavage sites, a gag-transframe region- pol protease polyprotein (gag-TFR-PR), a retroviral gag-pol polyprotein, and a non-retroviral protease capable of cleaving the protease cleavage sites. In some embodiments, the retroviral components of the XDP system are derived from a Orthoretrovirinae virus or a Spumaretrovirinae virus wherein the Orthoretrovirinae virus is selected from the group consisting of Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus, and the Spumaretrovirinae virus is selected from the group consisting of Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, and Spumavirus.

[0009] In some embodiments, the components of the XDP system are encoded on a single nucleic acid, on two nucleic acids, on three nucleic acids, on four nucleic acids, or on five nucleic acids, and the nucleic acids are configured according to any one of FIGS. 36-68. In some embodiments, the components of the XDP system are encoded by nucleic acids selected from the group of sequences of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, and 234-339 as set forth in Table 5.

[0010] In some embodiments, the components of the XDP system are capable of self assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed. In the foregoing embodiment, the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP. In a particular embodiment, wherein the therapeutic payload comprises a CasX and a guide RNA, the CasX and guide RNA are complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template is also encapsidated in the XDP. In another particular embodiment, the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

[0011] In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of an Alpharetrovirus gag polyprotein, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a P2A peptide, a P2B peptide, a P10 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC). In some embodiments of the XDP system, the nucleic acids further comprise sequences encoding one or more components selected from an HIV pi peptide, an HIV p6 peptide, a Gag-Pol polyprotein, one or more protease cleavage sites, a non-retroviral, heterologous protease capable of cleaving the cleavage sites, and a gag-transframe region-pol protease polyprotein.

[0012] In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of an Betaretrovirus gag polyprotein, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a PP21/24 peptide, a P12/P3/P8 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC). In some embodiments of the XDP system, the nucleic acids further comprise sequences encoding one or more components selected from an HIV pi peptide, an HIV p6 peptide, a Gag-Pol polyprotein, one or more protease cleavage sites, a non-retroviral, heterologous protease capable of cleaving the cleavage sites, and a gag-transframe region-pol protease polyprotein.

[0013] In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of a Deltaretrovirus gag polyprotein, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC). In some embodiments of the XDP system, the nucleic acids further comprise sequences encoding one or more components selected from an HIV pi peptide, an HIV p6 peptide, a Gag-Pol polyprotein, one or more protease cleavage sites, a non-retroviral, heterologous protease capable of cleaving the cleavage sites, and a gag-transframe region-pol protease polyprotein.

[0014] In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of a Epsilonretrovirus gag polyprotein, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a p20 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC). In some embodiments of the XDP system, the nucleic acids further comprise sequences encoding one or more components selected from an HIV pi peptide, an HIV p6 peptide, a Gag- Pol polyprotein, one or more protease cleavage sites, a non-retroviral, heterologous protease capable of cleaving the cleavage sites, and a gag-transframe region-pol protease polyprotein. [0015] In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of a Gammanretrovirus gag polyprotein, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a pl2 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC). In some embodiments of the XDP system, the nucleic acids further comprise sequences encoding one or more components selected from an HIV pi peptide, an HIV p6 peptide, a Gag- Pol polyprotein, one or more protease cleavage sites, a non-retroviral, heterologous protease capable of cleaving the cleavage sites, and a gag-transframe region-pol protease polyprotein. [0016] In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of a Lentivirus gag polyprotein, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a capsid polypeptide (CA), a p2 peptide, a nucleocapsid polypeptide (NC), a pi peptide, and a p6 peptide. In some embodiments of the XDP system, the nucleic acids further comprise sequences encoding one or more components selected from a Gag-Pol polyprotein, one or more protease cleavage sites, a non-retroviral, heterologous protease capable of cleaving the cleavage sites, and a gag-transframe region-pol protease polyprotein.

[0017] In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of a Spumaretrovirinae gag polyprotein, wherein the gag polyprotein comprises one or more components selected from the group consisting of a p68 Gag polypeptide and a p3 Gag polypeptide. In some embodiments of the XDP system, the nucleic acids further comprise sequences encoding one or more components selected from an HIV pi peptide, an HIV p6 peptide, a Gag-Pol polyprotein, one or more protease cleavage sites, a non- retroviral, heterologous protease capable of cleaving the cleavage sites, and a gag-transframe region-pol protease polyprotein.

[0018] In some embodiments of the CasX system, the CasX further comprises one or more NLS selected from the group of sequences consisting of PKKKRKV (SEQ ID NO: 130), KRPAATKKAGQAKKKK (SEQ ID NO: 131), PAAKRVKLD (SEQ ID NO: 132), RQRRNELKRSP (SEQ ID NO: 133),

NQ S SNF GPMKGGNF GGRS S GP Y GGGGQ YF AKPRNQGGY (SEQ ID NO: 134), RMRIZFKNKGKDTAELRRRRVEV S VELRKAKKDEQILKRRNV (SEQ ID NO: 135), VSRKRPRP (SEQ ID NO: 136), PPKKARED (SEQ ID NO: 137), PQPKKKPL (SEQ ID NO: 138), SALIKKKKKMAP (SEQ ID NO: 139), DRLRR (SEQ ID NO: 140), PKQKKRK (SEQ ID NO: 141), RKLKKKIKKL (SEQ ID NO: 142), REKKKFLKRR (SEQ ID NO: 143), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 144), RKCLQAGMNLEARKTKK (SEQ ID NO: 145), PRPRKIPR (SEQ ID NO: 146), PPRKKRTVV (SEQ ID NO: 147),

NL SKKKKRKREK (SEQ ID NO: 148), RRPSRPFRKP (SEQ ID NO: 149), KRPRSPSS (SEQ ID NO: 150), KRGINDRNFWRGENERKTR (SEQ ID NO: 151), PRPPKMARYDN (SEQ ID NO: 152), KRSFSKAF (SEQ ID NO: 153), KLKIKRPVK (SEQ ID NO: 154), PKTRRRPRRSQRKRPPT (SEQ ID NO: 156), RRKKRRPRRKKRR (SEQ ID NO: 159), PKKK SRKPKKK SRK (SEQ ID NO: 160), HKKKHPD AS VNF SEF SK (SEQ ID NO: 161), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 162), LSPSLSPLLSPSLSPL (SEQ ID NO: 163), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 164), PKRGRGRPKRGRGR (SEQ ID NO: 165), M SRRRK ANPTKL SENAKKL AKEVEN (SEQ ID NO: 157), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 155), and PKKKRKVPPPPKKKRKV (SEQ ID NO: 166), wherein the NLS are located at or near the N-terminus and/or the C-terminus.

[0019] In some embodiments of the XDP system, the non-retroviral, heterologous protease is selected from the group consisting of tobacco etch virus protease (TEV), potyvirus HC protease, potyvirus PI protease, PreScission (HRV3C protease), b virus NIa protease, B virus RNA-2- encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical vims) 3C-like protease, parsnip yellow fleck vims protease, 3C-like protease, heparin, cathepsin, thrombin, factor Xa, metalloproteinase, and enterokinase.

[0020] In other aspects, the present disclosure provides eukaryotic cells comprising the XDP system of any one of the foregoing embodiments, wherein the cell is a packaging cell. In some embodiments, the eukaryotic cell is selected from the group consisting of HEK293 cells, Lenti-X 293T cells, BHK cells, HepG2, Saos-2, HuH7, NSO cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO, NIH3T3 cells, COS, WI38, MRC5, A549, HeLa cells, CHO cells, and HT1080 cells.

[0021] In other aspects, the present disclosure provides methods of making an XDP comprising a therapeutic payload, the method comprising propagating the packaging cell of any of the embodiments under conditions such that XDPs are produced, and harvesting the XDPs produced by the packaging cell. The present disclosure further provides an XDP produced by the foregoing methods. In a particular embodiment, the XDP comprises a therapeutic payload of an RNP of a CasX and guide RNA and, optionally, a donor template of any of the embodiments disclosed herein.

[0022] In other aspects, the present disclosure provides methods of modifying a target nucleic acid sequence in a cell, the methods comprising contacting the cell with the XDP comprising an RNP of any of the embodiments disclosed herein, wherein said contacting comprises introducing into the cell the RNP comprising the CasX protein, the guide RNA, and, optionally, the donor template nucleic acid sequence, resulting in modification of the target nucleic acid sequence. In some cases, the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence. In other cases, the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence. In still other cases, the modification comprises insertion of the donor template into 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 XDP is 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. In some embodiments, the XDP is 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 routes and sub-retinal routes. In some embodiments, the therapeutically effective dose is at least about 1 x 10⁵ particles/kg, or at least about 1 x 10⁶ particles/kg, or at least about 1 x 10⁷ particles/kg, or at least about 1 x 10⁸ particles/kg, or at least about 1 x 10⁹ particles/kg, or at least about 1 x 10¹⁰ particles/kg, or at least about 1 x 10¹¹ particles/kg, or at least about 1 x 10¹² particles/kg, or at least about 1 x 10¹³ particles/kg, or at least about 1 x 10¹⁴ particles/kg, or at least about 1 x 10¹⁵ particles/kg, or at least about 1 x 10¹⁶ particles/kg. In some embodiments, the XDP is administered to the subject according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose of the XDP. In some embodiments, 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.

[0023] In another aspect, provided herein are XDP particles, and XDP systems, for use as a medicament for the treatment of a subject having a disease.

INCORPORATION BY REFERENCE

[0024] 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 PCT/US2020/036505, filed on June 5, 2020, and a U.S. provisional application entitled “Engineered CasX Systems”, filed on December 3, 2020, both applications which disclose CasX variants and gNA variants, are hereby incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS [0025] 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:

[0026] FIG. 1 shows an SDS-PAGE gel of StX2 purification fractions visualized by colloidal Coomassie staining, as described in Example 1. [0027] FIG. 2 shows the chromatogram from a size exclusion chromatography assay of the StX2, using of Superdex 200 16/600 pg Gel Filtration, as described in Example 1.

[0028] FIG. 3 shows an SDS-PAGE gel of StX2 purification fractions visualized by colloidal Coomassie staining, as described in Example 1.

[0029] FIG. 4 is a schematic showing the organization of the components in the pSTX34 plasmid used to assemble the CasX constructs, as described in Example 2.

[0030] FIG. 5 is a schematic showing the steps of generating the CasX 119 variant, as described in Example 2.

[0031] FIG. 6 shows an SDS-PAGE gel of purification samples, visualized on a Bio-Rad Stain-Free™ gel, as described in Example 2.

[0032] FIG. 7 shows the chromatogram of Superdex 200 16/600 pg Gel Filtration, as described in Example 2.

[0033] FIG. 8 shows an SDS-PAGE gel of gel filtration samples, stained with colloidal Coomassie, as described in Example 2.

[0034] FIG. 9 shows an SDS-PAGE gel of purification samples of CasX 438, visualized on a Bio-Rad Stain-Free™ gel, as described in Example 2.

[0035] FIG. 10 shows the chromatogram from a size exclusion chromatography assay of the CasX 438, using of Superdex 200 16/600 pg gel filtration, as described in Example 2.

[0036] FIG. 11 shows an SDS-PAGE gel of CasX 438 purification fractions visualized by colloidal Coomassie staining, as described in Example, as described in Example 2.

[0037] FIG. 12 shows an SDS-PAGE gel of purification samples of CasX 457, visualized on a Bio-Rad Stain-Free™ gel, as described in Example 2.

[0038] FIG. 13 shows the chromatogram from a size exclusion chromatography assay of the CasX 457, using of Superdex 200 16/600 pg gel filtration, as described in Example 2.

[0039] FIG. 14 shows an SDS-PAGE gel of CasX 457 purification fractions visualized by colloidal Coomassie staining, as described in Example 2.

[0040] FIG. 15 is a graph of the results of an assay for the quantification of active fractions of RNP formed by sgRNA174 and the CasX variants, as described in Example 9. Equimolar amounts of RNP and target were co-incubated and the amount of cleaved target was determined at the indicated timepoints. Mean and standard deviation of three independent replicates are shown for each timepoint. The biphasic fit of the combined replicates is shown. "2" refers to the reference CasX protein of SEQ ID NO:2. [0041] FIG. 16 shows the quantification of active fractions of RNP formed by CasX2 (reference CasX protein of SEQ ID NO:2) and the modified sgRNAs, as described in Example 9. Equimolar amounts of RNP and target were co-incubated and the amount of cleaved target was determined at the indicated timepoints. Mean and standard deviation of three independent replicates are shown for each timepoint. The biphasic fit of the combined replicates is shown. [0042] FIG. 17 shows the quantification of active fractions of RNP formed by CasX 491 and the modified sgRNAs under guide-limiting conditions, as described in Example 9. Equimolar amounts of RNP and target were co-incubated and the amount of cleaved target was determined at the indicated timepoints. The biphasic fit of the data is shown.

[0043] FIG. 18 shows the quantification of cleavage rates of RNP formed by sgRNA174 and the CasX variants, as described in Example 9. Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. Mean and standard deviation of three independent replicates are shown for each timepoint, except for 488 and 491 where a single replicate is shown. The monophasic fit of the combined replicates is shown.

[0044] FIG. 19 shows the quantification of cleavage rates of RNP formed by CasX2 and the sgRNA variants, as described in Example 9. Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. Mean and standard deviation of three independent replicates are shown for each timepoint. The monophasic fit of the combined replicates is shown.

[0045] FIG. 20 shows the quantification of initial velocities of RNP formed by CasX2 and the sgRNA variants, as described in Example 9. The first two time-points of the previous cleavage experiment were fit with a linear model to determine the initial cleavage velocity.

[0046] FIG. 21 shows the quantification of cleavage rates of RNP formed by CasX491 and the sgRNA variants, as described in Example 9. Target DNA was incubated with a 20-fold excess of the indicated RNP at 10°C and the amount of cleaved target was determined at the indicated time points. The monophasic fit of the timepoints is shown.

[0047] FIGS. 22A-D shows the quantification of cleavage rates of CasX variants on NTC PAMs, as described in Example 10. Target DNA substrates with identical spacers and the indicated PAM sequence were incubated with a 20-fold excess of the indicated RNP at 37°C and the amount of cleaved target was determined at the indicated time points. Monophasic fit of a single replicate is shown. FIG. 22A shows the results for sequences having a TTC PAM. FIG. 22B shows the results for sequences having a CTC PAM. FIG. 22C shows the results for sequences having a GTC PAM. FIG. 22D shows the results for sequences having a ATC PAM. [0048] FIG. 23 depicts the plasmids utilized in the creation of XDP comprising CasX, gNA, and pseudotyping proteins, as described in Example 13.

[0049] FIG. 24 is a schematic of the steps using in the creation of XDP, as described in Example 13.

[0050] FIG. 25 is a graph of the results of the editing of the dtTomato assay, as described in Example 16.

[0051] FIG. 26A shows the results of percentage editing in mouse tdTomato neural progenitor cells (NPCs) with XDPs pseudotyped with serial concentrations of VSV-G, as described in Example 17.

[0052] FIG. 26B shows the XDP titers determined by a commercially available Lenti-X p24 ELISA kit, as described in Example 17.

[0053] FIG. 27 shows the percentage of editing in mouse tdTomato NPCs with XDPs pseudotyped with different glycoproteins, as described in Example 17.

[0054] FIG. 28A shows the results of size distributions and viral titer comparisons of VSV-G pseudotyped XDP (both IX and 10X concentrated), rabies pseudotyped XDP and lentivirus (LV), as described in Example 17.

[0055] FIG. 28B shows the size comparisons between VSV-G XDP, LV and Rabies XDP, as described in Example 17.

[0056] FIG. 29 shows the results of percentage editing in mouse tdTomato NPCs with VSV-G pseudotyped XDPs carrying different RNPs, as described in Example 18.

[0057] FIG. 30 shows the percentage editing in mouse tdTomato NPCs with VSV-G pseudotyped XDPs with titrated amounts of Gag-Pol vs Gag-Stx (Stx construct), as described in Example 19.

[0058] FIG. 31 shows the titers for these different XDPs with varying amounts of Gag-Pol vs Gag-Stx constructs, as described in Example 19.

[0059] FIG. 32 shows the amount of guide RNA per XDP titer for different constructs as assessed by QPCR, as described in Example 19.

[0060] FIG. 33 shows the results of the relative knockout rates of B2M by XDPs containing two different B2M targeting spacers and one non targeting spacer, as described in Example 20. [0061] FIG. 34 shows representative SDS-PAGE and Western blot images of samples taken from throughout the centrifugation purification process for XDP particles, as described in Example 14.

[0062] FIG. 35 shows the results of an editing assay for XDP configured as version 7, version 122 and version 123, as described in Example 21.

[0063] FIG. 36A shows the schematic for the configuration of the components for version 1 XDP and the four plasmids used in the transfection to create the XDP.

[0064] FIG. 36B shows the schematic for the configuration of the components for version 2 XDP and the four plasmids used in the transfection to create the XDP.

[0065] FIG. 37A shows the schematic for the configuration of the components for version 3 XDP and the four plasmids used in the transfection to create the XDP.

[0066] FIG. 37B shows the schematic for the configuration of the components for version 4 XDP and the three plasmids used in the transfection to create the XDP.

[0067] FIG. 38A shows the schematic for the configuration of the components for version 5 XDP and the three plasmids used in the transfection to create the XDP.

[0068] FIG. 38B shows the schematic for the configuration of the components for version 6 XDP and the four plasmids used in the transfection to create the XDP.

[0069] FIG. 39A shows the schematic for the configuration of the components for version 7 XDP and the three plasmids used in the transfection to create the XDP.

[0070] FIG. 39B shows the schematic for the configuration of the components for version 8 XDP and the four plasmids used in the transfection to create the XDP.

[0071] FIG. 40 A shows the schematic for the configuration of the components for version 9 XDP and the three plasmids used in the transfection to create the XDP.

[0072] FIG. 40B shows the schematic for the configuration of the components for version 10 XDP and the three plasmids used in the transfection to create the XDP.

[0073] FIG. 41 A shows the schematic for the configuration of the components for version 11 XDP and the three plasmids used in the transfection to create the XDP.

[0074] FIG. 41B shows the schematic for the configuration of the components for version 12 XDP and the three plasmids used in the transfection to create the XDP.

[0075] FIG. 42A shows the schematic for the configuration of the components for version 13 XDP and the three plasmids used in the transfection to create the XDP. [0076] FIG. 42B shows the schematic for the configuration of the components for version 14 XDP and the three plasmids used in the transfection to create the XDP.

[0077] FIG. 43 A shows the schematic for the configuration of the components for version 15 XDP and the three plasmids used in the transfection to create the XDP.

[0078] FIG. 43B shows the schematic for the configuration of the components for version 16 XDP and the three plasmids used in the transfection to create the XDP.

[0079] FIG. 44A shows the schematic for the configuration of the components for version 24 XDP and the four plasmids used in the transfection to create the XDP.

[0080] FIG. 44B shows the schematic for the configuration of the components for version 25 XDP and the four plasmids used in the transfection to create the XDP.

[0081] FIG. 45A shows the schematic for the configuration of the components for version 26 XDP and the four plasmids used in the transfection to create the XDP.

[0082] FIG. 45B shows the schematic for the configuration of the components for version 27 XDP and the four plasmids used in the transfection to create the XDP.

[0083] FIG. 46A shows the schematic for the configuration of the components for version 31 XDP and the four plasmids used in the transfection to create the XDP.

[0084] FIG. 46B shows the schematic for the configuration of the components for version 32 XDP and the four plasmids used in the transfection to create the XDP.

[0085] FIG. 47 A shows the schematic for the configuration of the components for version 33 XDP and the four plasmids used in the transfection to create the XDP.

[0086] FIG. 47B shows the schematic for the configuration of the components for version 34 XDP and the four plasmids used in the transfection to create the XDP.

[0087] FIG. 48 A shows the schematic for the configuration of the components for version 35 XDP and the four plasmids used in the transfection to create the XDP.

[0088] FIG. 48B shows the schematic for the configuration of the components for version 36 XDP and the four plasmids used in the transfection to create the XDP.

[0089] FIG. 49 A shows the schematic for the configuration of the components for version 37 XDP and the four plasmids used in the transfection to create the XDP.

[0090] FIG. 49B shows the schematic for the configuration of the components for version 38 XDP and the four plasmids used in the transfection to create the XDP.

[0091] FIG. 50A shows the schematic for the configuration of the components for version 39 XDP and the four plasmids used in the transfection to create the XDP. [0092] FIG. 50B shows the schematic for the configuration of the components for version 40 XDP and the four plasmids used in the transfection to create the XDP.

[0093] FIG. 51 A shows the schematic for the configuration of the components for version 17 XDP and the three plasmids used in the transfection to create the XDP.

[0094] FIG. 5 IB shows the schematic for the configuration of the components for version 18 XDP and the three plasmids used in the transfection to create the XDP.

[0095] FIG. 52A shows the schematic for the configuration of the components for versions 44 and 45 XDP and the three plasmids used in the transfection to create the XDP.

[0096] FIG. 52B shows the schematic for the configuration of the components for versions 46, 47, 62, and 90 XDP and the three plasmids used in the transfection to create the XDP.

[0097] FIG. 53 A shows the schematic for the configuration of the components for versions 48, 49, and 63 XDP and the three plasmids used in the transfection to create the XDP.

[0098] FIG. 53B shows the schematic for the configuration of the components for version 50 XDP and the three plasmids used in the transfection to create the XDP.

[0099] FIG. 54A shows the schematic for the configuration of the components for versions 51 and 52 XDP and the three plasmids used in the transfection to create the XDP.

[00100] FIG. 54B shows the schematic for the configuration of the components for versions 53, 54, 55 and 91 XDP and the three plasmids used in the transfection to create the XDP.

[00101] FIG. 55A shows the schematic for the configuration of the components for versions 56- 61 and 92 XDP and the three plasmids used in the transfection to create the XDP.

[00102] FIG. 55B shows the schematic for the configuration of the components for versions 66a and 67a XDP and the three plasmids used in the transfection to create the XDP.

[00103] FIG. 56A shows the schematic for the configuration of the components for versions 66b and 67b XDP and the four plasmids used in the transfection to create the XDP.

[00104] FIG. 56B shows the schematic for the configuration of the components for versions 68a, 69a, 70a and 87a XDP and the three plasmids used in the transfection to create the XDP. [00105] FIG. 57A shows the schematic for the configuration of the components for versions 68b, 69b, 70b and 87b XDP and the four plasmids used in the transfection to create the XDP. [00106] FIG. 57B shows the schematic for the configuration of the components for versions 71a, 72a and 88a XDP and the three plasmids used in the transfection to create the XDP.

[00107] FIG. 58A shows the schematic for the configuration of the components for versions 71b, 72b and 88b XDP and the four plasmids used in the transfection to create the XDP. [00108] FIG. 58B shows the schematic for the configuration of the components for versions 73a XDP and the three plasmids used in the transfection to create the XDP.

[00109] FIG. 59A shows the schematic for the configuration of the components for version 73b XDP and the four plasmids used in the transfection to create the XDP.

[00110] FIG. 59B shows the schematic for the configuration of the components for versions 74a and 75a XDP and the three plasmids used in the transfection to create the XDP.

[00111] FIG. 60A shows the schematic for the configuration of the components for versions 74b and 75b XDP and the four plasmids used in the transfection to create the XDP.

[00112] FIG. 60B shows the schematic for the configuration of the components for versions 76a, 77a, 78a, and 79a XDP and the three plasmids used in the transfection to create the XDP. [00113] FIG. 61 A shows the schematic for the configuration of the components for versions 76b, 77b, 78b, and 79b XDP and the four plasmids used in the transfection to create the XDP. [00114] FIG. 6 IB shows the schematic for the configuration of the components for versions 80a, 81a, 82a, 83a, 84a, 85a and 86a XDP and the three plasmids used in the transfection to create the XDP.

[00115] FIG. 62A shows the schematic for the configuration of the components for versions 80b, 81b, 82b, 83b, 84b, 85b, and 86b XDP and the four plasmids used in the transfection to create the XDP.

[00116] FIG. 62B shows the schematic for the configuration of the components for versions 102 and 114 XDP and the three plasmids used in the transfection to create the XDP.

[00117] FIG. 63 A shows the schematic for the configuration of the components for versions

103, 108, and 109 XDP and the three plasmids used in the transfection to create the XDP. [00118] FIG. 63B shows the schematic for the configuration of the components for versions

104, 105, 115, 116 and 117 XDP and the three plasmids used in the transfection to create the XDP.

[00119] FIG. 64A shows the schematic for the configuration of the components for versions 106, 111, 112, 83b and 113 XDP and the three plasmids used in the transfection to create the XDP.

[00120] FIG. 64B shows the schematic for the configuration of the components for versions 107 and 110 XDP and the three plasmids used in the transfection to create the XDP.

[00121] FIG. 65 shows the schematic for the configuration of the components for version 118 XDP and the three plasmids used in the transfection to create the XDP. [00122] FIG. 66A shows the schematic for the configuration of the components for version 122 XDP and the three plasmids used in the transfection to create the XDP.

[00123] FIG. 66B shows the schematic for the configuration of the components for version 103 XDP and the three plasmids used in the transfection to create the XDP.

[00124] FIG. 67A shows the schematic for the configuration of the components for version 124 XDP and the three plasmids used in the transfection to create the XDP.

[00125] FIG. 67B shows the schematic for the configuration of the components for version 126 XDP and the three plasmids used in the transfection to create the XDP.

[00126] FIG. 68 shows the schematic for the configuration of the components for versions 128 XDP and the three plasmids used in the transfection to create the XDP.

[00127] FIGS. 69A and 69B show the results of editing assays of the various XDP versions, as described in Example 22.

[00128] FIG. 70 shows the results of editing assays of the various XDP versions, as described in Example 22.

[00129] FIGS. 71 A and 71B shows the results of editing assays of the various XDP versions, as described in Example 23.

[00130] FIG. 72 shows the results of editing assays of the various XDP versions, as described in Example 23.

[00131] FIGS. 73 A and 73B shows the results of editing assays of the various XDP versions, as described in Example 23.

[00132] FIG. 74 shows the results of editing assays of the various XDP versions, as described in Example 23.

[00133] FIGS. 75A and 75B shows the results of editing assays of the various XDP versions, as described in Example 25.

[00134] FIG. 76 shows the results of editing assays of the various XDP versions, as described in Example 25.

[00135] FIG. 77 shows the results of editing assays of the various XDP versions, as described in Example 26.

[00136] FIG. 78 shows the results of editing assays of the various XDP versions, as described in Example 26. DETAILED DESCRIPTION

[00137] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the 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.

[00138] 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

[00139] 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 single-stranded DNA; double- stranded DNA; multi -stranded DNA; single-stranded RNA; double-stranded RNA; multi- stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[00140] “Hybridizable” or “complementary” are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still hybridize to the target nucleic acid. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a 'bulge', ‘bubble’ and the like).

[00141] 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 regulatory 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.

[00142] 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.

[00143] 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.

[00144] The term “regulatory element” is used interchangeably herein with the term “regulatory sequence,” and is intended to include promoters, enhancers, and other expression regulatory elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Exemplary regulatory elements include a transcription promoter such as, but not limited to, CMV, CMV+intron A, SV40, RSV, HIV-Ltr, elongation factor 1 alpha (EFla), MMLV-ltr, internal ribosome entry site (IRES) or P2A peptide to permit translation of multiple genes from a single transcript, metallothionein, a transcription enhancer element, a transcription termination signal, polyadenylation sequences, sequences for optimization of initiation of translation, and translation termination sequences. In the case of systems utilized for exon skipping, regulatory elements include exonic splicing enhancers. 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.

[00145] The term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, transcription start site, TATA box, and/or B recognition element and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. A promoter can be proximal or distal to the gene to be transcribed. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties. A promoter of the present disclosure can include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter can be classified according to criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc.

[00146] 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 of base 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. [00147] “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).

[00148] 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.

[00149] 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.

[00150] 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.

[00151] “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 K_d=[L] [P]/[LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively. [00152] The disclosure provides compositions and methods useful for modifying a target nucleic acid. As used herein “modifying” includes but is not limited to cleaving, nicking, editing, deleting, knocking in, knocking out, and the like.

[00153] 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 "knock-down" 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.

[00154] 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.

[00155] 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- strand break.

[00156] As used herein "micro-homology 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. 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 homology) can be determined in a number of different manners. To determine sequence similarity, sequences can be aligned using the methods and computer programs that are known in the art, including 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).

[00157] The terms “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino 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 amino acid sequence.

[00158] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e., an “insert”, may be attached so as to bring about the replication or expression of the attached segment in a cell.

[00159] 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.

[00160] As used herein, a “mutation” refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides as compared to a wild-type or reference amino acid sequence or to a wild-type or reference nucleotide sequence.

[00161] 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.

[00162] 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.

[00163] The term “tropism” as used herein refers to preferential entry of the XDP into certain cell or tissue type(s) and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the XDP into the cell.

[00164] 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), which allows HIV to infect a wider range of cells because HIV envelope proteins target the virus mainly to CD4+ presenting cells.

[00165] The term “tropism factor” as used herein refers to components integrated into the surface of an XDP that provides tropism for a certain cell 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 markers.

[00166] 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.

[00167] 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.

[00168] The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[00169] 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.

[00170] 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.

[00171] 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.

[00172] 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.

I. General Methods

[00173] 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 ah, 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.

[00174] 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.

[00175] 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. [00176] 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.

[00177] 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 Use in Targeting Cells

[00178] In a first aspect, the present disclosure relates to particle delivery systems (XDP) designed to self-assemble particles comprising therapeutic payloads wherein the particles are designed for selective delivery to targeted cells. As used herein, the term “XDP” refers to a non replicating, self-assembling, non-naturally occurring multicomponent structure composed of one or more viral proteins, polyproteins, virally-derived peptides or polypeptides, such as, but not limited to, capsid, coat, shell, as well as tropism factors such as envelope glycoproteins derived from viruses, antibody fragments, receptors or ligand utilized for tropism to direct the XDP to target cells or tissues, with a lipid layer (derived from the host cell), wherein the XDP are capable of self-assembly in a host cell and encapsidating or encompassing a therapeutic payload. The XDP of present disclosure can be utilized to specifically and selectively deliver therapeutic payloads to target cells or tissues. The XDP 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 or organ for the treatment of a disease.

[00179] In some embodiments, the present disclosure provides XDP systems comprising one or more nucleic acids comprising sequences encoding the components of the XDP, the therapeutic payload, and tropism factors that, that, when introduced into an appropriate eukaryotic host cell, result in the expression of the individual XDP structural components, processing proteins, therapeutic payloads, and tropism factors that self-assemble into XDP particles that encapsidate the therapeutic payload, and that can be collected and purified for the methods and uses described herein.

[00180] In some embodiments, the therapeutic payloads packaged within the XDP comprise therapeutic proteins, described more fully below. In other embodiments, the therapeutic payloads packaged within XDP comprise therapeutic nucleic acids or nucleic acids that encode therapeutic proteins. In still other embodiments, the XDP comprise 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-Cas 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 Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2J, and CasX wherein the CRISPR nuclease and guide system 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; (iii) 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 XDP compositions, and methods to make the XDP compositions, designed to package ribonucleic acid particles (RNP) comprising CasX and guide RNA systems (CasX:gNA system) useful for the editing of nucleic acids in target cells, described more fully, below. Accordingly, the present disclosure provides XDP compositions, nucleic acids that encode the components of the XDP (both structural as well as gene-editing components), as well as methods of making and using the XDP. The nucleic acids, the components of the compositions, and the methods of making and using them, are described herein, below. a. XDP Components

[00181] XDP can be created in multiple forms and configurations (see, e.g., FIGS. 36-68) utilizing components derived from various sources and in different combinations.

[00182] The structural components of the XDP of the present disclosure are derived from members of the Retroviridae family of viruses, described more fully, below. The major structural component of retroviruses is the polyprotein Gag, which also typically contain protease cleavage sites that, upon action by the viral protease, processes the Gag into subcomponents that, in the case of the replication of the source virus, then self-assemble in the host cell to make the core inner shell of the virus. The expression of Gag alone is sufficient to mediate the assembly and release of virus-like particles (VLPs) from host cells. Gag proteins from all retroviruses contain an N-terminal membrane-binding matrix (MA) domain, a capsid (CA) domain (with two subdomains), and a nucleocapsid (NC) domain that are structurally similar across retroviral genera but differ greatly in sequence. Outside these core domains, Gag proteins vary among retroviruses, and other linkers and domains may be present (Shur, F., et al. The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the plO Domain in Assembly. J Virol. 89(20): 10294 (2015)). The assembly pathway of Gag into immature particles in the host cell is mediated by interactions between MA (which is responsible for targeting Gag polyprotein to the plasma membrane), between NC and RNA, and between CA domains (which, in the context of the present disclosure, assemble into the XDP capsid). For most retrovirus genera, assembly takes place on the plasma membrane, but for betaretroviruses the particles are assembled in the cytoplasm and then transported to the plasma membrane. In the context of the retroviruses, concomitant with, or shortly after, particle release, cleavage of Gag by the viral protease (PR) gives rise to separate MA, CA, and NC proteins, inducing a rearrangement of the internal viral structure, with CA forming the shell of the mature viral core. Full proteolytic cleavage of Gag into its individual domains is necessary for virus infectivity for the native viruses. However, it has been discovered that for self-assembly of XDP within a host cell comprising retroviral components that are then capable of being taken up by target cells and delivering the active therapeutic payload, the XDP does not require, in some configuration embodiments, cleavage of Gag; hence the omission of a protease and cleavage sites is dispensable in some embodiments, described more fully, below, including the Examples. [00183] In some embodiments, the present disclosure provides XDP comprising one or more structural components derived from a Retroviridae virus, a therapeutic payload (described more fully, below), and a tropism factor (described more fully, below). In some embodiments, the virus structural components are derived from a Orthoretrovirinae virus. In some embodiments, the Orthoretrovirinae virus is an Alpharetrovirus, a Betaretrovirus, a Deltaretrovirus, an Epsilonretrovirus , a Gammaretrovirus or a Lentivirus. In other embodiments, the virus structural components are derived from a Spumaretrovirinae virus. In some embodiments, the Spumaretrovirinae virus is a B ovaspumavirus, an Equispumavirus , a Felispumavirus , a Prosimiispumavirus or a Simiispumavirus . b. Retroviral Components

[00184] The Retroviridae family of viruses have different subfamilies, including Orthoretrovirinae, Spumaretrovirinae , and unclassified Retroviridae . Many retroviruses cause serious diseases in humans, other mammals, and birds. Human retroviruses include Human Immunodeficiency Virus 1 (HIV-1) and HIV-2, the cause of the disease AIDS, and human T- lymphotropic virus (HTLV) also cause disease in humans. The subfamily Orthoretrovirinae include the genera Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus , and Lentivirus. Members of Alpharetrovirus, including Avian leukosis virus and Rous sarcoma virus, can cause sarcomas, tumors, and anemia of wild and domestic birds. Examples of Betaretrovirus include mouse mammary tumor virus, Mason-Pfizer monkey virus, and enzootic nasal tumor virus. Examples of Deltaretrovirus include the bovine leukemia virus and the human T-lymphotropic viruses. Members of Epsilonretrovirus include Walleye dermal sarcoma virus, and Walleye epidermal hyperplasia virus 1 and 2. Members of Gammaretrovirus include murine leukemia virus, Maloney murine leukemia virus, and feline leukemia virus, as well as viruses that infect other animal species. Lentivirus is a genus of retroviruses that cause chronic and deadly diseases, including HIV-1 and HIV-2, the cause of the disease AIDS, and also includes Simian immunodeficiency virus. The subfamily Spumaretrovirinae include the genera Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus,

Simiispumavirus, and Spumavirus. Members of the Retroviridae have provided valuable research tools in molecular biology, and, in the context of the present disclosure, have been used in the generation of XDP for delivery systems. It has been discovered that the retroviral-derived structural components of XDP can be derived from each of the genera of Retroviridae , and that the resulting XDP are capable self-assembly in a host cell and encapsidating (or encompassing) therapeutic payloads that have utility in the targeted and selective delivery of the therapeutic payloads to target cells and tissues.

[00185] In some embodiments, the XDP retroviral components are derived from Alpharetrovirus , including but not limited to avian leukosis virus (ALV) and Rous sarcoma virus (RSV). In such embodiments, the present disclosure provides XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a p2A spacer peptide; ap2B spacer peptide; a plO spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), p2A, p2B, plO, a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-Pol polyprotein; a Gag-transframe region-Pol protease polyprotein; a cleavage site(s); and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, p2A, p2B, plO, and NC), and optionally the cleavage site and protease, are derived from an Alpharetrovirus , including but not limited to Avian leukosis virus and Rous sarcoma virus. The encoding sequences for these components are provided in Table 5, and the methods to create the encoding plasmids and produce the XDP in host cells are described herein, below. In some embodiments, the XDP comprises one or mor Q Alpharetrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, and 234 as set forth in Table 5, or a sequence having at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the XDP comprises one or more Alpharetrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234 as set forth in Table 5. The XDP having Alpharetrovirus components can be designed in various configurations, including the configurations of FIGS. 36-68, and may be encoded by one, two, three or four nucleic acids, described more fully, below. In some embodiments, the XDP comprise a subset of the components listed supra , such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. These alternative configurations are described more fully, below, as well as in the Examples. In a particular embodiment, the therapeutic payload is an RNP of a complexed CasX and gNA embodiment described herein, while the tropism factor is a viral glycoprotein embodiment described herein.

[00186] In some embodiments, the XDP viral components are derived from Betaretrovirus, including but not limited to mouse mammary tumor virus (MMTV), Mason-Pfizer monkey virus (MPMV), and enzootic nasal tumor virus (ENTV). In such embodiments, the present disclosure provides XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a pp21/24 spacer peptide; a p3-p8/pl2 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), pp21/24, p3-p8/pl2, a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-Pol polyprotein; a Gag-transframe region- Pol protease polyprotein; a cleavage site(s); and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, pp2124 spacer, p3-p8/pl2 spacer, andNC), and optionally the cleavage site and protease, are derived from an Betaretrovirus , including but not limited to mouse mammary tumor virus, Mason-Pfizer monkey virus, and enzootic nasal tumor virus. The encoding sequences for these components are provided in Table 5, and the methods to create the encoding plasmids and produce the XDP in host cells are described herein, below. In some embodiments, the XDP comprises one or more Betaretrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 235-257 as set forth in Table 5, or a sequence having at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the XDP comprises one or more Betaretrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 235-257 as set forth in Table 5. The XDP having Betaretrovirus components can be designed in various configurations, including the configurations of FIGS. 36-68, and may be encoded by one, two, three or four nucleic acids, described more fully, below. In some embodiments, the XDP comprise a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. These alternative configurations are described more fully, below, as well as in the Examples. In a particular embodiment, the therapeutic payload is an RNP of a complexed CasX and gNA embodiment described herein, while the tropism factor is a viral glycoprotein embodiment described herein.

[00187] In some embodiments, the XDP viral components are derived from Deltaretrovirus, including but not limited to bovine leukemia virus (BLV) and the human T-lymphotropic viruses (HTLV1). In such embodiments, the present disclosure provides XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-Pol polyprotein; a Gag-transframe region-Pol protease polyprotein; a cleavage site(s); and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, and NC), and optionally the cleavage site and protease, are derived from an Deltaretrovirus , including but not limited to bovine leukemia virus and the human T- lymphotropic viruses. The encoding sequences for these components are provided in Table 5, and the methods to create the encoding plasmids and produce the XDP in host cells are described herein, below. In some embodiments, the XDP comprises one or mor Q Deltaretrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 258-272 as set forth in Table 5, or a sequence having at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the XDP comprises one or more Deltaretrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 258-272 as set forth in Table 5. The XDP having Deltaretrovirus components can be designed in various configurations, including the configurations of FIGS. 36-68, and may be encoded by one, two, three or four nucleic acids, described more fully, below. In some embodiments, the XDP comprise a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. These alternative configurations are described more fully, below, as well as in the Examples. In a particular embodiment, the therapeutic payload is an RNP of a complexed CasX and gNA embodiment described herein, while the tropism factor is a viral glycoprotein embodiment described herein.

[00188] In some embodiments, the XDP viral components are derived from Epsilonretrovirus , including but not limited to Walleye dermal sarcoma virus (WDSV), and Walleye epidermal hyperplasia virus 1 and 2. In such embodiments, the present disclosure provides XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a p20 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), p20, a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-Pol polyprotein; a Gag-transframe region-Pol protease polyprotein; a cleavage site(s); and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, p20, and NC), and optionally the cleavage site and protease, are derived from an Epsilonretrovirus , including but not limited to Walleye dermal sarcoma virus, and Walleye epidermal hyperplasia virus 1 and 2. The encoding sequences for these components are provided in Table 5, and the methods to create the encoding plasmids and produce the XDP in host cells are described herein, below. In some embodiments, the XDP comprises one or more Epsilonretrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 273-277 as set forth in Table 5, or a sequence having at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the XDP comprises one or more Epsilonretrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 273-277 as set forth in Table 5. The XDP having Epsilonretrovirus components can be designed in various configurations, including the configurations of FIGS. 36- 68, and may be encoded by one, two, three or four nucleic acids, described more fully, below. In some embodiments, the XDP comprise a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. These alternative configurations are described more fully, below, as well as in the Examples. In a particular embodiment, the therapeutic payload is an RNP of a complexed CasX and gNA embodiment described herein, while the tropism factor is a viral glycoprotein embodiment described herein. [00189] In some embodiments, the XDP viral components are derived from Gammaretrovirus, including but not limited to murine leukemia virus (MLV), Maloney murine leukemia virus (MMLV), and feline leukemia virus (FLV). In such embodiments, the present disclosure provides XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a ppl2 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a ppl2 spacer, a capsid polypeptide (CA), a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-Pol polyprotein; a Gag-transframe region-Pol protease polyprotein; a cleavage site(s); and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, ppl2, CA, and NC), and optionally the cleavage site and protease, are derived from an Gammaretrovirus , including but not limited to Walleye dermal sarcoma virus, and Walleye epidermal hyperplasia virus 1 and 2. The encoding sequences for these components are provided in Table 5, and the methods to create the encoding plasmids and produce the XDP in host cells are described herein, below. In some embodiments, the XDP comprises one or more Gammaretrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 278-287 as set forth in Table 5, or a sequence having at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the XDP comprises one or more Gammaretrovirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 278-287 as set forth in Table 5. The XDP having Gammaretrovirus components can be designed in various configurations, including the configurations of FIGS. 36-68, and may be encoded by one, two, three or four nucleic acids, described more fully, below. In some embodiments, the XDP comprise a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. These alternative configurations are described more fully, below, as well as in the Examples. In a particular embodiment, the therapeutic payload is an RNP of a complexed CasX and gNA embodiment described herein, while the tropism factor is a viral glycoprotein embodiment described herein.

[00190] In some embodiments, the XDP viral components are derived from Lentivirus , including but not limited to HIV-1 and HIV-2, and Simian immunodeficiency virus (SIV). In such embodiments, the present disclosure provides XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a capsid (CA), a p2 spacer peptide, a nucleocapsid (NC), a pl/p6 spacer peptide; ); a Gag polyprotein comprising a matrix polypeptide (MA), CA, p2, NC, and pl/p6; a therapeutic payload; a tropism factor; a Gag-Pol polyprotein; a Gag-transframe region-Pol protease polyprotein; a cleavage site(s); and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, NC, and pl/p6), and optionally the cleavage site and protease, are derived from an Lentivirus , including but not limited to HIV-1, HIV-2, and Simian immunodeficiency virus (SIV). The encoding sequences for these components are provided in Table 5, and the methods to create the encoding plasmids and produce the XDP in host cells are described herein, below. In some embodiments, the XDP comprises one or more Lentivirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 288-312 and 334-339 as set forth in Table 5, or a sequence having at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the XDP comprises one or more Lentivirus structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 288-312 and 334-339 as set forth in Table 5. The XDP having Lentivirus components can be designed in various configurations, including the configurations of FIGS. 36-68, and may be encoded by one, two, three or four or more nucleic acids, described more fully, below. In some embodiments, the XDP comprise a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. These alternative configurations are described more fully, below, as well as in the Examples. In a particular embodiment, the therapeutic payload is an RNP of a complexed CasX and gNA embodiment described herein, while the tropism factor is a viral glycoprotein embodiment described herein.

[00191] In some embodiments, the XDP viral components are derived from Spumaretrovirinae, including but not limited to Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, and Spumavirus. In such cases, the present disclosure provides XDP wherein the XDP comprises components selected from the group consisting of: p68 Gag; a p3 Gag; a Gag polyprotein comprising of p68 Gag and p3 gag; a therapeutic payload; a tropism factor; a Gag-Pol polyprotein; a Gag-transframe region-Pol protease polyprotein; a cleavage site(s); and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., p68 AND p3p20), and optionally the cleavage site and protease, are derived from an Spumaretrovirinae including but not limited to Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, and Spumavirus. The encoding sequences for these components are provided in Table 5, and the methods to create the encoding plasmids and produce the XDP in host cells are described herein, below. In some embodiments, the XDP comprises one or more Spumaretrovirinae structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 313-333 as set forth in Table 5, or a sequence having at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the XDP comprises one or more Spumaretrovirinae structural components encoded by the sequences selected from the group consisting SEQ ID NOS: 313- 333 as set forth in Table 5. The XDP having Spumaretrovirus components can be designed in various configurations, including the configurations of FIGS. 36-68, and may be encoded by one, two, three or four nucleic acids, described more fully, below. In some embodiments, the XDP comprise a subset of the components listed in the paragraph, such as depicted in FIGS. 36- 68, which depict CasX and gNA as the therapeutic payloads. These alternative configurations are described more fully, below, as well as in the Examples. In a particular embodiment, the therapeutic payload is an RNP of a complexed CasX and gNA embodiment described herein, while the tropism factor is a viral glycoprotein embodiment described herein.

[00192] In other embodiments, the present disclosure provides XDP wherein the retroviral components of the XDP are selected from different genera of the Retroviridae. Thus the XDP can comprise two or more components selected from a matrix polypeptide (MA), a p2A spacer peptide, a p2B spacer peptide; a plO spacer peptide, a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a pp21/24 spacer peptide, a p3-P8 spacer peptide, a ppl2 spacer peptide, a p20 spacer peptide, a pl/p6 spacer peptide, a p68 Gag, a p3 Gag, a cleavage site(s), a Gag-Pol polyprotein; a Gag-transframe region-Pol protease polyprotein; and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites wherein the components are derived from Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, or Spumavirus..

[00193] In retroviral components derived from HIV-1, the accessory protein integrase (or its encoding nucleic acid) can be omitted from the XDP systems, as well as the HIV functional accessory genes vpr, vpx (HIV-2), which are dispensable for viral replication in vitro. Additionally, the nucleic acids of the XDP system do not require reverse transcriptase for the creation of the XDP compositions of the embodiments. Thus, in one embodiment, the HIV-1 Gag-Pol component of the XDP can be truncated to Gag linked to the transframe region (TFR) composed of the transframe octapeptide (TFP) and 48 amino acids of the p6pol, separated by a protease cleavage site, hereinafter referred to as Gag-TFR-PR, described more fully, below. c. Proteases

[00194] In some embodiments of the XDP systems, the protease capable of cleaving the protease cleavage sites is selected from a retroviral protease, including any of the genera of the Retroviridae. For example, the protease can be encoded by a sequence selected from the group consisting of SEQ ID NOS: 198, 234, 239, 245, 251, 257, 261, 266, 271, 276, 282, 287, 291,

296, 301, and 306 as set forth in Table 5, or a sequence having at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In other embodiments, the protease capable of cleaving the protease cleavage sites is a non-retroviral, heterologous protease selected from the group of proteases consisting of tobacco etch virus protease (TEV), potyvirus HC protease, potyvirus Plprotease, PreScission (HRV3C protease), b virus NIa protease, B virus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2 A protease, picoma 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, cathepsin, thrombin, factor Xa, metalloproteinases (including MMP-2, -3, -7, -9, -10, and -11), and enterokinase. In a particular embodiment, the protease capable of cleaving the protease cleavage sites is PreScission Protease; a fusion protein of human rhinovirus (HRV) 3C protease and glutathione S-transferase (GST). In another particular embodiment, the protease capable of cleaving the protease cleavage sites is tobacco etch virus protease (TEV), In another particular embodiment, the protease capable of cleaving the protease cleavage sites is HIV-1 protease. In the case of HIV-1 protease, the 99-amino acid protease (PR) of the precursor Gag— Pol polyprotein (which are encoded by overlapping open reading frames such that the synthesis of the of the Gag— Pol precursor results from a -1 frameshifting event) is flanked at its N-terminus by a transframe region (TFR) composed of the transframe octapeptide (TFP) and 48 amino acids of the p6pol, separated by a protease cleavage site. Cleavage at the p6pol-PR site to release a free N-terminus of protease is concomitant with the appearance of enzymatic activity and formation of a stable tertiary structure that is characteristic of the mature protease (Louis, JM. Et al. Autoprocessing of HIV-1 protease is tightly coupled to protein folding. Nat Struct Mol Biol 6, 868-875 (1999)). In some embodiments of the XDP systems, wherein the nucleic acid encodes all or a portion of the HIV-1 Gag-Pol polyprotein, the Gag-Pol sequence comprises the encoded TFR-PR to facilitate the-1 frameshifting event. In some cases, wherein the XDP system utilizes a component comprised of the Gag polyprotein and a portion of the pol polyprotein comprising the TFR and the protease, the component is referred to herein as “Gag-TFR-PR”, wherein the capability to facilitate the -1 frameshifting event is retained, along with the capability to produce the encoded protease. In non-limiting examples of nucleic acids encoding a Retroviral protease the can be incorporated into an encoding plasmid of the XDP system embodiments, representative sequences are provided in Table 5.

[00195] In a corresponding fashion, wherein protease cleavage sites are incorporated in the XDP systems, the protease cleavage sites utilized in the encoded proteins of the XDPs and their encoding sequences in the nucleic acids will correlate with the protease that is incorporated into the XDP system. In some embodiments, the protease cleavage site of the XDP component comprising all or a portion of a Gag polyprotein is located between the Gag polyprotein and the therapeutic payload such that upon maturation of the XDP particle, the therapeutic payload is not tethered to any component of the Gag polyprotein. In other embodiments, the protease cleavage site is incorporated between the individual components of the Gag polyprotein as well as between the Gag polyprotein and the therapeutic payload. In a representative embodiment, wherein the protease capable of cleaving the protease cleavage sites is TEV, the encoded TEV protease cleavage sites can have the sequences EXXYXQ(G/S) (SEQ ID NO: 17), ENLYFQG (SEQ ID NO: 18) or ENLYFQS (SEQ ID NO: 19), wherein X represents any amino acid and cleavage by TEV occurs between Q and G or Q and S. In another embodiment, wherein the protease is HIV-1 protease, the encoded HIV-1 cleavage sites can have the sequence SQNYPIVQ (SEQ ID NO: 20). In another embodiment, wherein the protease is PreScission, the protease cleavage sites include the core amino acid sequence Leu-Phe-Gln/Gly-Pro (SEQ ID NO: 1010), cleaving between the Gin and Gly residues. In one embodiment, the XDP comprising cleavage sites have protease cleavage sites that are identical. In another embodiment, the XDP comprising cleavage sites have protease cleavage sites that are different and are substrates for different proteases. In another embodiment, the XDP system can comprise a cleavage sequence that is susceptible to cleavage by two different proteases; e.g., HIV-1 and PreScission protease. In such cases, the nucleic acids encoding the XDP would include encoding sequences for both proteases. [00196] Additional protease cleavage sites are envisaged as within the scope of the XDP of the instant invention, and include, inter alia , SEQ ID NOS: 874-897, and 934-946. d. Protein and Nucleic Acid Therapeutic Payloads of the XDP Systems [00197] Protein therapeutic payloads suitable for inclusion in the XDP of the present disclosure include a diversity of categories of protein-based therapeutics, including, but not limited to cytokines (e.g., IFNs a, b, and g, 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-b), enzymes, receptors, microproteins, hormones (e.g., growth hormone, insulin), erythropoietin, 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 1 and Class 2 Type II, Type V, or Type VI) 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. Nucleic acid payloads suitable for inclusion in the XDP 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, nucleic acids utilized in gene therapy (e.g., guide RNAs utilized in CRISPR systems and donor templates), micro RNAs, ribozymes, RNA decoys and circular RNAs. In a particular embodiment, the protein payload of the XDP comprises a CasX variant protein of any of the embodiments described herein, including the CasX variants of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 and 388-397 as set forth in Tables 1, 7, 8, 9 and 11, while the nucleic acid payload comprises one or more guide RNAs of any of the embodiments described herein, including the gNA variants with a scaffold sequence of SEQ ID NOS: 597-781 as set forth in Table 3 and, optionally, a donor template. e. CRISPR Proteins of the XDP Systems

[00198] In some embodiments, the present disclosure provides XDP compositions and systems 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. In some embodiments, the CRISPR nuclease employed in the XDP systems is a Class 2 nuclease. In other embodiments, the CRISPR nuclease is a Class 2, Type V nuclease. Although members of Class 2, Type V CRISPR-Cas systems have differences, they share some common characteristics that distinguish them from the Cas9 systems. Firstly, the Type V nucleases possess a single RNA-guided RuvC domain-containing 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-rich PAM at 3’ side of target sequences. Type V nucleases generate staggered double-stranded breaks distal to the PAM sequence, unlike Cas9, which generates a blunt end in the proximal site close to the PAM. In addition, Type V nucleases degrade ssDNA in trans when activated by target dsDNA or ssDNA binding in cis. In some embodiments, the Type V nucleases utilized in the XDP embodiments recognize a 5’ TC PAM motif and produce staggered ends cleaved solely by the RuvC domain. In some embodiments, the XDP comprise a Class 2, Type V nuclease selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX. In a particular embodiment, the present disclosure provides XDP comprising a ribonucleoprotein (RNP) of a complexed CasX protein and one or more guide nucleic acids (gNA) that are specifically designed to modify a target nucleic acid sequence in eukaryotic cells. [00199] 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 in at least one domain relative to a naturally-occurring reference CasX protein. Reference CasX proteins include, but are not limited to those isolated or derived from Deltaproteobacter , Planctomycetes, or Candidatus (as described in US20180346927A1 and WO2018064371A1, incorporated herein by reference). Exemplary embodiments of CasX variants envisaged as being within the scope of the disclosure are described herein, below.

[00200] In some cases, a Type V reference CasX protein is isolated or derived from Deltaproteobacteria. In some embodiments, a CasX protein comprises a sequence at least 50% identical, 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 or 100% identical to a sequence of:

1 MEKRINKIRK KLSADNATKP VSRSGPMKTL LVRVMTDDLK KRLEKRRKKP EVMPQVISNN 61 AANNLRMLLD DYTKMKEAIL QVYWQEFKDD HVGLMCKFAQ PASKKIDQNK LKPEMDEKGN

121 LTTAGFACSQ CGQPLFVYKL EQVSEKGKAY TNYFGRCNVA EHEKLILLAQ LKPEKDSDEA

181 VTYSLGKFGQ RALDFYSIHV TKESTHPVKP LAQIAGNRYA SGPVGKALSD ACMGTIASFL

241 SKYQDIIIEH QKW KGNQKR LESLRELAGK ENLEYPSVTL PPQPHTKEGV DAYNEVIARV

301 RMWVNLNLWQ KLKLSRDDAK PLLRLKGFPS FPW ERRENE VDWWNTINEV KKLIDAKRDM

361 GRVFWSGVTA EKRNTILEGY NYLPNENDHK KREGSLENPK KPAKRQFGDL LLYLEKKYAG

421 DWGKVFDEAW ERIDKKIAGL TSHIEREEAR NAEDAQSKAV LTDWLRAKAS FVLERLKEMD

481 EKEFYACEIQ LQKWYGDLRG NPFAVEAENR W DISGFSIG SDGHSIQYRN LLAWKYLENG

541 KREFYLLMNY GKKGRIRFTD GTDIKKSGKW QGLLYGGGKA KVIDLTFDPD DEQLIILPLA

601 FGTRQGREFI WNDLLSLETG LIKLANGRVI EKTIYNKKIG RDEPALFVAL TFERREW DP

661 SNIKPVNLIG VDRGENIPAV IALTDPEGCP LPEFKDSSGG PTDILRIGEG YKEKQRAIQA

721 AKEVEQRRAG GYSRKFASKS RNLADDMVRN SARDLFYHAV THDAVLVFEN LSRGFGRQGK

781 RTEMTERQYT KMEDWLTAKL AYEGLTSKTY LSKTLAQYTS KTCSNCGFTI TTADYDGMLV

841 RLKKTSDGWA TTLNNKELKA EGQITYYNRY KRQTVEKELS AELDRLSEES GNNDISKWTK

901 GRRDEALFLL KKRFSHRPVQ EQFVCLDCGH EVHADEQAAL NIARSWLFLN SNSTEFKSYK

961 SGKQPFVGAW QAFYKRRLKE VWKPNA (SEQ ID NO: 1).

[00201] In some cases, a Type V reference CasX protein is isolated or derived from Planctomycetes. In some embodiments, a CasX protein comprises a sequence at least 50% identical, 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 or 100% identical to a sequence of: 1 MQEIKRINKI RRRLVKDSNT KKAGKTGPMK TLLVRVMTPD LRERLENLRK KPENIPQPIS

61 NTSRANLNKL LTDYTEMKKA ILHVYWEEFQ KDPVGLMSRV AQPAPKNIDQ RKLIPVKDGN

121 ERLTSSGFAC SQCCQPLYVY KLEQVNDKGK PHTNYFGRCN VSEHERLILL SPHKPEANDE

181 LVTYSLGKFG QRALDFYSIH VTRESNHPVK PLEQIGGNSC ASGPVGKALS DACMGAVASF

241 LTKYQDIILE HQKVIKKNEK RLANLKDIAS ANGLAFPKIT LPPQPHTKEG IEAYNNW AQ

301 IVIWVNLNLW QKLKIGRDEA KPLQRLKGFP SFPLVERQAN EVDWWDMVCN VKKLINEKKE

361 DGKVFWQNLA GYKRQEALLP YLSSEEDRKK GKKFARYQFG DLLLHLEKKH GEDWGKVYDE

421 AWERIDKKVE GLSKHIKLEE ERRSEDAQSK AALTDWLRAK ASFVIEGLKE ADKDEFCRCE

481 LKLQKWYGDL RGKPFAIEAE NSILDISGFS KQYNCAFIWQ KDGVKKLNLY LIINYFKGGK

541 LRFKKIKPEA FEANRFYTVI NKKSGEIVPM EVNFNFDDPN LIILPLAFGK RQGREFIWND

601 LLSLETGSLK LANGRVIEKT LYNRRTRQDE PALFVALTFE RREVLDSSNI KPMNLIGIDR

661 GENIPAVIAL TDPEGCPLSR FKDSLGNPTH ILRIGESYKE KQRTIQAAKE VEQRRAGGYS

721 RKYASKAKNL ADDMVRNTAR DLLYYAVTQD AMLIFENLSR GFGRQGKRTF MAERQYTRME

781 DWLTAKLAYE GLPSKTYLSK TLAQYTSKTC SNCGFTITSA DYDRVLEKLK KTATGWMTTI

841 NGKELKVEGQ ITYYNRYKRQ NW KDLSVEL DRLSEESVNN DISSWTKGRS GEALSLLKKR

901 FSHRPVQEKF VCLNCGFETH ADEQAALNIA RSWLFLRSQE YKKYQTNKTT GNTDKRAFVE

961 TWQSFYRKKL KEVWKPAV (SEQ ID NO: 2).

[00202] In some cases, a Type V reference CasX protein is isolated or derived from Candidatus Sungbacteria. In some embodiments, a CasX protein comprises a sequence at least 50% identical, 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 or 100% identical to a sequence of 1 MDNANKPSTK SLVNTTRISD HFGVTPGQVT RVFSFGIIPT KRQYAIIERW FAAVEAARER

61 LYGMLYAHFQ ENPPAYLKEK FSYETFFKGR PVLNGLRDID PTIMTSAVFT ALRHKAEGAM

121 AAFHTNHRRL FEEARKKMRE YAECLKANEA LLRGAADIDW DKIVNALRTR LNTCLAPEYD

181 AVIADFGALC AFRALIAETN ALKGAYNHAL NQMLPALVKV DEPEEAEESP RLRFFNGRIN

241 DLPKFPVAER ETPPDTETII RQLEDMARVI PDTAEILGYI HRIRHKAARR KPGSAVPLPQ

301 RVALYCAIRM ERNPEEDPST VAGHFLGEID RVCEKRRQGL VRTPFDSQIR ARYMDIISFR

361 ATLAHPDRWT EIQFLRSNAA SRRVRAETIS APFEGFSWTS NRTNPAPQYG MALAKDANAP

421 ADAPELCICL SPSSAAFSVR EKGGDLIYMR PTGGRRGKDN PGKEITWVPG SFDEYPASGV

481 ALKLRLYFGR SQARRMLTNK TWGLLSDNPR VFAANAELVG KKRNPQDRWK LFFHMVISGP

541 PPVEYLDFSS DVRSRARTVI GINRGEVNPL AYAW SVEDG QVLEEGLLGK KEYIDQLIET

601 RRRISEYQSR EQTPPRDLRQ RVRHLQDTVL GSARAKIHSL IAFWKGILAI ERLDDQFHGR

661 EQKIIPKKTY LANKTGFMNA LSFSGAVRVD KKGNPWGGMI EIYPGGISRT CTQCGTVWLA

721 RRPKNPGHRD AMW IPDIVD DAAATGFDNV DCDAGTVDYG ELFTLSREWV RLTPRYSRVM

781 RGTLGDLERA IRQGDDRKSR QMLELALEPQ PQWGQFFCHR CGFNGQSDVL AATNLARRAI

841 SLIRRLPDTD TPPTP (SEQ ID NO: 3).

[00203] In some embodiments of the XDP systems, the disclosure provides CasX variant proteins for use in the XDP comprising a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40 or at least 50 or more individual or sequential mutations relative to the sequence of a reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. These mutations can be insertions, deletions, amino acid substitutions, or any combinations thereof. In some embodiments, in addition to the aforementioned mutations, a CasX variant can further comprise a substitution of a portion or all of a domain from a heterologous reference CasX, and the substituted domain can further comprise one or more mutations. Suitable mutagenesis methods for generating CasX variant proteins of the disclosure may include, for example, Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping. In some embodiments, the CasX variants are designed, for example by selecting one or more desired mutations in a reference CasX. Any amino acid can be substituted for any other amino acid in the substitutions described herein. The substitution can be a conservative substitution (e.g., a basic amino acid is substituted for another basic amino acid). The substitution can be a non-conservative substitution (e.g., a basic amino acid is substituted for an acidic amino acid or vice versa). For example, a proline in a reference CasX protein can be substituted for any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine or valine to generate a CasX variant protein of the disclosure. In certain embodiments, the activity of a reference CasX protein is used as a benchmark against which the activity of one or more CasX variants are compared, thereby measuring improvements in function of the CasX variants.

[00204] In some embodiments, a CasX variant protein comprises at least one amino acid deletion relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises a deletion of 1-4 amino acids, 1-10 amino acids, 1-20 amino acids, 1-30 amino acids, 1-40 amino acids, 1-50 amino acids, 1-60 amino acids, 1-70 amino acids, 1-80 amino acids, 1-90 amino acids, 1-100 amino acids, 2-10 amino acids, 2-20 amino acids, 2-30 amino acids, 3-10 amino acids, 3-20 amino acids, 3-30 amino acids, 4-10 amino acids, 4-20 amino acids, 3-300 amino acids, 5-10 amino acids, 5-20 amino acids, 5-30 amino acids, 10-50 amino acids or 20-50 amino acids relative to a reference CasX protein. In some embodiments, a CasX protein comprises a deletion of at least about 100 consecutive amino acids relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or 100 consecutive amino acids relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids.

[00205] In some embodiments, a CasX variant protein comprises two or more deletions relative to a reference CasX protein, and the two or more deletions are not consecutive amino acids. For example, a first deletion may be in a first domain of the reference CasX protein, and a second deletion may be in a second domain of the reference CasX protein. In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 non-consecutive deletions relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises at least 20 non-consecutive deletions relative to a reference CasX protein. Each non-consecutive deletion may be of any length of amino acids described herein, e.g., 1-4 amino acids, 1-10 amino acids, and the like.

[00206] In some embodiments, the CasX variant protein comprises one or more amino acid insertions relative to the sequence of SEQ ID NOS: 1, 2, or 3. In some embodiments, a CasX variant protein comprises an insertion of 1 amino acid, an insertion of 2-3 consecutive or non- consecutive amino acids, 2-4 consecutive or non-consecutive amino acids, 2-5 consecutive or non-consecutive amino acids, 2-6 consecutive or non-consecutive amino acids, 2-7 consecutive or non-consecutive amino acids, 2-8 consecutive or non-consecutive amino acids, 2-9 consecutive or non-consecutive amino acids, 2-10 consecutive or non-consecutive amino acids,

2-20 consecutive or non-consecutive amino acids, 2-30 consecutive or non-consecutive amino acids, 2-40 consecutive or non-consecutive amino acids, 2-50 consecutive or non-consecutive amino acids, 2-60 consecutive or non-consecutive amino acids, 2-70 consecutive or non- consecutive amino acids, 2-80 consecutive or non-consecutive amino acids, 2-90 consecutive or non-consecutive amino acids, 2-100 consecutive or non-consecutive amino acids, 3-10 consecutive or non-consecutive amino acids, 3-20 consecutive or non-consecutive amino acids,

3-30 consecutive or non-consecutive amino acids, 4-10 consecutive or non-consecutive amino acids, 4-20 consecutive or non-consecutive amino acids, 3-300 consecutive or non-consecutive amino acids, 5-10 consecutive or non-consecutive amino acids, 5-20 consecutive or non- consecutive amino acids, 5-30 consecutive or non-consecutive amino acids, 10-50 consecutive or non-consecutive amino acids or 20-50 consecutive or non-consecutive amino acids relative to a reference CasX protein. In some embodiments, the CasX variant protein comprises an insertion of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive or non-consecutive amino acids. In some embodiments, a CasX variant protein comprises an insertion of at least about 100 consecutive or non-consecutive amino acids. Any amino acid, or combination of amino acids, can be inserted in the insertions described herein to generate a CasX variant protein.

[00207] Any permutation of the substitution, insertion and deletion embodiments described herein can be combined to generate a CasX variant protein of the disclosure. For example, a CasX variant protein can comprise at least one substitution and at least one deletion relative to a reference CasX protein sequence, at least one substitution and at least one insertion relative to a reference CasX protein sequence, at least one insertion and at least one deletion relative to a reference CasX protein sequence, or at least one substitution, one insertion and one deletion relative to a reference CasX protein sequence.

[00208] A CasX variant comprises some or all of the following domains: a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain, a helical II domain, an oligonucleotide binding domain (OBD), and a RuvC DNA cleavage domain (the latter which may be deleted in a catalytically dead CasX variant), described more fully, below.

In some embodiments, the at least one modification of the CasX variant protein comprises a deletion of at least a portion of one domain of the reference CasX protein, including the sequences of SEQ ID NOS: 1-3. In some embodiments, the deletion is in the NTSBD, TSLD, Helical I domain, Helical II domain, OBD, or RuvC DNA cleavage domain. In some embodiments, the CasX variant comprises at least one modification in the NTSB domain. In some embodiments, the CasX variant comprises at least one modification in the TSL domain. In some embodiments, the at least one modification in the TSL domain comprises an amino acid substitution of one or more of amino acids Y857, S890, or S932 of SEQ ID NO:2. In some embodiments, the CasX variant comprises at least one modification in the helical I domain. In some embodiments, the at least one modification in the helical I domain comprises an amino acid substitution of one or more of amino acids S219, L249, E259, Q252, E292, L307, or D318 of SEQ ID NO:2. In some embodiments, the CasX variant comprises at least one modification in the helical II domain. In some embodiments, the at least one modification in the helical II domain comprises an amino acid substitution of one or more of amino acids D361, L379, E385, E386, D387, F399, L404, R458, C477, or D489 of SEQ ID NO:2. In some embodiments, the CasX variant comprises at least one modification in the OBD domain. In some embodiments, the at least one modification in the OBD comprises an amino acid substitution of one or more of amino acids F536, E552, T620, or 1658 of SEQ ID NO:2. In some embodiments, the CasX variant comprises at least one modification in the RuvC DNA cleavage domain. In some embodiments, the at least one modification in the RuvC DNA cleavage domain comprises an amino acid substitution of one or more of amino acids K682, G695, A708, V711, D732, A739, D733, L742, V747, F755, M771, M779, W782, A788, G791, L792, P793, Y797, M799, Q804, S819, or Y857 or a deletion of amino acid P793 of SEQ ID NO:2.

[00209] In some embodiments, the CasX variant comprises at least one modification compared to the reference CasX sequence of SEQ ID NO:2 is selected from one or more of: (a) an amino acid substitution of L379R; (b) an amino acid substitution of A708K; (c) an amino acid substitution of T620P; (d) an amino acid substitution of E385P; (e) an amino acid substitution of Y857R; (f) an amino acid substitution of I658V; (g) an amino acid substitution of F399L; (h) an amino acid substitution of Q252K; (i) an amino acid substitution of L404K; and (j) an amino acid deletion of P793.

[00210] The CasX variant proteins of the disclosure have an enhanced ability to efficiently edit and/or bind target DNA, when complexed with a gNA as an RNP, utilizing PAM TC motif, including PAM sequences selected from TTC, ATC, GTC, or CTC, compared to an RNP of a reference CasX protein and reference gNA. In the foregoing, the PAM sequence is located at least 1 nucleotide 5’ to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a assay system compared to the editing efficiency and/or binding of an RNP comprising a reference CasX protein and reference gNA in a comparable assay system. In one embodiment, an RNP of a CasX variant and gNA 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 gNA 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 gNA 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 gNA 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 gNA 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 gNA 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 gNA 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 gNA 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 of any one of the CasX proteins of SEQ ID NOS: 1-3 and the gNA of Table 2 for the PAM sequences.

[00211] All variants that improve one or more functions or characteristics of the CasX variant protein when compared to a reference CasX protein described herein are envisaged as being within the scope of the disclosure. Exemplary improved characteristics of the CasX variant embodiments include, but are not limited to improved folding of the variant, improved binding affinity to the gNA, 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 DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, 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, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved proteimgNA (RNP) complex stability, improved protein solubility, improved proteimgNA (RNP) complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics, as described more fully, below. In some embodiments, the RNP of the CasX variant and the gNA variant exhibit one or more of the improved characteristics that are at least about 1.1 to about 100,000-fold improved relative to an RNP of the reference CasX protein of SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3 and the gNA of Table 2, when assayed in a comparable fashion. In other cases, the one or more improved characteristics of an RNP of the CasX variant and the gNA variant are at least about 1.1, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000-fold or more improved relative to an RNP of the reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 and the gNA of Table 2. In other cases, the one or more of the improved characteristics of an RNP of the CasX variant and the gNA variant are about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500- fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to an RNP of the reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 and the reference gNA of SEQ ID NOS: 4-16 as set forth in Table 2, when assayed in a comparable fashion. In other cases, the one or more improved characteristics of an RNP of the CasX variant and the gNA variant are about 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260- fold, 270-fold, 280-fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold improved relative to an RNP of the reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 and the gNA SEQ ID NOS: 4-16 as set forth in Table 2, when assayed in a comparable fashion. An exemplary improved characteristic includes improved editing efficiency. In some embodiments, an RNP comprising a CasX variant protein and a gNA of the disclosure, at a concentration of 20 pM or less, is capable of cleaving a double stranded DNA target with an efficiency of at least 80%. In some embodiments, the RNP at a concentration of 20 pM or less, is capable of cleaving a double stranded DNA target with an efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95%. In some embodiments, the RNP at a concentration of 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less, or 5 pM or less, is capable of cleaving a double stranded DNA target with an efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95%. The improved editing efficiency of the CasX variant, in combination with the gNA of the disclosure, make them well-suited for inclusion in the XDP of the disclosure

[00212] 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.

[00213] In some embodiments, the CasX variant protein comprises between 400 and 2000 amino acids, between 500 and 1500 amino acids, between 700 and 1200 amino acids, between 800 and 1100 amino acids or between 900 and 1000 amino acids.

[00214] In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous residues that form a channel in which gNA:target DNA complexing occurs. In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous residues that form an interface which binds with the gNA. For example, in some embodiments of a reference CasX protein, the helical I, helical II and OBD domains all contact or are in proximity to the gNA:target DNA complex, and one or more modifications to non-contiguous residues within any of these domains may improve function of the CasX variant protein.

[00215] In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous residues that form a channel which binds with the non target strand DNA. For example, a CasX variant protein can comprise one or more modifications to non-contiguous residues of the NTSBD. In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous residues that form an interface which binds with the PAM. For example, a CasX variant protein can comprise one or more modifications to non-contiguous residues of the helical I domain or OBD. In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous surface-exposed residues. As used herein, “surface-exposed residues” refers to amino acids on the surface of the CasX protein, or amino acids in which at least a portion of the amino acid, such as the backbone or a part of the side chain is on the surface of the protein. Surface exposed residues of cellular proteins such as CasX, which are exposed to an aqueous intracellular environment, are frequently selected from positively charged hydrophilic amino acids, for example arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. Thus, for example, in some embodiments of the variants provided herein, a region of surface exposed residues comprises one or more insertions, deletions, or substitutions compared to a reference CasX protein. In some embodiments, one or more positively charged residues are substituted for one or more other positively charged residues, or negatively charged residues, or uncharged residues, or any combinations thereof. In some embodiments, one or more amino acids residues for substitution are near bound nucleic acid, for example residues in the RuvC domain or helical I domain that contact target DNA, or residues in the OBD or helical II domain that bind the gNA, can be substituted for one or more positively charged or polar amino acids.

[00216] In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous residues that form a core through hydrophobic packing in a domain of the reference CasX protein. Without wishing to be bound by any theory, regions that form cores through hydrophobic packing are rich in hydrophobic amino acids such as valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and cysteine. For example, in some reference CasX proteins, RuvC domains comprise a hydrophobic pocket adjacent to the active site. In some embodiments, between 2 to 15 residues of the region are charged, polar, or base stacking. Charged amino acids (sometimes referred to herein as residues) may include, for example, arginine, lysine, aspartic acid, and glutamic acid, and the side chains of these amino acids may form salt bridges provided a bridge partner is also present. Polar amino acids may include, for example, glutamine, asparagine, histidine, serine, threonine, tyrosine, and cysteine. Polar amino acids can, in some embodiments, form hydrogen bonds as proton donors or acceptors, depending on the identity of their side chains. As used herein, “base-stacking” includes the interaction of aromatic side chains of an amino acid residue (such as tryptophan, tyrosine, phenylalanine, or histidine) with stacked nucleotide bases in a nucleic acid. Any modification to a region of non-contiguous amino acids that are in close spatial proximity to form a functional part of the CasX variant protein is envisaged as within the scope of the disclosure. f CasX Variant Proteins with Domains from Multiple Source Proteins [00217] Also contemplated within the scope of the disclosure are XDP comprising chimeric CasX proteins comprising protein domains from two or more different CasX proteins, such as two or more naturally occurring CasX proteins, or two or more CasX variant protein sequences as described herein. As used herein, a “chimeric CasX protein” refers to a CasX containing at least two domains isolated or derived from different sources, such as two naturally occurring proteins, which may, in some embodiments, be isolated from different species. For example, in some embodiments, a chimeric CasX protein comprises a first domain from a first CasX protein and a second domain from a second, different CasX protein. In some embodiments, the first domain can be selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD and RuvC domains. In some embodiments, the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD and RuvC domains with the second domain being different from the foregoing first domain. For example, a chimeric CasX protein may comprise an NTSB, TSL, helical I, helical II, OBD domains from a CasX protein of SEQ ID NO: 2, and a RuvC domain from a CasX protein of SEQ ID NO: 1, or vice versa. As a further example, a chimeric CasX protein may comprise an NTSB, TSL, helical II, OBD and RuvC domain from CasX protein of SEQ ID NO: 2, and a helical I domain from a CasX protein of SEQ ID NO: 1, or vice versa. Thus, in certain embodiments, a chimeric CasX protein may comprise an NTSB, TSL, helical II, OBD and RuvC domain from a first CasX protein, and a helical I domain from a second CasX protein. In some embodiments of the chimeric CasX proteins, the domains of the first CasX protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the domains of the second CasX protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the first and second CasX proteins are not the same. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 2. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 2 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3. In some embodiments, the CasX variant is selected of group consisting of CasX variants with sequences of SEQ ID NO: 102,

113, 114, 115, 103, 104, 105, 106, 107, 108, 109, and 110, as described in Table 1.

[00218] In some embodiments of the XDP systems, 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. In some embodiments, the first portion of a CasX domain comprises a sequence of SEQ ID NO: 1 and the second portion of a CasX domain comprises a sequence of SEQ ID NO: 2. In some embodiments, the first portion of the CasX domain comprises a sequence of SEQ ID NO: 1 and the second portion of the CasX domain comprises a sequence of SEQ ID NO: 3. In some embodiments, the first portion of the CasX domain comprises a sequence of SEQ ID NO: 2 and the second portion of the CasX domain comprises a sequence of SEQ ID NO: 3. In some embodiments, the at least one chimeric domain comprises a chimeric RuvC domain. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 661 to 824 of SEQ ID NO: 1 and amino acids 922 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, a chimeric RuvC domain comprises amino acids 648 to 812 of SEQ ID NO: 2 and amino acids 935 to 986 of SEQ ID NO: 1. In some embodiments, a CasX protein comprises a first domain from a first CasX protein and a second domain from a second CasX protein, and at least one chimeric domain comprising at least two parts isolated from different CasX proteins using the approach of the embodiments described in this paragraph. In the foregoing embodiments, the chimeric CasX proteins having domains or portions of domains derived from SEQ ID NOS: 1, 2 and 3, can further comprise amino acid insertions, deletions, or substitutions of any of the embodiments disclosed herein.

[00219] In some embodiments of the XDP systems, a CasX variant protein comprises a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397 as set forth in Tables

I, 7, 8, 9 or 11. In some embodiments, a CasX variant protein consists of a sequence of SEQ ID

NOS: 21-233 as set forth in Table 1. In other embodiments, a CasX variant protein 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 of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397 as set forth in Tables 1, 7, 8, 9 or

II. In other embodiments, a CasX variant protein comprises a sequence set forth in Table 1, and further comprises one or more NLS 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 1: CasX Variant Sequences * Where a number is indicated in the left column, it designates the CasX variant numerically; changes, where indicated, are relative to SEQ ID NO:2 g. CasX Fusion Proteins

[00220] Also contemplated within the scope of the disclosure are XDP comprising CasX variant proteins comprising a heterologous protein fused to the CasX. In some embodiments, the CasX variant protein is fused to one or more proteins or domains thereof that has a different activity of interest, resulting in a 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).

[00221] In some embodiments, a heterologous polypeptide (or heterologous amino acid such as a cysteine residue or a non-natural amino acid) can be inserted at one or more positions within a CasX protein to generate a CasX fusion protein utilized in the XDP systems. In other embodiments, a cysteine residue can be inserted at one or more positions within a CasX protein followed by conjugation of a heterologous polypeptide described below. In some alternative embodiments, a heterologous polypeptide or heterologous amino acid can be added at the N- or C-terminus of the CasX variant protein. In other embodiments, a heterologous polypeptide or heterologous amino acid can be inserted internally within the sequence of the CasX protein. [00222] A variety of heterologous polypeptides are suitable for inclusion in a CasX variant fusion protein utilized in the XDP systems of the disclosure. In some cases, the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA. For example, in some cases the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like). In some cases the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like). [00223] In some cases, a CasX fusion partner utilized in the XDP systems has enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransf erase 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).

[00224] In some cases, a CasX fusion partner utilized in the XDP systems 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).

[00225] Examples of proteins (or fragments thereof) that can be used as a CasX fusion partner utilized in the XDP systems to increase transcription include but are not limited to: transcriptional activators such as VP 16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, 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, ACTR, PI 60, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) di oxygenase 1 (TET1CD), TET1, DME, DML1, DML2,

ROS1, and the like.

[00226] Examples of proteins (or fragments thereof) that can be used as a CasX fusion partner in an XDP to decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); KOX1 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 methyltransferases such as Pr-SET7/8, SUV4- 20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID 1 A/RBP2, JARIDlB/PLU-1, JARID 1C/SMCX, JARIDID/SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[00227] In some cases, the CasX fusion partner utilized in the XDP 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., Fokl nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c- methyltransf erase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), 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) di oxygenase 1 (TET 1 CD), TET1, DME, DMLl, 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 APOBECl), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and/or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; 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).

[00228] In other cases, CasX variant protein of the present disclosure utilized in the XDP systems is fused to a polypeptide selected from: a domain for increasing transcription (e.g., a VP 16 domain, a VP64 domain), a domain for decreasing transcription (e.g., a KRAB domain, e.g., from the Koxl protein), a core catalytic domain of a histone acetyltransferase (e.g., histone acetyltransferase p300), a protein/domain that provides a detectable signal (e.g., a fluorescent protein such as GFP), a nuclease domain (e.g., a Fokl nuclease), and a base editor (e.g., cytidine deaminase such as APOBECl).

[00229] In still other cases, the CasX fusion partner utilized in the XDP systems has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the fusion partner include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT 1C and EHMT2), SUV39H2, ESET/SETDB 1, and the like, SET1A, SET IB, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8, SUV4- 20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARED 1 A/RBP2, JARIDlB/PLU-1, JARID1C/SMCX, JARIDID/SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core/fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HB01/MYST2, ITMOF/MYST1, SRC1, ACTR, PI 60, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., ITDAC1, ITDAC2, ITDAC3, ITDAC8, HDAC4, ITDAC5, ITDAC7, HDAC9, SIRTl, SIRT2, ITDAC11, and the like), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

[00230] Suitable chloroplast transit peptides include, but are not limited to:

M ASMIS S S AVTT V SRASRGQ S AAM APF GGLKSMT GFPVRKVNTDIT SIT SNGGR VKCMQVWPPIGKKKFETLSYLPPLTRDSRA (SEQ ID NO: 116);

M ASMIS S S AVTT V SRASRGQ SAAMAPF GGLKSMT GFPVRKVNTDIT SIT SNGGRVKS (SEQ ID NO: 117);

M AS SMLS S ATM VASP AQ ATM VAPFNGLK S S AAFP ATRK ANNDIT SIT SNGGRVNCMQ V WPPIEKKKFETL S YLPDLTD S GGRVN C (SEQ ID NO: 118;

M AQ V SRICN GV QNP SLISNL SK S S QRK SPL S V SLKTQQHPRA YPI S S S W GLKK S GMTLIG SELRPLKVMSSVSTAC (SEQ ID NO: 119);

M AQ V SRICN GVWNP SLISNL SK S S QRK SPL S V SLKTQQHPRA YPI S S S W GLKK S GMTLIG SELRPLKVMSSVSTAC (SEQ ID NO: 120);

M AQINNM AQGIQTLNPN SNFHKPQ VPKS S SFL VF GSKKLKN SAN SMLVLKKD SIFMQLF CSFRISASVATAC (SEQ ID NO: 121);

M AAL VT SQL AT SGTVL S VTDRFRRPGF QGLRPRNP AD AALGMRT VGAS A APKQ SRKPH RFDRRCL SM V V (SEQ ID NO: 122);

MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQ QRS VQRGSRRFP S VVV C (SEQ ID NO: 123);

MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQC (SEQ ID NO: 124);

MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVKCSAAVTPQASPVIS RSAAAA (SEQ ID NO: 125); and

MGA A AT SMQ SLKF SNRL VPP SRRL SP VPNN VT CNNLPK S AAP VRT VKC CAS S WN S TIN G AAATTNGASAASS (SEQ ID NO: 126).

[00231] 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: 127), 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: 128), or HHHHHHHHH (SEQ ID NO: 129).

[00232] Non-limiting examples of CasX fusion partners for use when targeting ssRNA target nucleic acids include (but are not limited to): splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; RNA-binding proteins; and the like. It is understood that a heterologous polypeptide can include the entire protein or in some cases can include a fragment of the protein (e.g., a functional domain).

[00233] A fusion partner can be any domain capable of interacting with ssRNA (which, for the purposes of this disclosure, includes intramolecular and/or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.), whether transiently or irreversibly, directly or indirectly, including but not limited to an effector domain selected from the group comprising; endonucleases (for example RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (for example CPSF, CstF, CFIm and CFIIm); exonucleases (for example XRN-1 or Exonuclease T); deadenylases (for example HNT3); proteins and protein domains responsible for nonsense mediated RNA decay (for example UPF1, UPF2, UPF3, UPF3b, RNP SI, Y14, DEK, REF2, and SRml60); proteins and protein domains responsible for stabilizing RNA (for example PABP); proteins and protein domains responsible for repressing translation (for example Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (for example Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (for example PAPl, GLD-2, and Star- PAP); proteins and protein domains responsible for polyuridinylation of RNA (for example Cl D1 and terminal uridylate transferase); proteins and protein domains responsible for RNA localization (for example from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (for example Rrp6); proteins and protein domains responsible for nuclear export of RNA (for example TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains responsible for repression of RNA splicing (for example PTB, Sam68, and hnRNP Al); proteins and protein domains responsible for stimulation of RNA splicing (for example serine/arginine-rich (SR) domains); proteins and protein domains responsible for reducing the efficiency of transcription (for example FUS (TLS)); and proteins and protein domains responsible for stimulating transcription (for example CDK7 and HIV Tat). Alternatively, the effector domain may be selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains having nonsense mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is a PUF RNA-binding domain, which is described in more detail in WO2012068627, which is hereby incorporated by reference in its entirety.

[00234] Some RNA splicing factors that can be used (in whole or as fragments thereof) as a CasX fusion partners in the XDP systems have modular organization, with separate sequence- specific RNA binding modules and splicing effector domains. For example, members of the serine/arginine-rich (SR) protein family contain N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP protein hnRNP A1 binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C- terminal glycine-rich domain. Some splicing factors can regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites. For example, ASF/SF2 can recognize ESEs and promote the use of intron proximal sites, whereas hnRNP A1 can bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions. The long splicing isoform Bcl- xL is a potent apoptosis inhibitor expressed in long-lived post mitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bcl-xS is a pro- apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). The ratio of the two Bcl-x splicing isoforms is regulated by multiple cis -elements that are located in either the core exon region or the exon extension region (i.e., between the two alternative 5' splice sites). For more examples, see W02010075303, which is hereby incorporated by reference in its entirety.

[00235] Further suitable CasX fusion partners utilized in the XDP systems include, but are not limited to, proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.).

[00236] 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).

[00237] In some cases, a CasX variant protein for use in the XDP 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. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C- terminus. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus. In some cases, a CasX variant protein includes (is fused to) between 1 and 10 NLSs (e.g., 1-9, 1- 8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2- 6, or 2-5 NLSs). In some cases, a CasX variant protein includes (is fused to) between 2 and 5 NLSs (e.g., 2-4, or 2-3 NLSs).

[00238] Non-limiting examples of NLSs include sequences derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 130); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 131); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 132) or RQRRNELKRSP (SEQ ID NO: 133); the hRNPAl M9 NLS having the sequence NQ S SNF GPMKGGNF GGRS S GP Y GGGGQ YF AKPRN Q GGY (SEQ ID NO: 134); the sequence

RMRIZFKNKGKDTAELRRRRVEV S VELRKAKKDEQILKRRNV (SEQ ID NO: 135) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 136) and PPKKARED (SEQ ID NO: 137) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 138) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 139) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 140) and PKQKKRK (SEQ ID NO: 141) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 142) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 143) of the mouse Mxl protein; the sequence KRKGDE VDGVDE V AKKK SKK (SEQ ID NO: 144) of the human poly(ADP-ribose) polymerase; the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 145) of the steroid hormone receptors (human) glucocorticoid; the sequence PRPRKIPR (SEQ ID NO: 146) of Borna disease virus P protein (BDV-P1); the sequence PPRKKRTVV (SEQ ID NO: 147) of hepatitis C virus nonstructural protein (HCV-NS5A); the sequence NLSKKKKRKREK (SEQ ID NO: 148) of LEF1; the sequence RRPSRPFRKP (SEQ ID NO: 149) of ORF57 simirae; the sequence KRPRSPSS (SEQ ID NO: 150) of EBV LANA; the sequence KRGINDRNFWRGENERKTR (SEQ ID NO: 151) of Influenza A protein; the sequence PRPPKMARYDN (SEQ ID NO: 152) of human RNA helicase A (RHA); the sequence KRSFSKAF (SEQ ID NO: 153) of nucleolar RNA helicase II; the sequence KLKIKRPVK (SEQ ID NO: 154) of TUS-protein; the sequence PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 155) associated with importin-alpha; the sequence PKTRRRPRRSQRKRPPT (SEQ ID NO: 156) from the Rex protein in HTLV-1; the sequence MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 157) from the EGL-13 protein of Caenorhabditis elegans; and the sequences KTRRRPRRSQRKRPPT (SEQ ID NO: 158), RRKKRRPRRKKRR (SEQ ID NO: 159),

PKKK SRKPKKK SRK (SEQ ID NO: 160), HKKKHPD AS VNF SEF SK (SEQ ID NO: 161), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 162), LSPSLSPLLSPSLSPL (SEQ ID NO: 163), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 164), PKRGRGRPKRGRGR (SEQ ID NO: 165), and PKKKRKVPPPPKKKRKV (SEQ ID NO: 166). In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of a reference or 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 reference or 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. [00239] In some cases, a reference or 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 reference or CasX variant fusion protein. In some cases, the PTD is inserted internally in the sequence of a reference or CasX variant fusion protein at a suitable insertion site. In some cases, a reference or 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 Y GRKKRRQRRR (SEQ ID NO: 167), RKKRRQRR (SEQ ID NO: 168); YARAAARQARA (SEQ ID NO: 169); THRLPRRRRRR (SEQ ID NO: 170); and GGRRARRRRRR (SEQ ID NO: 171); a polyarginine 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: 172)); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an 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); RRQRRT SKLMKR (SEQ ID NO: 173); Transportan GWTLN S AGYLLGRINLR AL AAL ARRIL (SEQ ID NO: 174);

RALAWE ARL ARAL ARAL ARHL ARAL ARALRCEA (SEQ ID NO: 175); and RQIRIWFQNRRMRWRR (SEQ ID NO: 176). 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 polycationic 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 polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus "activating" the ACPP to traverse the membrane. [00240] In some embodiments, a reference or CasX variant fusion protein can include a CasX protein that is linked to an internally inserted heterologous amino acid or heterologous polypeptide (a heterologous amino acid sequence) via a linker polypeptide (e.g., one or more linker polypeptides). In some embodiments, a reference or CasX variant fusion protein can be linked at the C-terminal and/or N-terminal end to a heterologous polypeptide (fusion partner) 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. These linkers are generally produced by using synthetic, linker encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine 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, glycine-serine polymer (including, for example, (GS)n, GSGGSn (SEQ ID NO: 177), GGSGGSn (SEQ ID NO: 178), and GGGSn (SEQ ID NO: 179), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, glycine-proline polymers, proline polymers and proline-alanine polymers. Example linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 180), GGSGG (SEQ ID NO: 181), GSGSG (SEQ ID NO: 182), GSGGG (SEQ ID NO: 183), GGGSG (SEQ ID NO: 184), GSSSG (SEQ ID NO: 185),GPGP (SEQ ID NO: 186), GGP, PPP, PPAPPA (SEQ ID NO: 187), PPPGPPP (SEQ ID NO: 188) and the like. 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. h. Guide Nucleic Acids of XDP Systems

[00241] In another aspect, the disclosure relates to XDP system components that encode or incorporate guide nucleic acids (gNA) of the CasX:gNA systems wherein the gNA comprises a targeting sequence engineered to be complementary to a target nucleic acid sequence to be edited. In some embodiments, the gNA is capable of forming a complex with a CRISPR protein that has specificity to a protospacer adjacent motif (PAM) sequence comprising a TC motif in the complementary non-target strand, and wherein the PAM sequence is located 1 nucleotide 5’ of the sequence in the non-target strand that is complementary to the target nucleic acid sequence in the target strand of the target nucleic acid. In some embodiments, the gNA is capable of forming a complex with a Class 2, Type V CRISPR nuclease. In a particular embodiment, the gNA is capable of forming a complex with a CasX nuclease.

[00242] Reference, or naturally-occurring gNA include, but are not limited to those isolated or derived from Deltaproteobacter , Planctomycetes, or Candidatus (as described in US20180346927A1 and WO2018064371A1, incorporated herein by reference), including the sequences of Table 2. In some embodiments of the XDP systems, the disclosure provides gNA variants having one or more modifications relative to a naturally-occurring gNA, the modified gNA hereinafter referred to as a “gNA variant”. In some cases, the encoded gNA variant comprises or consists of a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25 mutations relative to the sequence of a reference gNA.

These mutations can be insertions, deletions, nucleotide substitutions, or any combinations thereof. In some embodiments, the gNA variant is a ribonucleic acid molecule (“gRNA”). In other embodiments, the gNA variant is a deoxyribonucleic acid molecule (“gDNA”) in which uridine nucleotides have been replaced with thymidine. In some embodiments, the gNA is a chimera, and comprises both DNA and RNA.

[00243] It is envisioned that in some embodiments of the XDP system, multiple gNAs (e.g., two, three, four or more gNA) are delivered to the target cells or tissues in the XDP particles for the modification of a target nucleic acid. For example, when a deletion of a protein-encoding gene and/or regulatory element is desired, a pair of gNAs with targeting sequences to different regions of the target nucleic acid can be used in order to bind and cleave at two different sites within the gene or regulatory element, 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). For example, when an editing event designed to delete one or more mutant exons or a sequence of the target nucleic acid having two or more mutations that are distal to one another, a pair of gNAs can be incorporated into the XDP such that the CRISPR nuclease can bind and cleave at two different sites 5’ and 3’ of the exon(s) bearing the mutation(s) within the gene. In the context of nucleic acids, cleavage refers to the breakage of the covalent backbone of a nucleic acid molecule; either DNA or RNA, by the nuclease. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. In some embodiments, small indels introduced by the CasX:gNA systems of the embodiments described herein and cellular repair systems can restore the protein reading frame of the mutant gene (“refraining” strategy). When the refraining strategy is used, the cells may be contacted with a single gNA. In the case of deleting a long segment of the gene, the disclosure contemplates use of targeting sequences that flank the segment 5’ and 3’ such that it can be deleted or replaced with a donor template having the correct sequence. In other cases, when a deletion or a knock-down/knock-out of the HTT gene is desired, a pair of gNAs with targeting sequences to different or overlapping regions of the target nucleic acid sequence can be used in order to bind and the CasX to cleave at two different or overlapping sites within or proximal to the exon or regulatory element of the gene, which is then edited by non-homologous end joining (NHEJ), homology-directed repair (HDR, which can include, for example, insertion of a donor template to replace all or a portion of an HTT exon), homology-independent targeted integration (HITI), micro-homology mediated end joining (MMEJ), single strand annealing (SSA) or base excision repair (BER).

[00244] The gNA variants of the disclosure can be designed and created by a number of mutagenesis methods, which may include Deep Mutational Evolution (DME) (as described in U.S. patent application serial number PCT/US20/36506, incorporated by reference, herein), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping, in order to generate one or more gNA variants with enhanced or varied properties relative to the reference gNA. The activity of reference gNAs may be used as a benchmark against which the activity of gNA variants are compared, thereby measuring improvements in function or other characteristics of the gNA variants. In other embodiments, a reference gNA may be subjected to one or more deliberate, targeted mutations in order to produce a gNA variant, for example a rationally designed variant. [00245] The gNAs of the disclosure comprise two segments: a targeting sequence and a protein-binding segment. The targeting segment of a gNA includes a nucleotide sequence (referred to interchangeably as a guide sequence, a spacer, a targeter, or a targeting sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within the target nucleic acid sequence (e.g., a target ssRNA, a target ssDNA, a strand of a double stranded target DNA, etc.), described more fully below. The targeting sequence of a gNA is capable of binding to a target nucleic acid sequence, including a coding sequence, a complement of a coding sequence, a non-coding sequence, and to regulatory elements. The protein-binding segment (or “activator” or “protein-binding sequence”) interacts with (e.g., binds to) a CasX protein as a complex, forming an RNP (described more fully, below). The protein-binding segment is alternatively referred to herein as a “scaffold”, which is comprised of several regions, described more fully, below.

[00246] In the case of a dual guide RNA (dgRNA), the targeter and the activator portions each have a duplex-forming segment, where the duplex forming segment of the targeter and the duplex-forming segment of the activator have complementarity with one another and hybridize to one another to form a double stranded duplex (dsRNA duplex for a gRNA). When the gNA is 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. Thus, for example, a guide RNA (dgRNA or sgRNA) comprises a guide sequence and a duplex-forming segment of a crRNA, which can also be referred to as a crRNA repeat. A corresponding tracrRNA-like molecule (activator) also comprises a duplex-forming stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the guide RNA. Thus, a targeter and an activator, as a corresponding pair, hybridize to form a dual guide NA, referred to herein as a “dual guide NA”, a “dual-molecule gNA”, a “dgNA”, a “double-molecule guide NA”, or a “two-molecule guide NA”. 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 gNA and the target nucleic acid sequence. Thus, for example, the gNA 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 PAM motif or a PAM sequence, such as ATC, CTC, GTC, or TTC. Because the targeting sequence of a guide sequence hybridizes with a sequence of a target nucleic acid sequence, a targeter 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. Thus, in some cases, the sequence of a targeter may be a non-naturally occurring sequence. In other cases, the sequence of a targeter may be a naturally-occurring sequence, derived from the gene to be edited. In other embodiments, the activator and targeter of the gNA are covalently linked to one another (rather than hybridizing to one another) and comprise a single molecule, referred to herein as a “single-molecule gNA,” “one-molecule guide NA,” “single guide NA”, “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, a “single guide DNA”, a “single-molecule DNA”, or a “one-molecule guide DNA”, (“sgNA”, “sgRNA”, or a “sgDNA”). In some embodiments, the sgNA includes an “activator” or a “targeter” and thus can be an “activator-RNA” and a “targeter-RNA,” respectively.

[00247] Collectively, the assembled gNAs of the disclosure comprise four distinct regions, or domains: the RNA triplex, the scaffold stem, the extended stem, 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 gNA. The RNA triplex, the scaffold stem, and the extended stem, together, are referred to as the “scaffold” of the gNA. i. RNA Triplex

[00248] In some embodiments of the guide NAs provided herein (including reference sgNAs), there is a RNA-triplex, and the RNA triplex comprises the sequence of a UUU— nX(~4-15)— UUU stem loop (SEQ ID NO: 189) that ends with an AAAG after 2 intervening stem loops (the scaffold stem loop and the extended stem loop), forming a pseudoknot that may also extend past the triplex into a duplex pseudoknot. The UU-UUU-AAA sequence of the triplex forms as a nexus between the spacer, scaffold stem, and extended stem. In exemplary reference CasX sgNAs, the UUU-loop-UUU region is coded for first, then the scaffold stem loop, and then the extended stem loop, which is linked by the tetraloop, and then an AAAG closes off the triplex before becoming the spacer. j. Scaffold Stem Loop

[00249] In some embodiments of CasX sgNAs of the disclosure, the triplex region is followed by the scaffold stem loop. The scaffold stem loop is a region of the gNA that is bound by CasX protein (such as a reference or CasX variant protein). In some embodiments, the scaffold stem loop is a fairly short and stable stem loop. In some cases, the scaffold stem loop does not tolerate many changes, and requires some form of an RNA bubble. In some embodiments, the scaffold stem is necessary for CasX sgNA function. While it is perhaps analogous to the nexus stem of Cas9 as being a critical stem loop, the scaffold stem of a CasX sgNA, in some embodiments, has a necessary bulge (RNA bubble) that is different from many other stem loops found in CRISPR/Cas systems. In some embodiments, the presence of this bulge is conserved across sgNA that interact with different CasX proteins. An exemplary sequence encoding a scaffold stem loop sequence of a gNA comprises the sequence CCAGCGACTATGTCGTATGG (SEQ ID NO: 190). In other embodiments, the disclosure provides gNA variants wherein the scaffold stem loop is replaced with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends, such as, but not limited to stem loop sequences designated as MS2, Q b, U1 hairpin II, Uvsx, or PP7 stem loops, which can be used, in some cases, to facilitate transport out of the host cell nucleus. In some cases, the heterologous RNA stem loop of the gNA is capable of binding a protein, an RNA structure, a DNA sequence, or a small molecule, which can facilitate the binding of gNA to CasX. k. Extended Stem Loop

[00250] In some embodiments of the sgNAs of the disclosure, the scaffold stem loop is followed by the extended stem loop. In some embodiments, the extended stem comprises a synthetic tracr and crRNA fusion that is largely unbound by the CasX protein. In some embodiments, the extended stem loop can be highly malleable. In some embodiments, a single guide gRNA is made with a GAAA tetraloop linker or a GAGAAA linker between the tracr and crRNA in the extended stem loop. In some cases, the targeter and activator of a CasX sgNA are linked to one another by intervening nucleotides and the linker can have a length of from 3 to 20 nucleotides. In some embodiments of the CasX sgNAs of the disclosure, the extended stem is a large 32-bp loop that sits outside of the CasX protein in the ribonucleoprotein complex. An exemplary sequence encoding an extended stem loop sequence of a sgNA comprises GCGCTT ATTT ATCGGAGAGAAATCCGAT AAAT AAGAAGC (SEQ ID NO: 191). In some embodiments, the extended stem loop comprises a GAGAAA spacer sequence. In some embodiments, the disclosure provides gNA variants wherein the extended stem loop is replaced with an RNA stem loop sequence from a heterologous RNA source with proximal 5’ and 3’ ends, such as, but not limited to stem loop sequences designated MS2, QP, U1 hairpin II, Uvsx, or PP7 stem loops. In such cases, the heterologous RNA stem loop increases the stability of the gNA. In other embodiments, the disclosure provides gNA variants having an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,000 nucleotides, or at least 10-10,000, at least 10-1000, or at least 10-100 nucleotides. In some embodiments, the extended stem loop comprises a GAGAAA spacer sequence. 1. Targeting Sequence (a.k.a. Spacer)

[00251] In some embodiments of the gNAs of the disclosure utilized in the XDP systems, the extended stem loop is followed by a region that forms part of the triplex, and then the targeting sequence (or “spacer”) at the 3’ end of the gNA. 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, gNA targeting sequences of the disclosure have sequences complementarity to, and therefore can hybridize to, a portion of the HTT 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 gNA can be modified so that the gNA can target a desired sequence of any desired target nucleic acid sequence, so long as the PAM sequence location is taken into consideration. In some embodiments, the gNA scaffold is 5’ of the targeting sequence, with the targeting sequence on the 3’ end of the gNA. 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.

[00252] In some embodiments, the gNA of the XDP systems comprises a targeting sequence (a) complementary to a nucleic acid sequence encoding i) a target protein, which may be a wild-type sequence or may comprise one or more mutations or ii) the regulatory element of the protein, which may be a wild-type sequence; or (b) complementary to a complement of a nucleic acid sequence encoding a protein or its regulatory element, which may comprise one or more mutations. In some embodiments, the targeting sequence of the gNA 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 gNA is specific for a target gene exon. In some embodiments, the targeting sequence of a gNA is specific for a target gene intron. In some embodiments, the targeting sequence of the gNA is specific for a target gene intron-exon junction. In some embodiments, the targeting sequence of the gNA 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 gNA 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. [00253] In some embodiments, the targeting sequence of a gNA is specific for a regulatory element that regulates expression of a target gene. Such regulatory 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-regulatory 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 disclosure provides a gNA with a targeting sequence that hybridizes with target gene regulatory element. In the foregoing, the targets are those in which the encoding gene of the target is intended to be knocked out or knocked down such that the target protein comprising mutations is not expressed or is expressed at a lower level in a cell. In some embodiments, the disclosure provides a CasX:gNA system wherein the targeting sequence (or spacer) of the gNA is complementary to a nucleic acid sequence encoding the target protein, a portion of the target protein, a portion of a regulatory element, or the complement of a portion of a gene or a regulatory element for the target gene. In some embodiments, the targeting sequence has between 14 and 35 consecutive nucleotides. In some embodiments, the targeting sequence has 14, 15, 16, 18, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 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 nucleotides. In some embodiments, the targeting sequence consists of 15 nucleotides. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides and the targeting sequence can comprise 0 to 5, 0 to 4, 0 to 3, or 0 to 2 mismatches relative to the target nucleic acid sequence and retain sufficient binding specificity such that the RNP comprising the gNA comprising the targeting sequence can form a complementary bond with respect to the target nucleic acid.

[00254] In some embodiments, the CasX:gNA of the XDP system comprises a first gNA and further comprises a second (and optionally a third, fourth or fifth) gNA, wherein the second gNA has a targeting sequence complementary a different portion of the target nucleic acid or its complement compared to the targeting sequence of the first gNA. By selection of the targeting sequences of the gNA, defined regions of the target nucleic acid can be modified or edited using the CasX:gNA systems described herein. m. gNA scaffolds

[00255] With the exception of the targeting sequence region, the remaining regions of the gNA are referred to herein as the scaffold. In some embodiments, the gNA scaffolds are derived from naturally-occurring sequences, described below as reference gNA. In other embodiments, the gNA scaffolds are variants of reference gNA wherein mutations, insertions, deletions or domain substitutions are introduced to confer desirable properties on the gNA variant.

[00256] In some embodiments, a reference gRNA comprises a sequence isolated or derived from Deltaproteobacteria. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary CasX reference tracrRNA sequences isolated or derived from Deltaproteobacteria may include:

ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGU AUGGACGAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 6) and ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGU AUGGACGAAGCGCUUAUUUAUCGG (SEQ ID NO: 7). Exemplary crRNA sequences isolated or derived from Deltaproteobacter may comprise a sequence of CCGAUAAGUAAAACGCAUCAAAG (SEQ ID NO: 194). In some embodiments, a CasX reference gNA 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 or 100% identical to a sequence isolated or derived from Deltaproteobacter . In some embodiments, a reference guide RNA comprises a sequence isolated or derived from Planctomycetes. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary reference tracrRNA sequences isolated or derived from Planctomycetes may include: UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA UGGGUAAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 8) and

UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA UGGGUAAAGCGCUUAUUUAUCGG (SEQ ID NO: 9). Exemplary crRNA sequences isolated or derived from Planctomycetes may comprise a sequence of UCUCCGAUAAAUAAGAAGCAUCAAAG (SEQ ID NO: 197). In some embodiments, a CasX reference gNA 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 or 100% identical to a sequence isolated or derived from Planctomycetes.

[00257] In some embodiments, a reference gNA comprises a sequence isolated or derived from Candidatus Sungbacteria. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary CasX reference tracrRNA sequences isolated or derived from Candidatus Sungbacteria may comprise sequences of: GUUUACACACUCCCUCUCAUAGGGU (SEQ ID NO: 10), GUUUACACACUCCCUCUCAUGAGGU (SEQ ID M): 11), UUUUACAUACCCCCUCUCAUGGGAU (SEQ ID NO: 12) and

GUUU AC AC ACUCC CU CU C AU GGGGG (SEQ ID NO: 13). In some embodiments, a CasX reference guide RNA 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 or 100% identical to a sequence isolated or derived from Candidatus Sungbacteria. [00258] Table 2 provides the sequences of reference gRNAs tracr, cr and scaffold sequences.

In some embodiments, the disclosure provides gNA sequences wherein the gNA has a scaffold comprising a sequence having at least one nucleotide modification relative to a reference gNA sequence having a sequence of any one of SEQ ID NOS: 4-16 of Table 2. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gNA, or where a gNA is a gDNA or a chimera of RNA and DNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gNA sequence embodiments described herein, including the sequences of Table 2 and Table 3.

Table 2. Reference gRNA tracr and scaffold sequences n. gNA Variants

[00259] In another aspect, the disclosure relates to guide nucleic acid variants (referred to herein alternatively as “gNA variant” or “gRNA variant” when the nucleic acid variant comprises RNA), which comprise one or more modifications relative to a reference gRNA scaffold. As used herein, “scaffold” refers to all parts to the gNA necessary for gNA function with the exception of the spacer sequence.

[00260] In some embodiments, a gNA variant comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced regions relative to a reference gRNA sequence of the disclosure. In some embodiments, a mutation can occur in any region of a reference gRNA to produce a gNA variant. In some embodiments, the scaffold of the gNA variant sequence has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. [00261] In some embodiments, a gNA variant comprises one or more nucleotide changes within one or more regions of the reference gRNA that improve a characteristic relative to the reference gRNA. Exemplary regions 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 gNA variant scaffold comprises a scaffold stem loop having at least 60% sequence identity to SEQ ID NO:

14. In another embodiment, the gNA variant comprises a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 202). In another embodiment, the disclosure provides a gNA scaffold comprising, relative to SEQ ID NO:5, a C18G 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 gNA scaffold comprises the sequence

ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG U GGGU A A AGCU C C CU CUU C GG AGGG AGC AU C A A AG (SEQ ID NO: 734).

[00262] All gNA variants that have one or more improved functions or characteristics, or add one or more new functions when the variant gNA is compared to a reference gRNA described herein, are envisaged as within the scope of the disclosure. A representative example of such a gNA variant is guide 174 (SEQ ID NO: 734). In some embodiments, the gNA variant adds a new function to the RNP comprising the gNA variant. In some embodiments, the gNA variant has an improved characteristic selected from: improved stability; improved solubility; improved transcription of the gNA; improved resistance to nuclease activity; increased folding rate of the gNA; decreased side product formation during folding; increased productive folding; improved binding affinity to a CasX protein; improved binding affinity to a target DNA when complexed with a CasX protein; improved gene editing when complexed with a CasX protein; improved specificity of editing when complexed with a CasX protein; and improved ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target DNA when complexed with a CasX protein, or any combination thereof. In some cases, the one or more of the improved characteristics of the gNA variant is at least about 1.1 to about 100,000-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. In other cases, the one or more improved characteristics of the gNA variant is at least about 1.1, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000-fold or more improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. In other cases, the one or more of the improved characteristics of the gNA variant is about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250- fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. In other cases, the one or more improved characteristics of the gNA variant is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280- fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00263] In some embodiments, a gNA variant can be created by subjecting a reference gRNA to a one or more mutagenesis methods, such as the mutagenesis methods described herein, below, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping, in order to generate the gNA variants of the disclosure. The activity of reference gRNAs may be used as a benchmark against which the activity of gNA variants are compared, thereby measuring improvements in function of gNA variants. In other embodiments, a reference gRNA may be subjected to one or more deliberate, targeted mutations, substitutions, or domain swaps in order to produce a gNA variant, for example a rationally designed variant. Exemplary gRNA variants produced by such methods are described in the Examples and representative sequences of gNA scaffolds are presented in Table 3.

[00264] In some embodiments, the gNA variant comprises one or more modifications compared to a reference guide nucleic acid scaffold sequence, wherein the one or more modification is selected from: at least one nucleotide substitution in a region of the gNA variant; at least one nucleotide deletion in a region of the gNA variant; at least one nucleotide insertion in a region of the gNA variant; a substitution of all or a portion of a region of the gNA variant; a deletion of all or a portion of a region of the gNA variant; or any combination of the foregoing.

In some cases, the modification is a substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions. In other cases, the modification is a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions. In other cases, the modification is an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions. In other cases, the modification is a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends. In some cases, a gNA variant of the disclosure comprises two or more modifications in one region. In other cases, a gNA variant of the disclosure comprises modifications in two or more regions. In other cases, a gNA variant comprises any combination of the foregoing modifications described in this paragraph.

[00265] In some embodiments, a 5' G is added to a gNA variant sequence for expression in vivo, as transcription from a U6 promoter is more efficient and more consistent with regard to the start site when the +1 nucleotide is a G. In other embodiments, two 5' Gs are added to a gNA variant sequence for in vitro transcription to increase production efficiency, as T7 polymerase strongly prefers a G in the +1 position and a purine in the +2 position. In some cases, the 5’ G bases are added to the reference scaffolds of Table 2. In other cases, the 5’ G bases are added to the variant scaffolds of Table 3. [00266] Table 3 provides exemplary gNA variant scaffold sequences of the disclosure. In Table 3, (-) indicates a deletion at the specified position(s) relative to the reference sequence of SEQ ID NO: 5, (+) indicates an insertion of the specified base(s) at the position indicated relative to SEQ ID NO: 5, (:) indicates the range of bases at the specified starristop coordinates of a deletion or substitution relative to SEQ ID NO: 5, and multiple insertions, deletions or substitutions are separated by commas; e.g., A14C, T17G. In some embodiments, the gNA variant scaffold comprises any one of the sequences listed in Table 3, or SEQ ID NOS: 597-781, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gNA, or where a gNA is a gDNA or a chimera of RNA and DNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gNA sequence embodiments described herein.

Table 3. Exemplary gNA Variant Scaffold Sequences

[00267] In some embodiments, the gNA variant comprises a tracrRNA stem loop comprising the sequence -UUU-N4-25UUU- (SEQ ID NO: 203). For example, the gNA variant comprises a scaffold stem loop or a replacement thereof, flanked by two triplet U motifs that contribute to the triplex region. In some embodiments, the scaffold stem loop or replacement there of comprises at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides.

[00268] In some embodiments, the gNA variant comprises a crRNA sequence with -AAAG- in a location 5’ to the spacer region. In some embodiments, the -AAAG- sequence is immediately 5’ to the spacer region.

[00269] In some embodiments, the at least one nucleotide modification comprises at least one nucleotide deletion in the CasX variant gNA relative to the reference gRNA. In some embodiments, a gNA variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19 or 20 consecutive or non-consecutive nucleotides relative to a reference gRNA. In some embodiments, the at least one deletion comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gRNA. In some embodiments, the gNA variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotide deletions relative to the reference gRNA, and the deletions are not in consecutive nucleotides. In those embodiments where there are two or more non-consecutive deletions in the gNA variant relative to the reference gRNA, any length of deletions, and any combination of lengths of deletions, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA variant may comprise a first deletion of one nucleotide, and a second deletion of two nucleotides and the two deletions are not consecutive. In some embodiments, a gNA variant comprises at least two deletions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two deletions in the same region of the reference gRNA. For example, the regions may be the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5’ end of the gNA variant. Any deletion of any nucleotide in a reference gRNA is contemplated as within the scope of the disclosure.

[00270] In some embodiments, the at least one nucleotide modification comprises at least one nucleotide insertion. In some embodiments, a gNA variant comprises an insertion of 1, 2, 3, 4, 5,

6, 7, 8, 9 or 10 consecutive or non-consecutive nucleotides relative to a reference gRNA. In some embodiments, the at least one nucleotide insertion comprises an insertion of 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gRNA. In some embodiments, the gNA variant comprises 2 or more insertions relative to the reference gRNA, and the insertions are not consecutive. In those embodiments where there are two or more non-consecutive insertions in the gNA variant relative to the reference gRNA, any length of insertions, and any combination of lengths of insertions, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA variant may comprise a first insertion of one nucleotide, and a second insertion of two nucleotides and the two insertions are not consecutive. In some embodiments, a gNA variant comprises at least two insertions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two insertions in the same region of the reference gRNA. For example, the regions may be the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5’ end of the gNA variant. Any insertion of A, G, C, U (or T, in the corresponding DNA) or combinations thereof at any location in the reference gRNA is contemplated as within the scope of the disclosure.

[00271] In some embodiments, the at least one nucleotide modification comprises at least one nucleic acid substitution. In some embodiments, a gNA variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive or non-consecutive substituted nucleotides relative to a reference gRNA. In some embodiments, a gNA variant comprises 1-4 nucleotide substitutions relative to a reference gRNA. In some embodiments, the at least one substitution comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gRNA. In some embodiments, the gNA variant comprises 2 or more substitutions relative to the reference gRNA, and the substitutions are not consecutive. In those embodiments where there are two or more non- consecutive substitutions in the gNA variant relative to the reference gRNA, any length of substituted nucleotides, and any combination of lengths of substituted nucleotides, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA variant may comprise a first substitution of one nucleotide, and a second substitution of two nucleotides and the two substitutions are not consecutive. In some embodiments, a gNA variant comprises at least two substitutions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two substitutions in the same region of the reference gRNA. For example, the regions may be the triplex, the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5’ end of the gNA variant. Any substitution of A, G, C, U (or T, in the corresponding DNA) or combinations thereof at any location in the reference gRNA is contemplated as within the scope of the disclosure.

[00272] Any of the substitutions, insertions and deletions described herein can be combined to generate a gNA variant of the disclosure. For example, a gNA variant can comprise at least one substitution and at least one deletion relative to a reference gRNA, at least one substitution and at least one insertion relative to a reference gRNA, at least one insertion and at least one deletion relative to a reference gRNA, or at least one substitution, one insertion and one deletion relative to a reference gRNA.

[00273] In some embodiments, the gNA variant comprises a scaffold region at least 20% identical, at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% 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, or at least 99% identical to any one of SEQ ID NOS: 4-16. In some embodiments, the gNA variant comprises a scaffold region at least 60% homologous (or identical) to any one of SEQ ID NOS: 4-16.

[00274] In some embodiments, the gNA variant comprises a tracr stem loop at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% 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, or at least 99% identical to SEQ ID NO:

14. In some embodiments, the gNA variant comprises a tracr stem loop at least 60% homologous (or identical) to SEQ ID NO: 14.

[00275] In some embodiments, the gNA variant comprises an extended stem loop at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% 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, or at least 99% identical to SEQ ID NO:

15. In some embodiments, the gNA variant comprises an extended stem loop at least 60% homologous (or identical) to SEQ ID NO: 15.

[00276] In some embodiments, the gNA variant comprises an exogenous extended stem loop, with such differences from a reference gNA 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 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1,000 bp, at least 2,000 bp, at least 3,000 bp, at least 4,000 bp, at least 5,000 bp, at least 6,000 bp, at least 7,000 bp, at least 8,000 bp, at least 9,000 bp, at least 10,000 bp, at least 12,000 bp, at least 15,000 bp or at least 20,000 bp. In some embodiments, the gNA variant comprises an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,000 nucleotides. In some embodiments, the heterologous stem loop increases the stability of the gNA. 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 comprises an RNA stem loop or hairpin, for example a thermostable RNA such as MS2 (ACAUGAGGAUUACCCAUGU (SEQ ID NO: 204)), Qp (UGCAUGUCUAAGACAGCA (SEQ ID NO: 205)), U1 hairpin II

(AAUCCAUUGCACUCCGGAUU (SEQ ID NO: 206)), Uvsx (CCUCUUCGGAGG (SEQ ID NO: 207)), PP7 ( AGG AGUUU CU AU GG A A AC C CU (SEQ ID NO: 208)), Phage replication loop (AGGUGGGACGACCUCUCGGUCGUCCUAUCU (SEQ ID NO: 209)), Kissing loop a (UGCUCGCUCCGUUCGAGCA (SEQ ID NO: 210)), Kissing loop bl (UGCUCGACGCGUCCUCGAGCA (SEQ ID NO: 211)), Kissing loop_b2 (UGCUCGUUUGCGGCUACGAGCA (SEQ ID NO: 212)), G quadriplex M3q (AGGGAGGGAGGGAGAGG (SEQ ID NO: 213)), G quadriplex telomere basket (GGUUAGGGUUAGGGUUAGG (SEQ ID NO: 214)), Sarcin-ricin loop (CUGCUCAGUACGAGAGGAACCGCAG (SEQ ID NO: 215)) or Pseudoknots (UACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUAUACUUUGG AGUUUUAAAAUGUCUCUAAGUACA (SEQ ID NO: 216)). In some embodiments, an exogenous stem loop comprises a long non-coding RNA (lncRNA). As used herein, a lncRNA refers to a non-coding RNA that is longer than approximately 200 bp in length. In some embodiments, the 5’ and 3’ ends of the exogenous stem loop are base paired; i.e., interact to form a region of duplex RNA. In some embodiments, the 5’ and 3’ ends of the exogenous stem loop are base paired, and one or more regions between the 5’ and 3’ ends of the exogenous stem loop are not base paired. In some embodiments, the at least one nucleotide modification comprises: (a) substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (b) a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (c) an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (d) a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends; or any combination of (a)-(d).

[00277] In some embodiments, the gNA variant comprises a scaffold stem loop sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 202). In some embodiments, the gNA variant comprises a scaffold stem loop sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 202) and at least 1, 2, 3, 4, or 5 mismatches thereto.

[00278] In some embodiments, the gNA variant comprises an extended stem loop region comprising less than 32 nucleotides, less than 31 nucleotides, less than 30 nucleotides, less than 29 nucleotides, less than 28 nucleotides, less than 27 nucleotides, less than 26 nucleotides, less than 25 nucleotides, less than 24 nucleotides, less than 23 nucleotides, less than 22 nucleotides, less than 21 nucleotides, or less than 20 nucleotides. In some embodiments, the gNA variant comprises an extended stem loop region comprising less than 32 nucleotides. In some embodiments, the gNA variant further comprises a thermostable stem loop. [00279] In some embodiments, the gNA comprises an RNA binding domain. The RNA binding domain can be a retroviral Psi packaging element inserted into the gNA or is a stem loop with affinity to a protein selected from the group consisting of MS2, PP7, Qbeta, U1A, or phage R- loop, which can facilitate the binding of gNA to CasX. Similar RNA components with affinity to protein structures incorporated into the CasX include kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, and pseudoknots. It has been discovered that the incorporation of the Psi packaging element inserted into the guide RNA facilitates the packaging of the XDP particle due, in part, to the high affinity binding of Psi sequences for the Gag NC protein. Further, due to the affinity of the CasX for the gNA, resulting in an RNP, the incorporation of the RNP into the XDP is further facilitated.

[00280] In some embodiments, an sgRNA variant comprises a sequence of SEQ ID NOS: 597- 781 or a sequence having having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, an sgRNA variant comprises a sequence of SEQ ID NOS: 597-781. In some embodiments, an sgRNA variant comprises a sequence of SEQ ID NOS: 597-781 and a targeting sequence.

[00281] In some embodiments, a sgRNA variant comprises a sequence of SEQ ID NO: 600, SEQ ID NO: 602, SEQ ID NO: 659, SEQ ID NO: 603, SEQ ID NO: 660, SEQ ID NO: 661,

SEQ ID NO: 662, SEQ ID NO: 599, SEQ ID NO: 663, SEQ ID NO: 601, SEQ ID NO: 604,

SEQ ID NO: 608, SEQ ID NO: 656, SEQ ID NO: 666, SEQ ID NO: 610, SEQ ID NO: 667,

SEQ ID NO: 608, SEQ ID NO: 669, SEQ ID NO: 598, SEQ ID NO: 670, SEQ ID NO: 671,

SEQ ID NO: 605, SEQ ID NO: 672, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 736,

SEQ ID NO: 737, SEQ ID NO: 770, SEQ ID NO:771, SEQ ID NO: 775, or SEQ ID NO: 781. [00282] In some embodiments, the gNA variant comprises one or more additional changes to a sequence of any one of SEQ ID NOS: 732, 733, 734, 737, 740, 744, 745, or 755-781, or having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the gNA variant comprises one or more additional changes to a sequence of any one of SEQ ID NOs: 597-781. In some embodiments, the gNA variant comprises the sequence of any one of SEQ ID NOS:732, 733, 734, 737, 740, 744, 745, or 755-781. In some embodiments, the gNA variant scaffold consists of the sequence of any one of SEQ ID NOS:732, 733, 734, 737, 740, 744, 745, or 755-781, and further comprises a targeting sequence of any of the embodiments described herein.

[00283] In some embodiments, a sgRNA variant comprises one or more additional changes to a sequence of SEQ ID NO: 600, SEQ ID NO: 659, SEQ ID NO: 603, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 599, SEQ ID NO: 663, SEQ ID NO: 601, SEQ ID NO: 604, SEQ ID NO: 608, SEQ ID NO: 656, SEQ ID NO: 666, SEQ ID NO: 610, SEQ ID NO: 667, SEQ ID NO: 608, SEQ ID NO: 669, SEQ ID NO: 598, SEQ ID NO: 670, SEQ ID NO: 671,

SEQ ID NO: 605, SEQ ID NO: 672, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 736,

SEQ ID NO: 737, SEQ ID NO:770, SEQ ID NO:771, SEQ ID NO: 775, or SEQ ID NO: 781. [00284] In some embodiments of the gNA variants of the disclosure, the gNA variant comprises at least one modification, wherein the at least one modification compared to the reference guide scaffold of SEQ ID NO: 5 is selected from one or more of: (a) a C18G substitution in the triplex loop; (b) a G55 insertion in the stem bubble; (c) aUl deletion; (d) a modification of the extended stem loop wherein (i) a 6 nt loop and 13 loop-proximal base pairs are replaced by a Uvsx hairpin; and (ii) a deletion of A99 and a substitution of G65U that results in a loop-distal base that is fully base-paired. In some embodiments, the gNA variant comprises the sequence of any one of SEQ ID NOS: 732, 733, 734, 737, 740, 744, 745, or 755-781.

[00285] The gNA variants utilized in the XDP systems further comprises a spacer (or targeting sequence) region located at the 3’ end of the gNA, described more fully, supra, wherein the spacer is designed with a sequence that is complementary to a target nucleic acid to be edited. In some embodiments, the gNA variant comprises a targeting sequence of at least 14 to 30 nucleotides, wherein the sequence is complementary to the target nucleic acid to be edited. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides. In some embodiments, the gNA variant comprises a targeting sequence having 20 nucleotides. In some embodiments, the targeting sequence has 25 nucleotides. In some embodiments, the targeting sequence has 24 nucleotides.

In some embodiments, the targeting sequence has 23 nucleotides. In some embodiments, the targeting sequence has 22 nucleotides. In some embodiments, the targeting sequence has 21 nucleotides. In some embodiments, the targeting sequence has 20 nucleotides. In some embodiments, the targeting sequence has 19 nucleotides. In some embodiments, the targeting sequence has 18 nucleotides. In some embodiments, the targeting sequence has 17 nucleotides.

In some embodiments, the targeting sequence has 16 nucleotides. In some embodiments, the targeting sequence has 15 nucleotides. In some embodiments, the targeting sequence has 14 nucleotides. In some embodiments, the target nucleic acid comprises a PAM sequence located 5’ of the targeting sequence with at least a single nucleotide separating the PAM from the first nucleotide of the targeting sequence. In some embodiments, the PAM is located on the non- targeted strand of the target region, i.e. the strand that is complementary to the target nucleic acid. In some embodiments, the PAM sequence is a TC motif. In some embodiments, the PAM sequence is a ATC. In other embodiments, the PAM sequence is a TTC. In other embodiments, the PAM sequence is a GTC. In other embodiments, the PAM sequence is a CTC.

[00286] In some embodiments, the scaffold of the gNA variant is a variant comprising one or more additional changes to a sequence of a reference gRNA that comprises SEQ ID NO: 4 or SEQ ID NO: 5. In those embodiments where the scaffold of the reference gRNA is derived from SEQ ID NO: 4 or SEQ ID NO: 5, the one or more improved or added characteristics of the gNA variant are improved compared to the same characteristic in SEQ ID NO: 4 or SEQ ID NO: 5. [00287] In some embodiments of the XDP system, the scaffold of the gNA variant is part of an RNP with a CasX variant protein comprising any one of the sequences of SEQ ID NOS: 21- 233, 343-345, 350-353, 355-367 or 388-397, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In the foregoing embodiments, the gNA further comprises a targeting sequence. o. Chemically Modified gNA

[00288] In some embodiments, the disclosure relates to chemically-modified gNA. In some embodiments, the present disclosure provides a chemically-modified gNA that has guide RNA functionality and has reduced susceptibility to cleavage by a nuclease. A gNA that comprises any nucleotide other than the four canonical ribonucleotides A, C, G, and U, or a deoxynucleotide, is a chemically modified gNA. In some cases, a chemically-modified gNA comprises any backbone or internucleotide linkage other than a natural phosphodiester internucleotide linkage. In certain embodiments, the retained functionality includes the ability of the modified gNA to bind to a CasX of any of the embodiments described herein. In certain embodiments, the retained functionality includes the ability of the modified gNA to bind to a target nucleic acid sequence. In certain embodiments, the retained functionality includes targeting a CasX protein or the ability of a pre-complexed CasX protein-gNA to bind to a target nucleic acid sequence. In certain embodiments, the retained functionality includes the ability to nick a target polynucleotide by a CasX-gNA. In certain embodiments, the retained functionality includes the ability to cleave a target nucleic acid sequence by a CasX-gNA. In certain embodiments, the retained functionality is any other known function of a gNA in a CasX system with a CasX protein of the embodiments of the disclosure.

[00289] In some embodiments, the disclosure provides a chemically-modified gNA in which a nucleotide sugar modification is incorporated into the gNA selected from the group consisting of 2'-0 — Cl-4alkyl such as 2 '-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-0 — Cl -3 alkyl-0 — Cl- 3alkyl such as 2 '-m ethoxy ethyl (“2'-MOE”), 2'-fluoro (“2'-F”), 2'-amino (“2'-NH2”), 2'- arabinosyl (“2'-arabino”) nucleotide, 2'-F-arabinosyl (“2'-F-arabino”) nucleotide, 2'-locked nucleic acid (“LNA”) nucleotide, 2'-unlocked nucleic acid (“ULNA”) nucleotide, a sugar in L form (“L-sugar”), and 4'-thioribosyl nucleotide. In other embodiments, an internucleotide linkage modification incorporated into the guide RNA is selected from the group consisting of: phosphorothioate “P(S)” (P(S)), phosphonocarboxylate (P(CH2)nCOOR) such as phosphonoacetate “PACE” (P(CH2COO-)), thiophosphonocarboxylate ((S)P(CH2)nCOOR) such as thiophosphonoacetate “thioPACE” ((S)P(CH2)nCOO-)), alkylphosphonate (P(C1- 3alkyl) such as methylphosphonate — P(CH3), boranophosphonate (P(BH3)), and phosphorodithioate (P(S)2).

[00290] In certain embodiments, the disclosure provides a chemically-modified gNA in which a nucleobase (“base”) modification is incorporated into the gNA selected from the group consisting of: 2-thiouracil (“2-thioU”), 2-thiocytosine (“2-thioC”), 4-thiouracil (“4-thioU”), 6- thioguanine (“6-thioG”), 2-aminoadenine (“2-aminoA”), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5- methylcytosine (“5-methylC”), 5-methyluracil (“5-methylU”), 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5- ethynylcytosine, 5-ethynyluracil, 5-allyluracil (“5-allylU”), 5-allylcytosine (“5-allylC”), 5- aminoallyluracil (“5-aminoallylU”), 5-aminoallyl-cytosine (“5-aminoallylC”), an abasic nucleotide, Z base, P base, Unstructured Nucleic Acid (“UNA”), isoguanine (“isoG”), isocytosine (“isoC”), 5-methyl-2-pyrimidine, x(A,G,C,T) and y(A,G,C,T).

[00291] In other embodiments, the disclosure provides a chemically-modified gNA in which one or more isotopic modifications are introduced on the nucleotide sugar, the nucleobase, the phosphodiester linkage and/or the nucleotide phosphates, including nucleotides comprising one or more 15N, 13C, 14C, deuterium, 3H, 32P, 1251, 1311 atoms or other atoms or elements used as tracers.

[00292] In some embodiments, an “end” modification incorporated into the gNA is selected from the group consisting of: PEG (polyethyleneglycol), hydrocarbon linkers (including: heteroatom (0,S,N)-substituted hydrocarbon spacers; halo- substituted hydrocarbon spacers; keto-, carboxyl-, amido-, thionyl-, carbamoyl-, thionocarbamaoyl-containing hydrocarbon spacers), spermine linkers, dyes including fluorescent dyes (for example fluoresceins, rhodamines, cyanines) attached to linkers such as for example 6-fluorescein-hexyl, quenchers (for example dabcyl, BHQ) and other labels (for example biotin, digoxigenin, acridine, streptavidin, avidin, peptides and/or proteins). In some embodiments, an “end” modification comprises a conjugation (or ligation) of the gNA to another molecule comprising an oligonucleotide of deoxynucleotides and/or ribonucleotides, a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, a vitamin and/or other molecule. In certain embodiments, the disclosure provides a chemically-modified gNA in which an “end” modification (described above) is located internally in the gNA sequence via a linker such as, for example, a 2-(4-butylamidofluorescein)propane-l,3-diol bis(phosphodiester) linker, which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the gNA.

[00293] In some embodiments, the disclosure provides a chemically-modified gNA having an end modification comprising a terminal functional group such as an amine, a thiol (or sulfhydryl), a hydroxyl, a carboxyl, carbonyl, thionyl, thiocarbonyl, a carbamoyl, a thiocarbamoyl, a phoshoryl, an alkene, an alkyne, an halogen or a functional group-terminated linker that can be subsequently conjugated to a desired moiety selected from the group consisting of a fluorescent dye, a non-fluore scent label, a tag (for 14C, example biotin, avidin, streptavidin, or moiety containing an isotopic label such as ¹⁵N, ¹³C, deuterium, ³H, ³²P, ¹²⁵I and the like), an oligonucleotide (comprising deoxynucleotides and/or ribonucleotides, including an aptamer), an amino acid, a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, and a vitamin. The conjugation employs standard chemistry well-known in the art, including but not limited to coupling via N-hydroxysuccinimide, isothiocyanate, DCC (or DCI), and/or any other standard method as described in “Bioconjugate Techniques” by Greg T. Hermanson, Publisher Elsevier Science, 3^rd ed. (2013), the contents of which are incorporated herein by reference in its entirety. III. Tropism Factors and Pseudotyping of XDP Systems

[00294] In another aspect, the disclosure relates to the incorporation of tropism factors in the XDP to increase tropism and selectivity for target cells or tissues intended for gene editing. Tropism factors of the XDP 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 XDP particles enhances the ability of the XDP to selectively bind to and fuse with the cell membrane of a target cell bearing such target cell markers, increasing the therapeutic index and reducing unintended side effects of the therapeutic payload incorporated into the XDP.

[00295] In some embodiments, the XDP comprises one or more glycoproteins (GP) on the surface of the particle wherein the GP provides for enhanced or selective binding and fusion of the XDP to a target cell. In other embodiments, the XDP 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 XDP to a target cell. In other embodiments, the XDP comprises one or more cell surface receptors, including G-protein-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 XDP to a target cell. In some embodiments, the XDP comprises one or more ligands on the surface of the particle wherein the ligand provides for enhanced or selective binding and fusion of the XDP to a target cell bearing a receptor to the ligand on the cell surface. In still other embodiments, the XDP comprises a combinations 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 XDP to a target cell. [00296] 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 XDP to target particular cells or organs in a process known as pseudotyping. In some embodiments, the XDP 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 4 and in the Examples. In some embodiments, the viruses used to provide the glycoprotein include, but are not limited to Argentine hemorrhagic fever virus, Australian bat virus, Autographa californica multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Borna disease virus, Breda virus, Bunyamwera virus, Chandipura virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue 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 virus 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-lymphotro pic 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 virus, 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, rRotavirus, Rous sarcoma virus, rubella virus, Sabia-associated hemorrhagic fever virus, SARS-associated coronavirus (SARS-CoV), Sendai virus, Tacaribe virus, Thogotovirus, tick-borne encephalitis causing virus, varicella zoster virus (HHV3), varicella zoster virus (HHV3), variola major virus, variola minor virus, Venezuelan equine encephalitis virus, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus (VSV), glycoprotein G from vesicular stomatitis virus (VSV-G), Vesiculovirus, West Nile virus, western equine encephalitis virus, and Zika Virus. Non-limiting examples of glycoprotein sequences are provided in Table 4. In some embodiments, the XDP comprises one or more glycoprotein sequences of Table 4, 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 into the particle and exposed on the surface, providing tropism and enhanced selectivity for the XDP to the target cell to be edited.

Table 4: Glycoproteins for XDP

[00297] In some embodiments, the glycoprotein has a sequence selected from the group consisting of SEQ ID NOS: 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,

464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,

502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538,

540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576,

578, 580, 582, 584, 586, 588, 590, 592, 594 and 596 as set forth in Table 4, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity thereto. In some embodiments, the glycoprotein has a sequence selected from the group consisting of SEQ ID NOS: 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,

500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536,

538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574,

576, 578, 580, 582, 584, 586, 588, 590, 592, 594 and 596 as set forth in Table 4.

[00298] In some embodiments, the glycoprotein is incorporated into the XDP system by inclusion of a nucleic acid encoding the glycoprotein in a plasmid vector of the XDP system, described below. In some embodiments, the glycoprotein is encoded by a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569,

571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595 as set forth in Table 4, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity thereto. In some embodiments, the glycoprotein is encoded by a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561,

563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595 as set forth in Table 4.

[00299] In some embodiments, a XDP comprising a glycoprotein derived from an enveloped virus in a capsid of a XDP of the embodiments 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 XDP to a target cell compared to a XDP that does not have the glycoprotein. Representative examples demonstrating enhanced binding and uptake of XDP bearing glycoproteins to target cells leading to, in this case, enhance gene editing of target nucleic acid, are provided in the Examples, below. [00300] In some embodiments, the present disclosure provides XDP 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 XDP 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. Exemplary target cells include T cells, B cells, macrophages, liquid cancer cells (such as leukemia or myeloma cells), solid tumor cells, muscle cells, epithelial cells, endothelial cells, stem cells, dendritic cells, retinal cells, hepatic cells, cardiac cells, thyroid cells, neurons, glial cells, oligodendrocytes, Schwann cells, and pancreatic cells. Exemplary target organs include the brain, heart, liver, pancreas, lung, eye, stomach, small intestine, colon, and kidney. Exemplary tissues include skin, muscle, bone, epithelial, and connective tissue. 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, a XDP comprising an antibody fragment in a capsid of a XDP of the embodiments 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 XDP that does not have the antibody fragment. In the case of antibody fragments with affinity to cancer cell markers or receptors, the cancer cell markers or receptors can include, but not be limited to cluster of differentiation 19 (CD19), 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 A1 (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 cell 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 (BCMA), CD44V6, carcinoembryonic antigen (CEA), CEAC, CEA cell 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 (HER1), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), , Integrin B7, intercellular cell-adhesion molecule- 1 (ICAM-1), human telom erase 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 (L1CAM), Lysophosphatidic acid receptor 3 (LPAR3), melanoma-associated antigen 1 (MAGE-A1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16, cell surface associated (MUC16), 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. 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., alA, alb, ale, aid, a2a, a2b, a2c, a2d, bΐ, b2, b3), Dopaminergic (e.g., Dl, D2, D3, D4, D5), GABAergic (e.g., GABAA, GABABla, GABABlb, GABAB2, GAB AC), Glutaminergic (e.g., NMD A, AMP A, kainate, mGluRl, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7), Histaminergic (e.g., HI, 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., m, dΐ, d2, 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). [00301] In one embodiment, the antibody fragment is conjugated to the XDP after its production and isolation from the producing host cell. In another embodiment, the antibody fragment is produced as a part of the XDP capsid expressed by the producing host cell of the XDP system. In some cases, the present disclosure provides a nucleic acid comprising a sequence encoding the antibody fragment operably linked to the nucleic acid encoding the XDP capsid or other XDP components.

IV. Nucleic Acids Encoding XDP Systems

[00302] In another aspect, the present disclosure relates to nucleic acids encoding components of the XDP system and the incorporated therapeutic payloads, and the vectors that comprise the nucleic acids, as well as methods to make the nucleic acids and vectors.

[00303] In some embodiments, the present disclosure provides one or more nucleic acids encoding components including retroviral-derived XDP structural and processing components, therapeutic payloads, and tropism factors. The nucleic acids and vectors utilized for the key structural components and for processing and the assembly of XDP particles of the embodiments can be derived from a variety of viruses, such as retroviruses, including but not limited to Retroviridae family members Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses, Epsilonretroviruses, Spumaretrovirinae, or lentiviruses such as human immunodeficiency- 1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV) and the like.

[00304] In some embodiments, the nucleic acids encoding the XDP retroviral components are derived from Alpharetrovirus , including but not limited to avian leukosis virus (ALV) and Rous sarcoma virus (RSV). In some embodiments, the present disclosure provides nucleic acids encoding components selected from the group consisting of: a matrix polypeptide (MA); a p2A spacer peptide; ap2B spacer peptide; a plO spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), p2A, p2B, plO, a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-transframe region-Pol protease polyprotein; a protease cleavage site(s); and a protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, p2A, p2B, plO, andNC), and optionally the protease cleavage site and protease, are derived from an Alpharetrovirus , including but not limited to Avian leukosis virus and Rous sarcoma virus. In some embodiments, the encoding sequences for the Alpharetrovirus- derived components are selected from the group consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, and 234 as set forth in Table 5, 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 nucleic acids encode a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. In some embodiments of the foregoing, encoding nucleotides for protease cleavage sites are located between each of the individual components. In other cases, the protease cleavage sites are omitted. In a particular embodiment, an encoding sequence for a single protease cleavage site is located between the sequence encoding the nuclease and the linked retroviral component, which may be a retroviral sequence or a non-viral sequence, such as one that can be cleaved by TEV, PreScission Protease, or any of the other proteases disclosed herein. Representative configurations and sequences are presented in the Examples. In a particular embodiment, the encoded therapeutic payload is a CasX and gNA embodiment described herein, while the encoded tropism factor is a viral glycoprotein embodiment described herein.

[00305] In some embodiments, the nucleic acids encoding the XDP viral components are derived from Betaretrovirus, including but not limited to mouse mammary tumor virus (MMTV), Mason-Pfizer monkey virus (MPMV), and enzootic nasal tumor virus (ENTV). In such embodiments, the present disclosure provides nucleic acids encoding the XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a pp21/24 spacer peptide; a p3-P8/pl2 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), pp21/24, p3-8/pl2, a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-transframe region-Pol protease polyprotein; a protease cleavage site(s); and a protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, pp21/24 spacer, p3-p8/pl2 spacer, andNC), and optionally the protease cleavage site and protease, are derived from an Betaretrovirus , including but not limited to mouse mammary tumor virus, Mason-Pfizer monkey virus, and enzootic nasal tumor virus. In some embodiments, the encoding sequences for the Betaretrovirus- derived components are selected from the group consisting of SEQ ID NOS: 235-257 as set forth in Table 5, 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 nucleic acids encode a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. In some embodiments of the foregoing, encoding nucleotides for protease cleavage sites are located between each of the individual components. In other cases, the protease cleavage sites are omitted. In a particular embodiment, an encoding sequence for a single protease cleavage site is located between the sequence encoding the nuclease and the linked retroviral component, which may be a retroviral sequence or a non-viral sequence, such as one that can be cleaved by TEV, PreScission Protease, or any of the other proteases disclosed herein. Representative configurations and sequences are presented in the Examples. In a particular embodiment, the encoded therapeutic payload is a CasX and gNA embodiment described herein, while the encoded tropism factor is a viral glycoprotein embodiment described herein.

[00306] In some embodiments, the nucleic acids encoding the XDP viral components are derived from Deltaretrovirus, including but not limited to bovine leukemia virus (BLV) and the human T-lymphotropic viruses (HTLV1). In such embodiments, the present disclosure provides nucleic acids encoding the XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA)„ a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-transframe region-Pol protease polyprotein; a protease cleavage site(s); and a protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, and NC), and optionally the protease cleavage site and protease, are derived from an Deltaretrovirus , including but not limited to bovine leukemia virus and the human T- lymphotropic viruses. In some embodiments, the encoding sequences for the Deltaretrovirus- derived components are selected from the group consisting of the sequences SEQ ID NOS: 258- 272 as set forth in Table 5, 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 nucleic acids encode a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. In some embodiments of the foregoing, encoding nucleotides for protease cleavage sites are located between each of the individual components. In other cases, the protease cleavage sites are omitted. In a particular embodiment, an encoding sequence for a single protease cleavage site is located between the sequence encoding the nuclease and the linked retroviral component, which may be a retroviral sequence or a non-viral sequence, such as one that can be cleaved by TEV, PreScission Protease, or any of the other proteases disclosed herein. Representative configurations and sequences are presented in the Examples. In a particular embodiment, the encoded therapeutic payload is a CasX and gNA embodiment described herein, while the encoded tropism factor is a viral glycoprotein embodiment described herein.

[00307] In some embodiments, the nucleic acids encoding the XDP viral components are derived from Epsilonretrovirus , including but not limited to Walleye dermal sarcoma virus (WDSV), and Walleye epidermal hyperplasia virus 1 and 2. In such embodiments, the present disclosure provides nucleic acids encoding the XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a p20 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), p20, a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-transframe region-Pol protease polyprotein; a protease cleavage site(s); and a protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, p20, andNC), and optionally the protease cleavage site and protease, are derived from an Epsilonretrovirus , including but not limited to Walleye dermal sarcoma virus, and Walleye epidermal hyperplasia virus 1 and 2. In some embodiments, the encoding sequences for the Epsilonretrovirus-denvcd components are selected from the group consisting of the sequences of SEQ ID NOS: 273-277 as set forth in Table 5, 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 nucleic acids encode a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. In some embodiments of the foregoing, encoding nucleotides for protease cleavage sites are located between each of the individual components. In other cases, the protease cleavage sites are omitted. In a particular embodiment, an encoding sequence for a single protease cleavage site is located between the sequence encoding the nuclease and the linked retroviral component, which may be a retroviral sequence or a non-viral sequence, such as one that can be cleaved by TEV, PreScission Protease, or any of the other proteases disclosed herein. Representative configurations and sequences are presented in the Examples. In a particular embodiment, the encoded therapeutic payload is a CasX and gNA embodiment described herein, while the encoded tropism factor is a viral glycoprotein embodiment described herein.

[00308] In some embodiments, the nucleic acids encoding the XDP viral components are derived from Gammaretrovirus, including but not limited to murine leukemia virus (MLV), Maloney murine leukemia virus (MMLV), and feline leukemia virus (FLV). In such embodiments, the nucleic acids encoding the present disclosure provides XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a ppl2 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a ppl2 spacer, a capsid polypeptide (CA), a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-transframe region-Pol protease polyprotein; a protease cleavage site(s); and a protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, ppl2, CA, and NC), and optionally the protease cleavage site and protease, are derived from an Gammaretrovirus , including but not limited to Walleye dermal sarcoma virus, and Walleye epidermal hyperplasia virus 1 and 2. In some embodiments, the encoding sequences for the Gammaretrovirus-denved components are selected from the group consisting of the sequences of SEQ ID NOS: 278-287 as set forth in Table 5, 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 nucleic acids encode a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. In some embodiments of the foregoing, encoding nucleotides for protease cleavage sites are located between each of the individual components. In other cases, the protease cleavage sites are omitted. In a particular embodiment, an encoding sequence for a single protease cleavage site is located between the sequence encoding the nuclease and the linked retroviral component, which may be a retroviral sequence or a non-viral sequence, such as one that can be cleaved by TEV, PreScission Protease, or any of the other proteases disclosed herein. Representative configurations and sequences are presented in the Examples. In a particular embodiment, the encoded therapeutic payload is a CasX and gNA embodiment described herein, while the encoded tropism factor is a viral glycoprotein embodiment described herein.

[00309] In some embodiments, the nucleic acids encoding the XDP viral components are derived from Lentivirus , including but not limited to HIV-1 and HIV-2, and Simian immunodeficiency virus (SIV). In such embodiments, the present disclosure provides nucleic acids encoding the XDP wherein the XDP comprises components selected from the group consisting of: a matrix polypeptide (MA); a capsid (CA), a p2 spacer peptide, a nucleocapsid (NC), a pl/p6 spacer peptide; ); a Gag polyprotein comprising a matrix polypeptide (MA), CA, P2, NC, and pl/p6; a therapeutic payload; a tropism factor; a Gag-transframe region-Pol protease polyprotein; a protease cleavage site(s); and a protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, NC, and pl/p6), and optionally the protease cleavage site and protease, are derived from an Lentivirus , including but not limited to HIV-1, HIV-2, and Simian immunodeficiency virus (SIV). In some embodiments, the encoding sequences for the Lentivirus- derived components are selected from the group consisting of the sequences of SEQ ID NOS: 288-312 and 334-339 as set forth in Table 5, 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 nucleic acids encode a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. In some embodiments of the foregoing, encoding nucleotides for protease cleavage sites are located between each of the individual components. In other cases, the protease cleavage sites are omitted. In a particular embodiment, an encoding sequence for a single protease cleavage site is located between the sequence encoding the nuclease and the linked retroviral component, which may be a retroviral sequence or a non-viral sequence, such as one that can be cleaved by TEV, PreScission Protease, or any of the other proteases disclosed herein. Representative configurations and sequences are presented in the Examples. In a particular embodiment, the encoded therapeutic payload is a CasX and gNA embodiment described herein, while the encoded tropism factor is a viral glycoprotein embodiment described herein.

[00310] In some embodiments, the nucleic acids encoding the XDP viral components are derived from Spumaretrovirinae, including but not limited to Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, and Spumavirus. In such cases, the present disclosure provides nucleic acids encoding the XDP wherein the XDP comprises components selected from the group consisting of: P68 Gag; a p3 Gag; a Gag polyprotein comprising of P68 Gag and p3 gag; a therapeutic payload; a tropism factor; a Gag-transframe region-Pol protease polyprotein; a protease cleavage site(s); and a protease capable of cleaving the protease cleavage sites. In the forgoing embodiment, Gag components (e.g., MA, CA, p20, and NC), and optionally the protease cleavage site and protease, are derived from an Spumaretrovirinae including but not limited to Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, and Spumavirus. In some embodiments, the encoding sequences for the Sumaretrovirinae- derived components are selected from the group consisting of the sequences of SEQ ID NOS: 313-333 as set forth in Table 5, 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 nucleic acids encode a subset of the components listed in the paragraph, such as depicted in FIGS. 36-68, which depict CasX and gNA as the therapeutic payloads. In some embodiments of the foregoing, encoding nucleotides for protease cleavage sites are located between each of the individual components. In other cases, the protease cleavage sites are omitted. In a particular embodiment, an encoding sequence for a single protease cleavage site is located between the sequence encoding the nuclease and the linked retroviral component, which may be a retroviral sequence or a non-viral sequence, such as one that can be cleaved by TEV, PreScission Protease, or any of the other proteases disclosed herein. Representative configurations and sequences are presented in the Examples. In a particular embodiment, the encoded therapeutic payload is a CasX and gNA embodiment described herein, while the encoded tropism factor is a viral glycoprotein embodiment described herein.

[00311] In other embodiments, the present disclosure provides nucleic acids encoding the XDP wherein the retroviral components of the XDP are selected from different genera of the Retroviridae. Thus the nucleic acids encoding the XDP can comprise two or more components selected from a matrix polypeptide (MA), a p2A spacer peptide, a p2B spacer peptide; a plO spacer peptide, a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a pp21/24 spacer peptide, a p3-p8 spacer peptide, a ppl2 spacer peptide, a p20 spacer peptide, a pl/p6 spacer peptide, a p68 Gag, a p3 Gag, a cleave site(s), and a protease capable of cleaving the protease cleavage sites wherein the components are derived from two or more of Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, or Spumavirus.

[00312] In retroviral components derived from HIV-1, the accessory protein integrase (or its encoding nucleic acid) can be omitted from the XDP systems, as well as the HIV functional accessory genes vpr, vpx (HIV-2), which are dispensable for viral replication in vitro. Additionally, the nucleic acids of the XDP system do not require reverse transcriptase for the creation of the XDP compositions of the embodiments. Thus, in one embodiment, the HIV-1 Gag-Pol component of the XDP can be truncated to Gag linked to the transframe region (TFR) composed of the transframe octapeptide (TFP) and 48 amino acids of the p6pol, separated by a protease cleavage site, hereinafter referred to as Gag-TFR-PR, described more fully, below.

Table 5: Retroviral structural component encoding DNA sequences

* Wild-type sequence (optionally incorporated, depending on configuration) [00313] 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 XDP, wherein the tropism factor confers an increased ability of the XDP to bind and fuse with the membrane of a target cell or tissue. In one embodiment, the tropism factor is a glycoprotein, wherein the encoding nucleic acid is selected from the group consisting of the sequences of Table 4, 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 acids encoding an antibody fragment, wherein the antibody fragment has specific binding affinity for a target cell marker or receptor on a target cell 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 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 cell or tissue. By inclusion of the nucleic acids encoding for the tropism factors, it will be understood that the resulting XDP will have increased selectivity for the target cell or tissue, resulting in an increased therapeutic index and reduced off-target effects.

[00314] The present disclosure further provides nucleic acids encoding or comprising the therapeutic payloads incorporated into the XDP. Exemplary therapeutic payloads have been described herein, supra. In some embodiments, the therapeutic payload of the XDP 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 Table 1, 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. Representative examples of such nucleic acids are presented in Tables 6-8, 11 and 16 of the Examples, which disclose nucleic acids of SEQ ID NOS: 354, 340-342, 346-349, 378-387 and 426-431. In another particular embodiment of the foregoing, the disclosure provides nucleic acids encoding the gNA variants of SEQ ID NO: 597- 781 set forth in Table 3, 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, and wherein the gNA further comprises a targeting sequence complementary to a target nucleic acid.

[00315] In some embodiments of the disclosure, the components of the XDP systems are encoded by one, two, three, four, five or more nucleic acids (see FIGS. 36-68, which are schematics of the representative plasmids and XDP configurations), which can encode single components or multiple components that are operably linked to (under the control of) regulatory elements operable in a eukaryotic cell and appropriate for the component to be expressed. It will be understood that in the descriptions of the XDP system configurations, the absolute order of the components encoded within a nucleic acid may be varied in order to take advantage of the placement of the regulatory elements, cleavage sequences, etc., such that each component can be expressed and/or utilized in the assembly of the XDP in an optimal fashion, as would be understood by one of ordinary skill in the art. For example, where a nucleic acid encodes the Gag polyprotein, the therapeutic payload, and a protease cleavage site, the order (5’ to 3’) may be Gag-cleavage site-therapeutic payload or it may be therapeutic payload-cleavage site-gag, and it is intended that the same would apply for any combination of components encoded in a single nucleic acid. Representative regulatory elements are described herein.

[00316] In some embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the XDP system selected from two or more of a retroviral Gag polyprotein (all or portions thereof), a protease cleavage site, a therapeutic payload, a Gag-Pol polyprotein, and a tropism factor, wherein the components are encoded on one, two, three, or four individual nucleic acids. In some embodiments of the foregoing, the components are encoded on a single nucleic acid. In some embodiments of the foregoing, a first nucleic acid encodes the Gag polyprotein (or portions thereof) and the CasX protein as the therapeutic payload with, optionally, an intervening protease cleavage site between the two components, and a second nucleic acid encodes the Gag-Pol polyprotein (or portions thereof), the tropism factor and the gNA. In another embodiment of the foregoing, a first nucleic acid encodes the Gag polyprotein (or portions thereof) and the CasX protein as the therapeutic payload with, optionally, and intervening protease cleavage site separating the two components, a second nucleic acid encodes the Gag-Pol polyprotein, and a third nucleic acid encodes the tropism factor and the gNA. In another embodiment, a first nucleic acid encodes the Gag polyprotein (or portions thereof) and the CasX protein as the therapeutic payload with, optionally, an intervening protease cleavage site separating the two components, a second nucleic acid encodes the tropism factor, a third nucleic acid encodes the Gag-Pol polyprotein (or portions thereof), and a fourth nucleic acid encodes the gNA. In some cases, the protease cleavage sites are omitted. In other cases, protease cleavage sites are located between each component of the Gag polyprotein and, optionally, the therapeutic payload. Representative examples of the encoding nucleic acids of the foregoing embodiments are presented in the Examples.

[00317] In other embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the XDP system comprising the Gag-TFR-PR polyprotein (or portions thereof), the protease cleavage site, the CasX protein as the therapeutic payload, the gNA, and the tropism factor, wherein the components are encoded on one, two, or three individual nucleic acids. In some embodiments of the foregoing, the components are encoded on a single nucleic acid. In another embodiment of the foregoing, a first nucleic acid encodes the Gag-TFR-PR polyprotein and the CasX protein as the therapeutic payload with an intervening protease cleavage site separating the two components, and a second nucleic acid encodes the tropism factor and the gNA. In another embodiment, a first nucleic acid encodes the Gag-TFR-PR polyprotein and the CasX protein as the therapeutic payload with an intervening protease cleavage site separating the two components, a second nucleic acid encodes the tropism factor, and a third nucleic acid encodes the gNA. In some embodiments of the foregoing, protease cleavage sites are located between each component of the Gag polyprotein and, optionally, the CasX protein. Representative examples of the encoding nucleic acids of the foregoing embodiments are presented in the Examples (see Tables 16, 17, 19, 20, 22, 23, 24, 27, 30, 33 and 36 and the sequences contained therein).

[00318] In other embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the XDP system comprising the Gag polyprotein (or portions thereof), the protease cleavage site, the protease, the CasX protein, the gNA and the tropism factor wherein the components are encoded on one, two, or three individual nucleic acids. In some embodiments of the foregoing, the components are encoded on a single nucleic acid. In another embodiment of the foregoing, a first nucleic acid encodes the Gag polyprotein, the protease, the CasX protein, and intervening protease cleavage sites located between the components, and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA. In another embodiment of the foregoing, a first nucleic acid encodes the Gag polyprotein, the protease, the CasX protein and intervening protease cleavage sites between the components, a second nucleic acid encodes the tropism factor; and a third nucleic acid encodes one or more gNA.

[00319] In other embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the XDP system comprising the Gag-Pol polyprotein, the CasX protein, the protease cleavage site, the gNA, the RNA binding domain, and the tropism factor, wherein the components are encoded on one, two, or three individual nucleic acids. In some embodiments of the foregoing, the components are encoded on a single nucleic acid. In another case of the foregoing, a first nucleic acid encodes the Gag-Pol polyprotein and the CasX with an intervening protease cleavage site between the two components, and a second nucleic acid encodes the tropism factor, the gNA and the RNA binding domain. In another case of the foregoing, a first nucleic acid encodes the Gag-Pol polyprotein and the CasX with an intervening protease cleavage site between the two components, and a second nucleic acid encodes the tropism factor, and a third nucleic acid encodes the gNA and the RNA binding domain.

[00320] In some embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the XDP system comprising the Gag-Pol polyprotein, the CasX protein, the protease cleavage site, the tropism factor, and the gNA, wherein the components are encoded on one, two, or three individual nucleic acids. In some embodiments of the foregoing, the components are encoded on a single nucleic acid. In another case of the foregoing, a first nucleic acid encodes the first nucleic acid encodes the Gag-Pol polyprotein and the CasX with an intervening protease cleavage site between the two components, and a second nucleic acid encodes the tropism factor and the gNA. In another case, a first nucleic acid encodes the Gag-Pol polyprotein and the CasX with an intervening protease cleavage site between the two components, a second nucleic acid encodes the tropism factor, and a third nucleic acid encodes the gNA.

[00321] In other embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the XDP system comprising the MA, the CasX protein, the protease, the protease cleavage site, the gNA, and the tropism factor, wherein the components are encoded on one, two, three, or four individual nucleic acids. In some embodiments of the foregoing, the components are encoded on a single nucleic acid. In other cases of the foregoing, a first nucleic acid encodes the first nucleic acid encodes the MA, the CasX protein, the protease, and intervening protease cleavage sites between the three components, and a second nucleic acid encodes the tropism factor and the gNA. In other cases, a first nucleic acid encodes the MA, the CasX protein the protease, and intervening protease cleavage sites between the three components, a second nucleic acid encodes the tropism factor; and a third nucleic acid encodes the gNA. In other cases, a first nucleic acid encodes the MA and the CasX protein with an intervening protease cleavage site between the two components, a second nucleic acid encodes the tropism factor, a third nucleic acid encodes the gNA, and a fourth nucleic acid encodes the protease. In the foregoing embodiments, the first nucleic acid can further encode a CA component linked to the MA by an additional intervening protease cleavage site. In some embodiments of the foregoing, the protease and protease cleavage sites are omitted.

[00322] In some embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the XDP system comprising the Gag polyprotein (all or portions thereof), the CasX protein, the protease, the protease cleavage site, the gNA, the tropism factor, and the Gag-Pol polyprotein (all or portions thereof), 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 Gag polyprotein, the CasX protein, the protease, and intervening protease cleavage sites between the three components, and a second nucleic acid encodes the Gag-Pol polyprotein, the tropism factor, and the gNA. In other embodiments, a first nucleic acid encodes the Gag polyprotein and the CasX protein with an intervening protease cleavage site between the two components, a second nucleic acid encodes the protease, and a third nucleic acid encodes the tropism factor, the gNA, and the Gag-Pol polyprotein. In other embodiments, a first nucleic acid encodes the Gag polyprotein, and the CasX protein with an intervening protease cleavage site between the two components, a second nucleic acid encodes the protease, a third nucleic acid encodes the tropism factor, and a fourth nucleic acid encodes the gNA and the Gag-Pol polyprotein. In some embodiments of the foregoing, the protease and protease cleavage sites are omitted.

[00323] In other embodiments, the XDP system is encoded by a portion or all of a sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 426-436, 784- 823, 828-873, 880-933, 947-1009 as set forth in Tables 16, 17, 19, 20, 22, 23, 24, 27, 30, 33, or 36, or a sequence having 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 thereto.

[00324] In some embodiments, the nucleic acids encoding the XDP 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 HIT! 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, 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 wild-type 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, the donor template ranges in size from 10-10,000 nucleotides. In other embodiments, the donor template ranges in size from 100-1,000 nucleotides. In some embodiments, the donor template is a single-stranded DNA template or a single stranded RNA template. In other embodiments, the donor template is a double-stranded DNA template. In another embodiment of the XDP system, the donor template nucleic acid is incorporated in the first nucleic acid of the XDP system. In another embodiment of the XDP system, the donor template nucleic acid is incorporated in the second nucleic acid. In another embodiment of the XDP system, the donor template nucleic acid is incorporated in the third nucleic acid. In another embodiment of the XDP system, the donor template nucleic acid is incorporated in the fourth or a fifth nucleic acid.

[00325] In some embodiments, each of the individual nucleic acids are incorporated into plasmid vectors appropriate for transfection into a eukaryotic packaging cell, examples of which are detailed more fully, below, such that the XDP system will involve one, two, three, four, or five plasmids, as depicted in FIGS. 36-68. In each case, the nucleotide sequence encoding the components of the XDP system are operably linked to (under the control of) regulatory elements operable in a eukaryotic cell and appropriate for the component to be expressed. Exemplary regulatory elements include a transcription promoter (e.g., CMV, CMV+intron A, SV40, RSV, HIV-Ltr, MMLV-ltr, and metallothionein), a transcription enhancer element, a transcription termination signal, internal ribosome entry site (IRES) or p2A peptide to permit translation of multiple genes from a single transcript, polyadenylation sequences to promote downstream transcriptional termination, sequences for optimization of initiation of translation, and translation termination sequences. In some cases the promoter is a constitutive promoter, such as a CMV promoter, CAGG, PGK, U6 (for RNA pol III, which synthesizes shRNAs), elongation factor 1 alpha (EF1 -alpha), or HI. In one embodiment, a constitutive promoter, such as the human cytomegalovirus immediate early (HCMV-IE) enhancer/promoter is used to compensate for the regulation of transcription normally provided by tat. In other cases, the promoter can be an inducible promoter such as, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, heat shock promoter, or tetracycline-regulated promoter (TRE), or a negative inducible pLac promoter. Any strong promoter known to those skilled in the art can be used for driving the expression of the nucleic acid. In the case of the nucleic acid encoding the lentiviral packaging components, the vector can be a psPax2 (detailed in the Examples, SEQ ID NO: 430) or pMDLg/pRRE plasmid. In the case of the nucleic acid encoding the VSV-G pseudotyping viral envelope glycoprotein, the vector can be a pMD2.G plasmid.

[00326] The vectors of the embodiments may also comprise a polyadenylation signal, which may be downstream, for example, of the therapeutic payload, such as the CasX sequence. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human u- globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA). [00327] The vectors of the embodiments may also comprise an enhancer upstream of the therapeutic payload, such as the CasX sequence or gNA sequence. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV, or EBV. Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94/016737, the contents of each are fully incorporated by reference. The vector may also comprise a mammalian origin of replication in order to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The vector may also comprise a regulatory element, which may be well suited for gene expression in a mammalian or human cell into which the vector is administered. The vector may also comprise a reporter gene, such as green fluorescent protein (“GFP”) and/or a selectable marker, such as hygromycin (“Hygro”).

[00328] In embodiments involving the use of HIV-based vectors, the vectors can include additional sequences encoding factors or accessory proteins that assist in the replication of viral proteins. In one embodiment, the HIV-based vector comprises a sequence encoding tat, a protein involved in the activation of RNA Polymerase II, and that stimulates transcription and translation (Das, A., et al. The HIV-1 Tat Protein Has a Versatile Role in Activating Viral Transcription. J Virol. 85(18): 9506 (2011)). In another embodiment, the HIV-based vector comprises a sequence encoding Rev, an RNA binding protein that is critical in the nuclear export of intron-containing HIV-1 RNA (Pollard, V., et al. The HIV-1 Rev protein. Ann Rev Microbiol. 52:491 (1998)). In another embodiment, the HIV-based vector comprises a sequence encoding viral infectivity factor (Vif), an accessory proteins essential for viral replication that disrupts the antiviral activity of the mammalian enzyme APOBEC by targeting it for ubiquitination and cellular degradation (Yang, G., et al. Viral infectivity factor: a novel therapeutic strategy to block HIV-1 replication. Minireviw Med Chem 13(7): 1047 (2013)). In another embodiment, the HIV-based vector comprises a sequence encoding Viral protein U (Vpu), an accessory protein essential for suppressing the antiviral activity of host cell restriction factors as well as the efficient release of viral particles from infected cells (Gonzalez, M. Vpu Protein: The Viroporin Encoded by HIV-1. Viruses 7:4352 (2015). In another embodiment, the HIV-based vector comprises a sequence encoding Negative Factor (Net), an accessory protein essential for both evading host adaptive cell-mediated immunity as well as enhancing infectivity in the target cell (Basmaciogullari, S., et al. The activity of Nef on HIV-1 infectivity. Frontiers Microbiol 5:232 (2014). In another embodiment, the HIV-based vector comprises a sequence encoding Viral protein R (VpR), an accessory protein important for its interactions with a number of cellular proteins that impact viral replication in addition to a potential role in restricting host anti-viral pathways (Zhao, Richard Y, and Michael I Bukrinsky. HIV-1 accessory proteins: VpR. Methods Mol Biol 1087:125 (2014). In some embodiments, the HIV-based vector comprises a sequence encoding any combination of tat, Vif, Rev, Vpu, Nef, and VpR.

[00329] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding a matrix polypeptide (MA), a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a pl/p6 polypeptide and a CasX polypeptide. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, pl/p6 operably linked, for example by a ribosomal frameshift, to a protease (PRO), a reverse transcriptase (RT) and an integrase (INT). In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00330] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, a NC, pl/p6 and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, pl/p6, CasX and PRO. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00331] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding a matrix polypeptide (MA), a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a pl/p6 polypeptide and a CasX polypeptide. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, pl/p6 operably linked, for example by a ribosomal frameshift, to a CasX polypeptide, and PRO. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00332] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6 operably linked, for example by a ribosomal frameshift, to PRO, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00333] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, CasX and PRO. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00334] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, CasX and PRO. In some embodiments, the third nucleic acid comprises, from 5’ to 3’, sequence encoding MA, CA, NC and pl/p6. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00335] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00336] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA. [00337] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00338] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pi and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00339] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, CasX, and pl/p6 operably linked, for example by a ribosomal frameshift, to PRO. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00340] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, CasX, and pl/p6 operably linked, for example by a ribosomal frameshift, to PRO. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00341] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CasX, and pl/p6 operably linked, for example by a ribosomal frameshift, to PRO. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00342] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CasX, and PRO. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00343] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, CasX, and PRO. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00344] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, tev cleavage sequence (TCS), and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, pl/p6, TCS and a TEV protease (TEV). In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00345] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, TCS, and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, pl/p6, PreScission cleavage sequence (PCS) and a PreScission protease (PSP). In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00346] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, TCS, and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, pl/p6, PCS and a PreScission protease (PSP). In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00347] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, PCS, and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, pl/p6, PCS and PSP. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00348] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, pl/p6, PCS, and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, pl/p6, PCS and TEV. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00349] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00350] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, PI and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00351] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, CasX and Pl/p6 operably linked, for example by a ribosomal frameshift, to PRO. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00352] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, CasX and Pl/p6 operably linked, for example by a ribosomal frameshift, to PRO. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00353] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CasX, NC, and Pl/p6 operably linked, for example by a ribosomal frameshift, to PRO. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00354] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CasX and Pl/p6 operably linked, for example by a ribosomal frameshift, to PRO. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00355] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, NC, CasX and PRO. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00356] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, CasX and PRO. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00357] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00358] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, MA, CA, NC, and pl/p6. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00359] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00360] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00361] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, the Alpharetrovirus gag polyprotein components P2A, P2B, and P10, as well as CA, NC, PRO and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00362] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, pp21/24, P12/P3/P8, CA, NC operably linked, for example by a ribosomal frameshift, to PRO, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00363] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, NC operably linked, for example by a ribosomal frameshift, to PRO, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00364] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, p20, CA, NC, PRO, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00365] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, ppl2, CA, NC, PRO, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00366] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, P6 operably linked, for example by a ribosomal frameshift, to PRO, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00367] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding p68- Gag operably linked, for example by a ribosomal frameshift, to PRO, and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00368] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, P2A, P2B, P10, CA and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA. [00369] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, P2A, P2B, P10, CA and CasX. In some embodiments the second nucleic acid comprises, from 5’ to 3’, MA, P2A, P2B, P10, CA, NC, PRO and CasX. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00370] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, pp21/24, P12/P3/P8, CA and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00371] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, pp21/24, P12/P3/P8, CA and CasX. In some embodiments the second nucleic acid comprises, from 5’ to 3’, MA, pp21/24, P12/P3/P8, CA, NC operably linked, for example by a ribosomal frameshift, to PRO and CasX. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00372] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00373] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC operably linked, for example by a ribosomal frameshift, to PRO and CasX. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00374] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, p20, CA and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00375] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, p20, CA and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, sequences encoding MA, p20, CA, NC operably linked, for example by a ribosomal frameshift, to PRO and CasX. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00376] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, ppl2, CA and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00377] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, ppl2, CA and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, sequences encoding MA, ppl2, CA, NC, PRO and CasX. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00378] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00379] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, sequences encoding MA, CA, NC, P6 operably linked, for example by a ribosomal frameshift, to PRO and CasX. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00380] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, p68-Gag, p3-Gag and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00381] In some embodiments, the XDP system of the disclosure comprises four nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, sequences encoding p68- Gag, p3-Gag and CasX. In some embodiments, the second nucleic acid comprises, from 5’ to 3’, sequences encoding p68-Gag, p3-Gag operably linked, for example by a ribosomal frameshift, to PRO and CasX. In some embodiments, the third nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the fourth nucleic acid comprises a sequence encoding a gNA.

[00382] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, P2A, P2B, P10, CA, NC and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00383] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, CA, NC and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00384] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, CA, NC, p6 and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00385] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, pp21/24, P12/P3/P8, CA, NC and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00386] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, ppl2, CA, NC and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00387] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, p20, CA, NC and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00388] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, CA, pl/p6 and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00389] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, CA, NC, pl/p6, pl/p6 and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00390] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, CA, NC, CasX and pl/p6. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA.

[00391] In some embodiments, the XDP system of the disclosure comprises three nucleic acids. In some embodiments, the first nucleic acid comprises, from 5’ to 3’, MA, CA, NC, P2, pl/p6 and CasX. In some embodiments, the second nucleic acid comprises a sequence encoding a glycoprotein, for example VSV-G. In some embodiments, the third nucleic acid comprises a sequence encoding a gNA. [00392] In any of the foregoing, any of the components may be separated by sequences encoding protease cleavage sites, self-cleaving polypeptides, or internal ribosome entry sites, or any combination thereof.

V. XDP Packaging Cells

[00393] In another aspect, the present disclosure relates to packaging cells utilized in the production of XDP. As used herein, the term “packaging 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 (e.g., Gag, pol, etc.) which are necessary or useful for the correct packaging of XDP particles. In the embodiments, the cell line can be any cell line suitable for the production of XDP, 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. coli. Exemplary yeast cell types may include Saccharomyces cerevisiae. Also suitable for use as packaging cells are insect cell lines, such as Spodoptera frugiperda sf9 cells. Exemplary mammalian cell types may include mouse, hamster, and human primary cells, as we 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, 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 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. In some embodiments, the eukaryotic cell is modified by one or more mutations one or more mutations to reduce expression of a cell surface marker that could be incorporated into the XDP. Such markers can include receptors or proteins capable of being bound by MHC receptors or that would otherwise trigger an immune response in a subject. [00394] In the embodiments of the XDP system, vectors are introduced into the packaging cell that encode the particular therapeutic payload (e.g., a CasX:gNA designed for editing target nucleic acid), as well as the other viral-derived structural components, detailed above, (e.g., the Gag polyprotein, the pol polyprotein, 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 cell. [00395] In some embodiments, the vectors comprising the nucleic acids of the XDP system are introduced into the cell via transfection, transduction, lipofection or electroporation to generate a packaging 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 Mirus 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.

[00396] 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.

[00397] Assembly and release of XDP with the encapsidated therapeutic payload from the transfected host cell can be mediated by the viral structural protein, Gag. Human immunodeficiency virus type 1 (HIV-1) Gag is synthesized as a precursor polyprotein, Pr55^gag. This polyprotein is comprised of four major structural domains, which are cleaved by the viral protease into pl7 matrix (MA), p24 capsid (CA), p7 nucleocapsid (NC), and p6, during or immediately after the budding process (Adamson CS., and Freed EO. Human immunodeficiency virus type 1 assembly, release, and maturation. Adv. Pharmacol. 55:347 (2007)). Utilizing an HIV-1 system, it is sufficient to express the p55 Gag protein to allow the efficient production of XDPs from cells (Gheysen et ah, Assembly and release of HIV-1 precursor Pr55Gag virus-like particles from recombinant baculovirus-infected insect cells. Cell. 59(1): 103 (1989)). In the context of the uncleaved Pr55^Gag, MA constitutes the N-terminal domain of the Gag protein and is essential for membrane binding and localization of the Gag precursor to the plasma membrane. CA and NC domains promote Gag multimerization through direct protein-protein interactions and indirect RNA-mediated interactions, respectively. Inclusion of the late domain motif within p6 can promote release of XDP particles from the cell surface. Upon expression, the Gag polypeptide is targeted to the cell membrane and incorporated in the XDP during membrane budding. During or shortly after virus budding from the host cell, the HIV-1 protease cleaves Pr55^gag into the mature Gag proteins pl7 matrix (MA), p24 capsid (CA), p7 nucleocapsid (NC), and p6. The proteolytic processing of Gag results in a major transformation in XDP structure: MA remains associated with the inner face of the viral membrane, whereas CA condenses to form a shell around the NC complex (if incorporated). This rearrangement produces a morphological transition to a particle with a conical core characteristic similar to an infectious virion.

[00398] It has been discovered that components derived, in part, from retroviruses can be utilized to create XDP within packaging cells for delivery of the therapeutic payload to the target cells. In one embodiment, the packaging cell transformed with the XDP system plasmids produce XDP that facilitate delivery of the encapsidated RNP of a CasX:gNA system to cells to effect editing of target nucleic acid.

VI. XDP Expression Systems and Methods of Producing XDP

[00399] In another aspect, the present disclosure provides a recombinant expression system for use in the production of XDP in a selected host packaging cell, comprising an expression cassette comprising the nucleic acids of the XDP system described herein operably linked to regulatory 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 regulatory elements include a transcription promoter such as, but not limited to, CMV, CMV+intron A, SV40, RSV, HIV-Ltr, elongation factor 1 alpha (EFla), MMLV-ltr, internal ribosome entry site (IRES) or p2A peptide to permit translation of multiple genes from a single transcript, metallothionein, a transcription enhancer element, a transcription termination signal, polyadenylation 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.

[00400] In some embodiments, the present disclosure provides methods of making an XDP comprising a therapeutic payload (e.g., an RNP of a CasX protein and a gNA), the method comprising propagating the packaging cell of the embodiments described herein comprising the expression cassettes or the integrated nucleic acids encoding the XDP systems of any one of the embodiments described herein under conditions such that XDPs are produced with the encapsidated therapeutic payload, followed by harvesting the XDPs produced by the packaging cell, as described below or in the Examples. In some embodiments, the packaging cell produces XDP comprising RNP of a CasX and gNA and, optionally, a donor template for the editing of the target nucleic acid by HDR.

[00401] The packaging 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, 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, CHO cells, and HT1080 cells), an insect cell (e.g., Trichoplusia ni (Tn5) or Sf9), a bacterial cell, a plant cell, a yeast cell, an antigen presenting cell (e.g., primary, immortalized or tumor-derived lymphoid cells such as macrophages, monocytes, dendritic cells, B-cells, T-cells, stem cells, and progenitor cells thereof). Packaging cells 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 cell can be modified to reduce or eliminate cell surface markers or receptors that would otherwise be incorporated into the XDP, thereby reducing an immune response to the cell surface markers or receptors by the subject receiving an administration of the XDP.

[00402] The introduction of the vectors into the packaging 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 Mirus 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.

[00403] In one embodiment, XDP are produced by the incubation of the transfected packaging cells in appropriate growth medium for 48 to 96 hours and are collected by filtration of the growth medium through a 0.45 micron filter. In some cases, the XDP can be further concentrated by centrifugation in a 10% or a 10-30% density gradient sucrose buffer. In other cases, the XDP can be concentrated by column chromatography, such as by use of an ion- exchange resin or a size exclusion resin.

VII. Applications

[00404] The XDP systems comprising CasX proteins and guides provided herein are useful in methods for modifying target nucleic acids in cells. In the XDP systems of modifying a target nucleic acid, the method utilizes any of the embodiments of the CasX:gNA 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. In other cases, the method knocks-out the expression of the mutant protein. In still other cases, the method results in the correction of the mutation in the target nucleic acid, resulting in the expression of functional protein.

[00405] In some embodiments, the method comprises contacting the cells comprising the target nucleic acid with an effective dose of XDPs comprising RNPs of CasX protein and a guide nucleic acid (gNA) 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. In another embodiment, the XDP further comprises a donor template wherein the contacting of the cell with the XDP 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 in the target nucleic acid 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 nucleic acid gene. [00406] 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 XDPs wherein the cell is modified in vitro or ex vivo.

[00407] 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 XDP is administered to a subject. The method has the advantage over viral delivery systems in that the RNP are comparatively short-lived relative to the nucleic acids delivered in viral systems such as AAV. A further advantage of the XDP system is the ability to match the system to specific cell types by manipulating the tropism of the XDP. In some embodiments, the half-life of the delivered RNP is about 24h, 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 XDP results in the improvement of one, two, or more symptoms, clinical parameters or endpoints associated with the disease in the subject. [00408] In some embodiments, the subject administered the XDP 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 one embodiment of the method, the XDP is administered to the subject at a dose of at least about 1 x 10⁵ XDP particles/kg, or at least about 1 x 10⁶ particles/kg, or at least about 1 x 10⁷ particles/kg, or at least about 1 x 10⁸ particles/kg, or at least about 1 x 10⁹ particles/kg, or at least about 1 x 10¹⁰ particles/kg, or at least about 1 x 10¹¹ particles/kg, or at least about 1 x 10¹² particles/kg, or at least about 1 x 10¹³ particles/kg, or at least about 1 x 10¹⁴ particles/kg, or at least about 1 x 10¹⁵ parti cles/kg, or at least about 1 x 10¹⁶ particles/kg. In other embodiments, the VLP is administered to the subject at a dose of at least about 1 x 10⁵ particles/kg to at least about 1 x 10¹⁶ particles/kg. In another embodiment, the VLP is administered to the subject at a dose of at least about 1 x 10⁵ particles/kg to about 1 x 10¹⁶ particles/kg, or at least about 1 x 10⁶ particles/kg to about 1 x 10¹⁵ particles/kg, or at least about 1 x 10⁷ particles/kg to about 1 x 10¹⁴ particles/kg. In other embodiments, the VLP is administered to the subject at a dose of at least about 1 x 10⁵ particles/kg to at least about 1 x 10¹⁶ particles/kg. In one embodiment, the XDP is administered by a route of administration selected from the group consisting of subcutaneous, intradermal, intraneural, intranodal, intramedullary, intramuscular, intravenous, intra-arterial, intracerebroventricular, intracisternal, intrathecal, intracranial, intralumbar, intratracheal, intraosseous, inhalatory, intracontralateral striatum, intraocular, intravitreal, intralymphatical, intraperitoneal routes and sub-retinal routes. [00409] 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 an XDP 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.

VIII. Kits and Articles of Manufacture

[00410] In another aspect, provided herein are kits comprising the compositions of the embodiments described herein. In some embodiments, the kit comprises an XDP comprising a therapeutic payload 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 and a gNA.

[00411] 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. IX. Exemplary Embodiments

[00412] The following exemplary embodiments, are provided by way of example only.

[00413] In some embodiments, the XDP system comprises an editing efficiency of at least 75%, at least 80%, at least 85%, at least 87%, at least 90% or at least 91% as per the editing assay dilution in Table 25, or at least 70%, at least 75%, at least 80% or at least 85% as per the editing assay dilution of Table 26. In some embodiments, the XDP system comprises version 44, encoded by plasmid pXDP40 (SEQ ID NO: 882) as described in Table 24. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00414] In some embodiments, the XDP system comprises an editing efficiency of at least 25%, at least 30%, at least 35% or at least 37% as per the editing assay dilution in Table 25 or at least 5%, at least 10% or at least 13% as per the editing assay dilution of Table 26. In some embodiments, the XDP system comprises version 63, encoded by plasmid pXDP62 (SEQ ID NO: 904) as described in Table 24. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00415] In some embodiments, the XDP system comprises an editing efficiency of at least 60%, at least 65%, at least 70%, at least 75% or at least 77% as per the editing assay dilution in Table 28, or at least 20%, at least 25%, at least 30% or at least 32% as per the editing assay dilution of Table 29. In some embodiments, the XDP system comprises version 74a, encoded by plasmid pXDP72 (SEQ ID NO:917) as described in Table 27. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00416] In some embodiments, the XDP system comprises an editing efficiency of at least at least 50%, at least 55%, at least 60%, at least 65% or at least 67% as per the editing assay dilution in Table 28, or at least 25%, at least 30%, at least 35% or at least 38% as per the editing assay dilution of Table 29. In some embodiments, the XDP system comprises version 75a, encoded by plasmid pXDP73 (SEQ ID NO:918) as described in Table 27. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00417] In some embodiments, the XDP system comprises an editing efficiency of at least 75%, at least 80%, at least 85%, at least 87%, at least 90% or at least 91% as per the editing assay dilution in Table 31, or at least 70%, at least 75%, at least 80% or at least 85% as per the editing assay dilution of Table 32. In some embodiments, the XDP system comprises version 44, encoded by plasmid pXDP40 (SEQ ID NO: 949) as described in Table 30. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00418] In some embodiments, the XDP system comprises an editing efficiency of at least 25%, at least 30%, at least 35% or at least 37% as per the editing assay dilution in Table 31 or at least 5%, at least 10% or at least 13% as per the editing assay dilution of Table 32. In some embodiments, the XDP system comprises version 63, encoded by plasmid pXDP62 (SEQ ID NO: 971) as described in Table 30. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00419] In some embodiments, the XDP system comprises an editing efficiency of at least 75%, at least 80%, at least 85%, at least 87%, at least 90% or at least 94% as per the editing assay dilution in Table 34 or at least 75%, at least 80%, at least 85%, at least 87%, at least 90% or at least 95% as per the editing assay dilution of Table 35. In some embodiments, the XDP system comprises version 102, encoded by plasmid pXDP127 (SEQ ID NO: 976) as described in Table 33. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00420] In some embodiments, the XDP system comprises an editing efficiency of at least 70%, at least 75%, at least 80% or at least 84% as per the editing assay dilution in Table 34 or at least 70%, at least 75%, or at least 80% as per the editing assay dilution of Table 35. In some embodiments, the XDP system comprises version 7, encoded by plasmid pXDP0017. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00421] In some embodiments, the XDP system comprises an editing efficiency of at least at least 25%, at least 25%, at least 30% or at least 33% as per the editing assay dilution in Table 37 or at least 1.8 % as per the editing assay dilution of Table 38. In some embodiments, the XDP system comprises version 66B, encoded by plasmid pXDP78 + pXDP54. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00422] In some embodiments, the XDP system comprises an editing efficiency of at least 10%, at least 15%, at least 20% or at least 21% as per the editing assay dilution in Table 37 or at least 5%, at least 7% or at least 9% as per the editing assay dilution of Table 38. In some embodiments, the XDP system comprises version 87B, encoded by plasmids pXDP83 + pXDP59. In some embodiments, the XDP system comprises a VSV glycoprotein as encoded by pGP2, and an sgRNA.

[00423] Editing efficiency may be measured by any known method or assay in the art. A person of skill in the art would know how to identify and use such assays. In some embodiments, the editing efficiency may be measured as %TDT positive cells, for example as shown in FIG. 69- 70.

[00424] In some embodiments, an XDP system comprises one or more plasmids or elements in an arrangement resulting in an increased editing efficiency compared an XDP system not comprising said arrangement. In some embodiments, the XDP system may have an increased editing efficiency of at least 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% compared to an XDP system not comprising the same elements and/or arrangement.

[00425] In some embodiments, an XDP system may be derived from Alpharetroviruses (avian leukosis virus (ALV) and rous sarcoma virus (RSV)), and encoded by the three plasmids encoding the Gag-protease-CasX, the glycoprotein (VSV-G), and the guide RNA (sgRNA). The elements of the structural plasmid may be arranged as: MA, P2A, P2B, P10, CA, NC, Pro and CasX (FIG. 52A). In an exemplary embodiment, the XDP system version 44 comprises elements of a structural plasmid arranged as: MA, P2A, P2B, P10, CA, NC, Pro and CasX (FIG 52A), wherein version 44 has an increased editing efficiency of at least 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,

99% or 100% compared to an XDP not comprising the same elements and/or arrangement. [00426] In some embodiments, an XDP system may be encoded by the three plasmids as shown in FIG. 53 A. The elements of the structural plasmid may be arranged as: MA, CA, NC, Pro and CasX. In an exemplary embodiment, the XDP system version 63 comprises elements of a structural plasmid arranged as: MA, CA, NC, Pro and CasX, wherein version 63 has an increased editing efficiency of at least 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% compared to an XDP not comprising the same elements and/or arrangement.

[00427] In some embodiments, an XDP system may be derived from Gammaretroviruses (FLV and MMLV), and encoded by the three plasmids as shown in FIG. 59B. The elements of the structural plasmid may be arranged as: MA, ppl2, CA, and CasX. In an exemplary embodiment, the XDP system version 74a comprises elements of a structural plasmid arranged as: MA, ppl2, CA, and CasX, wherein version 74a has an increased editing efficiency of at least 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,

85%, 90%, 95%, 99% or 100% compared to an XDP not comprising the same elements and/or arrangement.

[00428] In some embodiments, an XDP system may be derived from Alpharetroviruses (avian leukosis virus (ALV) and rous sarcoma virus (RSV) and encoded by the three plasmids as shown in FIG. 62B. The elements of the structural plasmid may be arranged as: MA, P2A, P2B, P10, CA, NC, and CasX. In an exemplary embodiment, the XDP system version 102 comprises elements of a structural plasmid arranged as: MA, P2A, P2B, P10, CA, NC, and CasX, wherein version 102 has an increased editing efficiency of at least 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% compared to an XDP not comprising the same elements and/or arrangement.

[00429] In some embodiments, an XDP system may be encoded by three plasmids as shown in FIG. 39A. The elements of the structural plasmid may be arranged as: MA, CA, NC, pl/p6, and CasX. In an exemplary embodiment, the XDP system version 7 comprises elements of a structural plasmid arranged as: MA, CA, NC, pl/p6, and CasX, wherein version 7 has an increased editing efficiency of at least 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% compared to an XDP not comprising the same elements and/or arrangement.

[00430] In some embodiments, an XDP system may be encoded by the four plasmids as shown in FIG. 56A. The elements of structural plasmid 1 may be arranged as: MA, P2A, P2B, P10, CA, and CasX, and elements of structural plasmid 2 may be arranged as: MA, P2A, P2B, P10, CA, NC, Pro, and CasX. In an exemplary embodiment, the XDP system version 66B comprises elements of a structural plasmid 1 arranged as: MA, P2A, P2B, P10, CA, and CasX, and elements of structural plasmid 2 arranged as: MA, P2A, P2B, P10, CA, NC, Pro, and CasX, wherein version 66B has an increased editing efficiency of at least 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,

99% or 100% compared to an XDP not comprising the same elements and/or arrangement. [00431] In some embodiments, an XDP system may be encoded by the four plasmids as shown in FIG. 57A. The elements of structural plasmid 1 may be arranged as: MA, pp21/24,

P12/P3/P8, CA, and CasX, and elements of structural plasmid 2 may be arranged as: MA, pp21/24, P12/P3/P8, CA, NC, Pro, and CasX. In an exemplary embodiment, the XDP system version 87B comprises elements of a structural plasmid larranged as: MA, pp21/24, P12/P3/P8, CA, and CasX, and elements of structural plasmid 2 arranged as: MA, pp21/24, P12/P3/P8, CA, NC, Pro, and CasX, wherein version 87B has an increased editing efficiency of at least 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% compared to an XDP not comprising the same elements and/or arrangement.

[00432] The XDP systems disclosed herein may be derived from the Retroviridae virus family, including Othoretrovirinae (Lentivirus, Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus), and Spumaretrovirinae . Exemplary XDP system versions and their corresponding virus are shown in Tables 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38.

X. Enumerated Embodiments

[00433] The invention may be defined by reference to the following sets of enumerated, illustrative embodiments:

Set

[00434] Embodiment 1-1. A CasX delivery particle (CasX XDP) system comprising: a. a first nucleic acid comprising a sequence encoding a fusion polypeptide that comprises: i) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC); ii) a CasX protein; and iii) a protease cleavage site between the gag polyprotein and the CasX protein; b. a second nucleic acid comprising a sequence encoding a guide RNA; c. a third nucleic acid comprising a sequence encoding a fusion polypeptide that comprises: i) a gag polyprotein; and ii) a pol polyprotein comprising at least a protease capable of cleaving the protease cleavage site between the CasX protein and the gag polyprotein; and d. a fourth nucleic acid, comprising a sequence encoding a pseudotyping viral envelope glycoprotein or an antibody fragment that provides for binding and fusion of the XDP to a target cell.

[00435] Embodiment 1-2. A CasX delivery particle (CasX XDP) system comprising: a. a first nucleic acid comprising a sequence encoding a fusion polypeptide that comprises: i) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC); ii) a CasX protein; iii) a protease cleavage site between the gag polyprotein and the CasX protein; and iv) a protease capable of cleaving the protease cleavage site between the CasX protein and the gag polyprotein; b. a second nucleic acid comprising a sequence encoding a guide RNA; and c. a third nucleic acid, comprising a sequence encoding a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell.

[00436] Embodiment 1-3. A CasX delivery particle (CasX XDP) system comprising: a. a first nucleic acid comprising a sequence encoding a fusion polypeptide that comprises: i) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC); ii) a CasX protein; and iii) a protease cleavage site between the gag polyprotein and the CasX protein; b. a second nucleic acid comprising a sequence encoding a guide RNA; c. a third nucleic acid comprising a sequence encoding a protease capable of cleaving the protease cleavage site between the CasX protein and the gag polyprotein; and d. a fourth nucleic acid, comprising a sequence encoding a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell.

[00437] Embodiment 1-4. A CasX delivery particle (CasX XDP) system comprising: a. a first nucleic acid comprising a sequence encoding i) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC); and ii) a chimeric RNA comprising a guide RNA and a retroviral Psi packaging element inserted into the guide RNA; b. a second nucleic acid comprising a sequence encoding a Cas X protein; and c. a third nucleic acid, comprising a sequence encoding a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell. [00438] Embodiment 1-5. A CasX delivery particle (CasX XDP) system comprising: a. a first nucleic acid comprising a sequence encoding: i) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC); ii) an RNA binding domain protein; and iii) an optional protease cleavage site between the gag polyprotein and the RNA binding domain protein; b. a second nucleic acid comprising a sequence encoding a guide RNA and a CasX protein; c. a third nucleic acid comprising a sequence encoding a protease capable of cleaving the protease cleavage site between the gag polyprotein and the RNA binding domain protein; and d. a fourth nucleic acid, comprising a sequence encoding a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell.

[00439] Embodiment 1-6. The XDP system of embodiment 5, wherein the RNA binding domain protein is selected from the group consisting of MS2, PP7 or Qbeta, U1A, phage replication loop, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, and pseudoknots.

[00440] Embodiment 1-7. The XDP system of any one of embodiments 1-3, comprising all or a portion of any one of the nucleic acid sequences of Table 8 or Table 9.

[00441] Embodiment 1-8. The XDP system of any one of the preceding embodiments of Set I, wherein the gag polypeptide comprises one or more protease cleavage sites between the matrix polypeptide (MA) and the capsid polypeptide (CA) and/or between the capsid polypeptide (CA) and the nucleocapsid polypeptide (NC), wherein the one or more protease cleave sites are capable of being cleaved by the protease.

[00442] Embodiment 1-9. The XDP system of any one of the preceding embodiments of Set I, wherein the protease is selected from the group of proteases consisting of HIV- 1 protease, tobacco etch virus protease (TEV), potyvirus HC protease, potyvirus PI protease, PreScission, b virus NIa protease, B virus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2 A protease, picoma 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, cathepsin, thrombin, factor Xa, metalloproteinases MMP-2, -3, -7, -9, - 10, and -11, and enterokinase. [00443] Embodiment 1-10. The XDP system of embodiment 1, wherein the pol polyprotein is a retroviral polyprotein.

[00444] Embodiment 1-11. The XDP system of embodiment 10, wherein the retrovirus is an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, a epsilonretrovirus, or a lentivirus.

[00445] Embodiment 1-12. The XDP system of embodiment 11, wherein the lentivirus is a human immunodeficiency virus (HIV).

[00446] Embodiment 1-13. The XDP system of any one of the preceding embodiments of Set I, wherein the gag polyprotein is a retroviral polyprotein.

[00447] Embodiment 1-14. The XDP system of embodiment 13, wherein the gag polyprotein is derived from a alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, a epsilonretrovirus, or a lentivirus.

[00448] Embodiment 1-15. The XDP system of embodiment 14, wherein the gag polyprotein is a lentiviral polyprotein.

[00449] Embodiment 1-16. The XDP system of embodiment 15, wherein the lentiviral gag polypeptide is an HIV-1 gag polyprotein.

[00450] Embodiment 1-17. The XDP system of any one of embodiments 13-16, wherein the gag polypeptide further comprises a p6 polypeptide.

[00451] Embodiment 1-18. The XDP system of embodiment 16 or embodiment 17, wherein the HIV-1 gag polypeptide comprises a MA polypeptide, a CA polypeptide, a p2 polypeptide, an NC polypeptide, a pi polypeptide, and a p6 polypeptide, and wherein the HIV gag polyprotein comprises one or more protease cleavage sites located between one or more of: a. the MA polypeptide and the CA polypeptide; b. the CA polypeptide and the p2 polypeptide; c. the p2 polypeptide and the NC polypeptide; d. the NC polypeptide and the pi polypeptide; and e. the pi polypeptide and the p6 polypeptide.

[00452] Embodiment 1-19. The XDP system of embodiment 18, wherein the protease capable of cleaving the protease cleavage site is selected from the group of proteases consisting of HIV-1 protease, tobacco etch virus protease (TEV), poty virus HC protease, poty virus PI protease, PreScission, b virus NIa protease, B virus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2 A protease, rhinovirus 2 A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, cathepsin, thrombin, factor Xa, metalloproteinases MMP-2, -3, -7, -9, -10, and -11, and enterokinase.

[00453] Embodiment 1-20. The XDP system of embodiment 19, wherein the protease capable of cleaving the protease cleavage site is HIV-1 protease.

[00454] Embodiment 1-21. The XDP system of any one of the preceding embodiments of Set I, further comprising a nucleic acid encoding a retroviral packaging signal and further comprising a donor template nucleic acid complementary to a target nucleic acid.

[00455] Embodiment 1-22. The XDP system of embodiment 21, wherein the donor template nucleic acid sequence comprises at least a portion of a target nucleic acid gene or a regulatory element of the target nucleic acid gene.

[00456] Embodiment 1-23. The XDP system of embodiment 21 or embodiment 22, wherein the donor template nucleic acid sequence comprises a corrective sequence for a mutation in the target nucleic acid gene or regulatory element of the target nucleic acid gene.

[00457] Embodiment 1-24. The XDP system of embodiment 21 or embodiment 22, wherein the donor template nucleic acid sequence comprises a mutation compared to the target nucleic acid gene or regulatory element of the target nucleic acid gene.

[00458] Embodiment 1-25. The XDP system of embodiment 24, where the mutation is an insertion, a deletion, or a substitution of one or more nucleotides in the donor template nucleic acid sequence.

[00459] Embodiment 1-26. The XDP system of any one of the preceding embodiments of Set I, wherein the guide RNA is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[00460] Embodiment 1-27. The XDP system of embodiment 26, wherein the guide RNA scaffold sequence has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group of sequences consisting of SEQ ID NOS: 4, 5, and 597-781.

[00461] Embodiment 1-28. The XDP system of embodiment 26 or embodiment 27, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides. [00462] Embodiment 1-29. The XDP system of embodiment 28, wherein the targeting sequence of the guide RNA consists of 20 nucleotides.

[00463] Embodiment 1-30. The XDP system of embodiment 28, wherein the targeting sequence of the guide RNA consists of 19 nucleotides.

[00464] Embodiment 1-31. The XDP system of embodiment 28, wherein the targeting sequence of the guide RNA consists of 18 nucleotides.

[00465] Embodiment 1-32. The XDP system of embodiment 28, wherein the targeting sequence of the guide RNA consists of 17 nucleotides.

[00466] Embodiment 1-33. The XDP system of embodiment 28, wherein the targeting sequence of the guide RNA consists of 16 nucleotides.

[00467] Embodiment 1-34. The XDP system of embodiment 28, wherein the targeting sequence of the guide RNA consists of 15 nucleotides.

[00468] Embodiment 1-35. The XDP system of any one of the preceding embodiments of Set I, wherein the guide RNA further comprises one or more ribozymes.

[00469] Embodiment 1-36. The XDP system of embodiment 35, wherein the one or more ribozymes are independently fused to a terminus of the guide RNA.

[00470] Embodiment 1-37. The XDP system of embodiment 35 or embodiment 36, wherein at least one of the one or more ribozymes are a hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme. [00471] Embodiment 1-38. The XDP system of any one of the preceding embodiments of Set I, wherein the guide RNA is chemically modified.

[00472] Embodiment 1-39. The XDP system of any one of the preceding embodiments of Set I, wherein the CasX protein comprises a sequence having at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, 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%, or at least 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 1.

[00473] Embodiment 1-40. The XDP system of any one of the preceding embodiments of Set I, wherein the CasX protein has binding affinity for a protospacer adjacent motif (PAM) sequence selected from the group consisting of TTC, ATC, GTC, and CTC.

[00474] Embodiment 1-41. The XDP system of any one of the preceding embodiments of Set I, wherein the CasX protein further comprises one or more nuclear localization signals (NLS). [00475] Embodiment 1-42. The XDP system of embodiment 41, wherein the one or more NLS are selected from the group of sequences consisting of SEQ ID NOS: 130-166.

[00476] Embodiment 1-43. The CasX variant of embodiment 41 or embodiment 42, wherein the one or more NLS are expressed at the C-terminus of the CasX protein.

[00477] Embodiment 1-44. The CasX variant of embodiment 41 or embodiment 42, wherein the one or more NLS are expressed at the N-terminus of the CasX protein.

[00478] Embodiment 1-45. The CasX variant of embodiment 41 or embodiment 42, wherein the one or more NLS are expressed at the N-terminus and C-terminus of the CasX protein. [00479] Embodiment 1-46. The XDP system of any one of the preceding embodiments of Set I, wherein the CasX protein comprises a nuclease domain having nickase activity.

[00480] Embodiment 1-47. The XDP system of any one of embodiments 1-45, wherein the CasX protein comprises a nuclease domain having double-stranded cleavage activity.

[00481] Embodiment 1-48. The XDP system of any one of embodiments 1-45, wherein the CasX protein is a catalytically inactive CasX (dCasX) protein, and wherein the dCasX and the guide RNA retain the ability to bind to the target nucleic acid.

[00482] Embodiment 1-49. The XDP system of embodiment 48, wherein the dCasX comprises a mutation at residues: a. D672, E769, and/or D935 corresponding to the CasX protein of SEQ ID NO: 1; or b. D659, E756 and/or D922 corresponding to the CasX protein of SEQ ID NO: 2.

[00483] Embodiment 1-50. The XDP system of embodiment 49, wherein the mutation is a substitution of alanine for the residue.

[00484] Embodiment 1-51. The XDP system of any one of the preceding embodiments of Set I, wherein the envelope glycoprotein is derived from an enveloped virus selected from the group consisting of influenza A, influenza B, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, Norwalk virus, enteric adenovirus, parvovirus, Dengue fever virus, monkey pox, Mononegavirales, rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1, European bat virus 2,

Australian bat virus, Ephemerovirus, Vesiculovirus, vesicular stomatitis virus (VSV), herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesvirus (HHV), human herpesvirus type 6, human herpesvirus type 8, human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Crimean-Congo hemorrhagic fever virus, Hantavirus, Rift Valley fever virus, Ebola hemorrhagic fever virus, Marburg hemorrhagic fever virus, Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, tick-borne encephalitis causing virus, Hendra virus, Nipah virus, variola major virus, variola minor virus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS- associated coronavirus (SARS-CoV), and West Nile virus.

[00485] Embodiment 1-52. The XDP system of embodiment 51, wherein the envelope glycoprotein is derived from vesicular stomatitis virus (VSV).

[00486] Embodiment 1-53. The XDP system of any one of embodiments 1-50, wherein the antibody fragment has binding affinity for a cell surface marker or receptor of a target cell. [00487] Embodiment 1-54. The XDP system of embodiment 53, wherein the antibody fragment is a scFv.

[00488] Embodiment 1-55. A eukaryotic cell comprising the XDP system of any one of the preceding embodiments of Set I.

[00489] Embodiment 1-56. The eukaryotic cell of embodiment 54, wherein the cell is a packaging cell.

[00490] Embodiment 1-57. The eukaryotic cell of embodiment 55 or embodiment 56, wherein the eukaryotic cell is selected from the group consisting of HEK293 cells, Lenti-X 293T cells, BHK cells, HepG2, Saos-2, HuH7, NS0 cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO, NIH3T3 cells, COS, WI38, MRC5, A549, HeLa cells, CHO cells, or HT1080 cells.

[00491] Embodiment 1-58. The eukaryotic cell of embodiment 56 or embodiment 57, wherein the packaging cell comprises one or more mutations to reduce expression of a cell surface marker.

[00492] Embodiment 1-59. A method of making an XDP comprising a CasX protein, the method comprising: a. introducing the XDP system of any one of embodiments 1-54 into the packaging cell of any one of embodiments 56-58; b. propagating the packaging cell under conditions such that XDPs are produced; and c. harvesting the XDPs produced by the packaging cell.

[00493] Embodiment 1-60. An XDP produced by the method of embodiment 59. [00494] Embodiment 1-61. An XDP comprising: a. a retroviral capsid (CA), matrix, (MA), and nucleocapsid (NC) polypeptides b. a pseudotyping viral envelope glycoprotein or an antibody fragment that provides for binding and fusion to a target cell; and c. a CasX protein and a guide RNA associated together in a ribonuclear protein complex (RNP) within the XDP.

[00495] Embodiment 1-62. The XDP of embodiment 61, comprising the CasX of any one of embodiments 39-50 and the guide RNA of any one of embodiments 26-38.

[00496] Embodiment 1-63. The XDP of embodiment 61, wherein the pseudotyping viral envelope glycoprotein is derived from the packaging cell of embodiment 57 or embodiment 58 or a nucleic acid encoding the glycoprotein introduced into the packaging cell.

[00497] Embodiment 1-64. The XDP of embodiment 60-63, further comprising a donor template nucleic acid sequence of any one of embodiments 21-25.

[00498] Embodiment 1-65. A method of method of modifying a target nucleic acid sequence in a cell, the method comprising contacting the cell with the XDP of any one of embodiments 60-64, wherein said contacting comprises introducing into the cell the CasX, the guide RNA, and, optionally, the donor template nucleic acid sequence, resulting in modification of the target nucleic acid sequence.

[00499] Embodiment 1-66. The method of embodiment 65, wherein the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence. [00500] Embodiment 1-67. The method of embodiment 65, wherein the modification comprises introducing a double-stranded break in the target nucleic acid sequence.

[00501] Embodiment 1-68. The method of any one of embodiments 65-67, wherein the modification comprises insertion of the donor template into the target nucleic acid sequence. [00502] Embodiment 1-69. The method of any one of embodiments 65-68, wherein the cell is modified in vitro.

[00503] Embodiment 1-70. The method of any one of embodiments 65-68, wherein the cell is modified in vivo.

[00504] Embodiment 1-71. The method of embodiment 70, wherein the XDP is administered to a subject.

[00505] Embodiment 1-72. The method of embodiment 71, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human. [00506] Embodiment 1-73. The method of embodiment 71 or embodiment 72, wherein the XDP is administered by a route of administration selected from the group consisting of intravenous, intracerebroventricular, intracistemal, intrathecal, intracranial, lumbar, intratracheal, intraosseous, inhalatory, intracontralateral striatum, intraocular, intravitreal, and sub-retinal routes.

[00507] Embodiment 1-74. The method of any one of embodiments 71-73, wherein the XDP is administered to the subject using a therapeutically effective dose.

[00508] Embodiment 1-75. The method of embodiment 74, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles, or at least about 1 x 10⁶ particles, or at least about 1 x 10⁷ particles, or at least about 1 x 10⁸ particles, or at least about 1 x 10⁹ particles, or at least about 1 x 10¹⁰ particles, or at least about 1 x 10¹¹ particles, or at least about 1 x 10¹² particles, or at least about 1 x 10¹³ particles, or at least about 1 x 10¹⁴ particles, or at least about 1 x 10¹⁵ particles, or at least about 1 x 10¹⁶ particles.

Set

[00509] Embodiment II- 1. A CasX delivery particle (XDP) system comprising one or more nucleic acids comprising sequences encoding components selected from: a. a matrix polypeptide (MA); b. a capsid polypeptide (CA); c. a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC); d. a CasX protein; e. a guide nucleic acid (gNA); f. a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell; g. an RNA binding domain; h. a protease cleavage site; i. a gag-transframe region-pol protease polyprotein (gag-TFR-PR); j . a gag-pol polyprotein; and k. a protease capable of cleaving the protease cleavage sites.

[00510] Embodiment II-2. The XDP system of Embodiment II- 1, wherein the encoded components comprise the gag polyprotein, the protease cleavage site, the CasX protein, the gag- pol polyprotein, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on two, three, or four individual nucleic acids. [00511] Embodiment II-3. The XDP system of Embodiment II-2, wherein a. a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the gag-pol polyprotein, the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; b. a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the gag-pol polyprotein; and a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; or c. a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; a third nucleic acid encodes the gag-pol polyprotein; and a fourth nucleic acid encodes the gNA.

[00512] Embodiment II-4. The XDP system of Embodiment II- 1, wherein the encoded components are selected from the gag-TFR-PR polyprotein, the protease cleavage site, the CasX protein, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00513] Embodiment II-5. The XDP system of Embodiment II-4, wherein a. the components are encoded on a single nucleic acid; b. a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; c. a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00514] Embodiment II-6. The XDP system of Embodiment II- 1, wherein the encoded components are selected from the gag polyprotein, the protease cleavage site, the protease, the CasX protein, the gNA and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00515] Embodiment II-7. The XDP system of Embodiment II-6, wherein a. the components are encoded on a single nucleic acid; b. a first nucleic acid encodes the gag polyprotein, the protease, the CasX protein, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; c. a first nucleic acid encodes the gag polyprotein, the protease, the CasX protein and intervening protease cleavage sites between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00516] Embodiment II-8. The XDP system of Embodiment II- 1, wherein the encoded components are selected from the gag-pol polyprotein, the CasX protein, the protease cleavage site, the gNA, the RNA binding domain, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00517] Embodiment II-9. The XDP system of Embodiment II-8, wherein a. the components are encoded on a single nucleic acid; b. a first nucleic acid encodes the gag-pol polyprotein,, the CasX protein, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the RNA binding domain; or c. a first nucleic acid encodes the gag-pol polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA and the RNA binding domain.

[00518] Embodiment II- 10. The XDP system of Embodiment II- 1, wherein the encoded components are selected from the gag-TFR-PR polyprotein, the CasX protein, the protease cleavage site, the gNA, the RNA binding domain, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00519] Embodiment II- 11. The XDP system of Embodiment II- 10, wherein a. the components are encoded on a single nucleic acid; b. a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the RNA binding domain; or c. a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA and the RNA binding domain.

[00520] Embodiment 11-12. The XDP system of any one of Embodiments II-8-11, wherein the RNA binding domain is a retroviral Psi packaging element inserted into the gNA or is a protein selected from the group consisting of MS2, PP7 or Qbeta, U1A, phage replication loop, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, and pseudoknots.

[00521] Embodiment 11-13. The XDP system of Embodiment II- 1, wherein the encoded components are selected from the gag-pol polyprotein, the CasX protein, the protease cleavage site, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gNA, wherein the components are encoded on one, two, or three individual nucleic acids.

[00522] Embodiment 11-14. The XDP system of Embodiment 11-13, wherein a. the components are encoded on a single nucleic acid; b. a first nucleic acid encodes the gag-pol polyprotein, an intervening protease cleavage site, the CasX protein; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; or c. a first nucleic acid encodes the gag-pol polyprotein, an intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00523] Embodiment 11-15. The XDP system of Embodiment II- 1, wherein the encoded components are selected from the MA, the CasX protein, the protease, the protease cleavage site, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, three, or four individual nucleic acids.

[00524] Embodiment 11-16. The XDP system of Embodiment 11-15, wherein a. the components are encoded on a single nucleic acid; b. a first nucleic acid encodes the MA, the CasX protein, the protease, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; c. a first nucleic acid encodes the MA, the CasX protein the protease, and intervening protease cleavage sites between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA; or d. a first nucleic acid encodes the MA, an intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; a third nucleic acid encodes the gNA; and a fourth nucleic acid encodes the protease. [00525] Embodiment 11-17. The XDP system of Embodiment 11-15 or Embodiment 11-16, further comprising the CA component linked between the MA and the CasX protein components with intervening protease cleavage sites.

[00526] Embodiment 11-18. The XDP system of Embodiment II- 1, wherein the encoded components are selected from the gag polyprotein, the CasX protein, the protease, the protease cleavage site, the gNA, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gag-pol polyprotein, wherein the components are encoded on two, three, or four individual nucleic acids.

[00527] Embodiment 11-19. The XDP system of Embodiment 11-18, wherein a. a first nucleic acid encodes the gag polyprotein, the CasX protein, the protease, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the gag-pol polyprotein, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gNA; or b. a first nucleic acid encodes the gag polyprotein, the intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the protease; and a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the gag-pol polyprotein; or c. a first nucleic acid encodes the gag polyprotein, the intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the protease; a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a fourth nucleic acid encodes the gNA and the gag-pol polyprotein.

[00528] Embodiment 11-20. The XDP system of Embodiment II-2 or Embodiment II-3, comprising all or a portion of any one of the nucleic acid sequences of Table 6.

[00529] Embodiment 11-21. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the MA, the CA, the gag-TFR-PR polyprotein, the gag polyprotein, and the gag-pol polyprotein are derived from a retrovirus. [00530] Embodiment 11-22. The XDP system of Embodiment 11-21, wherein the retrovirus is selected from the group consisting of an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, an epsilonretrovirus, and a lentivirus.

[00531] Embodiment 11-23. The XDP system of Embodiment 11-22, wherein the lentivirus is selected from the group consisting of human immunodeficiency- 1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[00532] Embodiment 11-24. The XDP system of Embodiment 11-23, wherein the lentivirus is HIV-1 or SIV.

[00533] Embodiment 11-25. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the gag polypeptide further comprises a p6 polypeptide.

[00534] Embodiment 11-26. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the gag polypeptide comprises a MA polypeptide, a CA polypeptide, a p2 polypeptide, an NC polypeptide, a pi polypeptide, and a p6 polypeptide, and wherein the gag polyprotein comprises one or more protease cleavage sites located between one or more of: a. the MA polypeptide and the CA polypeptide; b. the CA polypeptide and the p2 polypeptide; c. the p2 polypeptide and the NC polypeptide; d. the NC polypeptide and the pi polypeptide; and e. the pi polypeptide and the p6 polypeptide.

[00535] Embodiment 11-27. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the protease capable of cleaving the protease cleavage site is selected from the group of proteases consisting of HIV-1 protease, tobacco etch virus protease (TEV), potyvirus HC protease, potyvirus PI protease, PreScission, b virus NIa protease, B virus RNA-2- encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, cathepsin, thrombin, factor Xa, metalloproteinase-2 (MMP-2), MMP -3, MMP-7, MMP-9, MMP-10, MMP-11, and enterokinase.

[00536] Embodiment 11-28. The XDP system of Embodiment 11-27, wherein the protease capable of cleaving the protease cleavage site is HIV-1 protease. [00537] Embodiment 11-29. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the pseudotyping viral envelope glycoprotein is derived from an enveloped virus selected from the group consisting of Argentine hemorrhagic fever virus, Australian bat virus, Autographa californica multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Borna disease virus, Breda virus, Bunyamwera virus, Chandipura virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue 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 virus 2, 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-lymphotro pic 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 virus, 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 virus, rubella virus, Sabia-associated hemorrhagic fever virus, SARS-associated coronavirus (SARS-CoV), Sendai virus, Tacaribe virus, Thogotovirus, tick-borne encephalitis causing virus, varicella zoster virus (HHV3), varicella zoster virus (HHV3), variola major virus, variola minor virus, Venezuelan equine encephalitis virus, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus (VSV), Vesiculovirus, West Nile virus, western equine encephalitis virus, and Zika Virus.

[00538] Embodiment 11-30. The XDP system of Embodiment 11-29, wherein the pseudotyping viral envelope glycoprotein is derived from vesicular stomatitis virus (VSV). [00539] Embodiment II-31. The XDP system of any one of Embodiments II- 1-29, wherein the pseudotyping viral envelope glycoprotein comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 4.

[00540] Embodiment 11-32. The XDP system of any one of Embodiments II- 1-28, wherein the antibody fragment has binding affinity for a cell surface marker or receptor of a target cell. [00541] Embodiment 11-33. The XDP system of Embodiment 11-32, wherein the antibody fragment is a scFv.

[00542] Embodiment 11-34. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the gNA is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[00543] Embodiment 11-35. The XDP system of Embodiment 11-29, wherein the guide RNA scaffold sequence has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group of sequences consisting of SEQ ID NOS: 4, 5, and 2101-2241.

[00544] Embodiment 11-36. The XDP system of Embodiment 11-29 or Embodiment II- Embodiment 11-35, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[00545] Embodiment 11-37. The XDP system of Embodiment 11-36, wherein the targeting sequence of the guide RNA consists of 20 nucleotides.

[00546] Embodiment 11-38. The XDP system of Embodiment 11-36, wherein the targeting sequence of the guide RNA consists of 19 nucleotides.

[00547] Embodiment 11-39. The XDP system of Embodiment 11-36, wherein the targeting sequence of the guide RNA consists of 18 nucleotides.

[00548] Embodiment 11-40. The XDP system of Embodiment 11-36, wherein the targeting sequence of the guide RNA consists of 17 nucleotides.

[00549] Embodiment 11-41. The XDP system of Embodiment 11-36, wherein the targeting sequence of the guide RNA consists of 16 nucleotides.

[00550] Embodiment 11-42. The XDP system of Embodiment 11-36, wherein the targeting sequence of the guide RNA consists of 15 nucleotides. [00551] Embodiment 11-43. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the guide RNA further comprises one or more ribozymes.

[00552] Embodiment 11-44. The XDP system of Embodiment 11-43, wherein the one or more ribozymes are independently fused to a terminus of the guide RNA.

[00553] Embodiment 11-45. The XDP system of Embodiment 11-43 or Embodiment 11-44, wherein at least one of the one or more ribozymes is a hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

[00554] Embodiment 11-46. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the guide RNA is chemically modified.

[00555] Embodiment 11-47. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the CasX protein comprises a sequence having at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, 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%, or at least 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 1.

[00556] Embodiment 11-48. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the CasX protein has binding affinity for a protospacer adjacent motif (PAM) sequence selected from the group consisting of TTC, ATC, GTC, and CTC.

[00557] Embodiment 11-49. The XDP system of Embodiment 11-48, wherein the binding affinity of the CasX protein for the PAM sequence is at least 1.5-fold greater compared to the binding affinity of any one of the CasX proteins of SEQ ID NOS: 1-3 for the PAM sequences. [00558] Embodiment 11-50. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the CasX protein further comprises one or more nuclear localization signals (NLS).

[00559] Embodiment II-51. The XDP system of Embodiment 11-50, wherein the one or more NLS are selected from the group of sequences consisting of PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP,

NQ S SNF GPMKGGNF GGRS S GP Y GGGGQ YF AKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEV S VELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDE VDGVDE V AKKK SKK, RKCLQAGMNLEARKTKK, PRPRKIPR, PPRKKRTVV, NLSKKKKRKREK, RRPSRPFRKP, KRPRSPSS, KRGINDRNFWRGENERKTR, PRPPKMARYDN, KRSFSKAF, KLKIKRPVK, PKTRRRPRRSQRKRPPT, RRKKRRPRRKKRR, PKKK SRKPKKK SRK, HKKKHPDASVNFSEFSK, QRPGPYDRPQRPGPYDRP, LSPSLSPLLSPSLSPL, RGKGGKGLGKGGAKRHRK, PKRGRGRPKRGRGR, and MSRRRKANPTKLSENAKKLAKEVEN.

[00560] Embodiment 11-52. The CasX variant of Embodiment 11-50 or Embodiment 11-51, wherein the one or more NLS are fused to the C-terminus of the CasX protein.

[00561] Embodiment 11-53. The CasX variant of Embodiment 11-50 or Embodiment 11-51, wherein the one or more NLS are fused to the N-terminus of the CasX protein.

[00562] Embodiment 11-54. The CasX variant of Embodiment 11-50 or Embodiment 11-51, wherein the one or more NLS are fused to the N-terminus and C-terminus of the CasX protein. [00563] Embodiment 11-55. The XDP system of any one of the preceding embodiments of Set I of Set II, wherein the CasX protein comprises a nuclease domain having nickase activity. [00564] Embodiment 11-56. The XDP system of any one of Embodiments II- 1-54, wherein the CasX protein comprises a nuclease domain having double-stranded cleavage activity.

[00565] Embodiment 11-57. The XDP system of any one of the preceding embodiments of Set I of Set II, further comprising a nucleic acid encoding a retroviral packaging signal.

[00566] Embodiment 11-58. The XDP system of any one of the preceding embodiments of Set I of Set II, further comprising a donor template nucleic acid complementary to a target nucleic acid.

[00567] Embodiment 11-59. The XDP system of Embodiment 11-58, wherein the donor template comprises two homologous arms complementary to sequences flanking a cleavage site in the target nucleic acid.

[00568] Embodiment 11-60. The XDP system of Embodiment 11-58 or Embodiment 11-59, wherein the donor template nucleic acid sequence comprises a corrective sequence for a mutation in the target nucleic acid.

[00569] Embodiment 11-61. The XDP system of Embodiment 11-58 or Embodiment 11-59, wherein the donor template nucleic acid sequence comprises a mutation compared to the target nucleic acid. [00570] Embodiment 11-62. The XDP system of Embodiment 11-61, where the mutation is an insertion, a deletion, or a substitution of one or more nucleotides in the donor template nucleic acid sequence.

[00571] Embodiment 11-63. The XDP system of any one of Embodiments II- 1-54, wherein the CasX protein is a catalytically inactive CasX (dCasX) protein, and wherein the dCasX and the guide RNA retain the ability to bind to the target nucleic acid.

[00572] Embodiment 11-64. The XDP system of Embodiment 11-63, wherein the dCasX comprises a mutation at residues: a. D672, E769, and/or D935 corresponding to the CasX protein of SEQ ID NO: 1; or b. D659, E756 and/or D922 corresponding to the CasX protein of SEQ ID NO: 2.

[00573] Embodiment 11-65. The XDP system of Embodiment 11-64, wherein the mutation is a substitution of alanine for the residue.

[00574] Embodiment 11-66. A eukaryotic cell comprising the XDP system of any one of the preceding embodiments of Set I of Set II.

[00575] Embodiment 11-67. The eukaryotic cell of Embodiment 11-66, wherein the cell is a packaging cell.

[00576] Embodiment 11-68. The eukaryotic cell of any one of Embodiments 11-66 or Embodiment 11-67, wherein the eukaryotic cell is selected from the group consisting of HEK293 cells, Lenti-X 293T cells, BHK cells, HepG2, Saos-2, HuH7, NS0 cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO, NIH3T3 cells, COS, WI38, MRC5, A549, HeLa cells, CHO cells, and HT1080 cells.

[00577] Embodiment 11-69. The eukaryotic cell of Embodiment 11-67 or Embodiment 11-68, wherein the packaging cell comprises one or more mutations to reduce expression of a cell surface marker.

[00578] Embodiment 11-70. The eukaryotic cell of any one of Embodiments 11-66-69, wherein all or a portion of the nucleic acids encoding the XDP system of any one of Embodiments II- 1- 56 are integrated into the genome of the eukaryotic cell.

[00579] Embodiment 11-71. A method of making an XDP comprising a CasX protein and a gNA, the method comprising: a. propagating the packaging cell of any one of Embodiments 11-67-70 under conditions such that XDPs are produced; and b. harvesting the XDPs produced by the packaging cell.

[00580] Embodiment 11-72. An XDP produced by the method of Embodiment 11-71.

[00581] Embodiment 11-73. An XDP comprising one or more components selected from: a. a matrix polypeptide (MA); b. a capsid polypeptide (CA); c. a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC); d. a CasX protein; e. a guide nucleic acid (gNA); f. a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell; and g. an RNA binding domain;

[00582] Embodiment 11-74. The XDP of Embodiment 11-73, wherein the XDP comprises a. the matrix polypeptide (MA); b. the pseudotyping viral envelope glycoprotein or antibody fragment; and c. the CasX and the gNA contained within the XDP.

[00583] Embodiment 11-75. The XDP of Embodiment 11-74, further comprising the capsid polypeptide (CA).

[00584] Embodiment 11-76. The XDP of Embodiment 11-74 or Embodiment 11-75, further comprising the nucleocapsid polypeptide (NC).

[00585] Embodiment 11-77. The XDP of any one of Embodiments 11-74-76, further comprising an RNA binding domain.

[00586] Embodiment 11-78. The XDP of Embodiment 11-77, wherein the RNA binding domain is a retroviral Psi packaging element inserted into the gNA or is a protein selected from the group consisting of MS2, PP7 or Qbeta, U1A, phage replication loop, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, and pseudoknots.

[00587] Embodiment 11-79. The XDP of any one of Embodiments 11-74-78, wherein the CasX and the gNA are associated together in a ribonuclear protein complex (RNP) within the XDP. [00588] Embodiment 11-80. The XDP of any one of Embodiments 11-74-79, comprising the CasX of any one of Embodiments 11-47-65 and the guide RNA of any one of Embodiments II- 34-46. [00589] Embodiment 11-81. The XDP of any one of Embodiments 11-74-80, wherein the pseudotyping viral envelope glycoprotein comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 4.

[00590] Embodiment 11-82. The XDP of any one of Embodiments 11-73-80, wherein the pseudotyping viral envelope glycoprotein is derived from an enveloped virus selected from the group consisting of influenza A, influenza B, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, Norwalk virus, enteric adenovirus, parvovirus, Dengue fever virus, monkey pox, Mononegavirales, rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1, European bat virus 2,

Australian bat virus, Ephemerovirus, Vesiculovirus, vesicular stomatitis virus (VSV), herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesvirus (HHV), human herpesvirus type 6, human herpesvirus type 8, human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Crimean-Congo hemorrhagic fever virus, Hantavirus, Rift Valley fever virus, Ebola hemorrhagic fever virus, Marburg hemorrhagic fever virus, Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, tick-borne encephalitis causing virus, Hendra virus, Nipah virus, variola major virus, variola minor virus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS- associated coronavirus (SARS-CoV), and West Nile virus.

[00591] Embodiment 11-83. The XDP of any one of Embodiments 11-73-82, further comprising the donor template nucleic acid sequence of any one of Embodiments 11-58-62.

[00592] Embodiment 11-84. A method of method of modifying a target nucleic acid sequence in a cell, the method comprising contacting the cell with the XDP of any one of Embodiments II- 73-83, wherein said contacting comprises introducing into the cell the CasX protein, the guide RNA, and, optionally, the donor template nucleic acid sequence, resulting in modification of the target nucleic acid sequence.

[00593] Embodiment 11-85. The method of Embodiment 11-84, wherein the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence. [00594] Embodiment 11-86. The method of Embodiment 11-84, wherein the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence. [00595] Embodiment 11-87. The method of any one of Embodiments 11-84-86, wherein the modification comprises insertion of the donor template into the target nucleic acid sequence. [00596] Embodiment 11-88. The method of any one of Embodiments 11-84-87, wherein the cell is modified in vitro.

[00597] Embodiment 11-89. The method of any one of Embodiments 11-84-87, wherein the cell is modified in vivo.

[00598] Embodiment 11-90. The method of Embodiment 11-89, wherein the XDP is administered to a subject.

[00599] Embodiment 11-91. The method of Embodiment 11-90, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human. [00600] Embodiment 11-92. The method of Embodiment 11-90 or Embodiment 11-91, wherein the XDP is 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 routes and sub-retinal routes.

[00601] Embodiment 11-93. The method of any one of Embodiments 11-90-92, wherein the XDP is administered to the subject using a therapeutically effective dose.

[00602] Embodiment 11-94. The method of Embodiment 11-93, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles, or at least about 1 x 10⁶ particles, or at least about 1 x 10⁷ particles, or at least about 1 x 10⁸ particles, or at least about 1 x 10⁹ particles, or at least about 1 x 10¹⁰ particles, or at least about 1 x 10¹¹ particles, or at least about 1 x 10¹² particles, or at least about 1 x 10¹³ particles, or at least about 1 x 10¹⁴ particles, or at least about 1 x 10¹⁵ particles, or at least about 1 x 10¹⁶ particles.

[00603] Embodiment 11-95. A method for introducing a CasX and gNA RNP into a cell having a target nucleic acid, comprising contacting the cell with the XDP of any one of Embodiments 11-79-83, such that the RNP enters the cell.

[00604] Embodiment 11-96. The method of Embodiment 11-95, wherein the RNP binds to the target nucleic acid. [00605] Embodiment 11-97. The method of Embodiment 11-96, wherein the target nucleic acid is cleaved by the CasX.

[00606] Embodiment 11-98. The method of any one of Embodiments 11-95-97, wherein the cell is modified in vitro.

[00607] Embodiment 11-99. The method of any one of Embodiments 11-95-97, wherein the cell is modified in vivo.

[00608] Embodiment II- 100. The method of Embodiment 11-99, wherein the XDP is administered to a subject.

[00609] Embodiment II- 101. The method of Embodiment II- 100, wherein the subj ect is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human. [00610] Embodiment 11-102. The method of any one of Embodiments II-99-101, wherein the XDP is administered to the subject using a therapeutically effective dose.

[00611] Embodiment II- 103. The method of Embodiment 11-102, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles, or at least about 1 x 10⁶ particles, or at least about 1 x 10⁷ particles, or at least about 1 x 10⁸ particles, or at least about 1 x 10⁹ particles, or at least about 1 x 10¹⁰ particles, or at least about 1 x 10¹¹ particles, or at least about 1 x 10¹² particles, or at least about 1 x 10¹³ particles, or at least about 1 x 10¹⁴ particles, or at least about 1 x 10¹⁵ particles, or at least about 1 x 10¹⁶ particles.

Set III

[00612] Embodiment III- 1. A CasX delivery particle (XDP) system comprising one or more nucleic acids comprising sequences encoding components selected from:

(a) a matrix polypeptide (MA);

(b) a capsid polypeptide (CA);

(c) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC);

(d) a CasX protein;

(e) a guide nucleic acid (gNA);

(f) a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell;

(g) an RNA binding domain;

(h) a protease cleavage site; (i) a gag-transframe region-pol protease polyprotein (gag-TFR-PR);

(j) a gag-pol polyprotein; and

(k) a protease capable of cleaving the protease cleavage sites.

[00613] Embodiment III-2. The XDP system of Embodiment III- 1 , wherein the encoded components comprise the gag polyprotein, the protease cleavage site, the CasX protein, the gag- pol polyprotein, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on two, three, or four individual nucleic acids. Embodiment III-3. The XDP system of Embodiment III-2, wherein

(a) a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the gag-pol polyprotein, the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA;

(b) a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the gag-pol polyprotein; and a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; or

(c) a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; a third nucleic acid encodes the gag-pol polyprotein; and a fourth nucleic acid encodes the gNA.

[00614] Embodiment III-4. The XDP system of Embodiment III- 1 , wherein the encoded components are selected from the gag-TFR-PR polyprotein, the protease cleavage site, the CasX protein, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00615] Embodiment III-5. The XDP system of Embodiment III-4, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA;

(c) a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00616] Embodiment III-6. The XDP system of Embodiment III- 1 , wherein the encoded components are selected from the gag polyprotein, the protease cleavage site, the protease, the CasX protein, the gNA and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00617] Embodiment III-7. The XDP system of Embodiment III-6, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag polyprotein, the protease, the CasX protein, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA;

(c) a first nucleic acid encodes the gag polyprotein, the protease, the CasX protein and intervening protease cleavage sites between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00618] Embodiment III-8. The XDP system of Embodiment III- 1 , wherein the encoded components are selected from the gag-pol polyprotein, the CasX protein, the protease cleavage site, the gNA, the RNA binding domain, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00619] Embodiment III-9. The XDP system of Embodiment III-8, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag-pol polyprotein, the CasX protein, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the RNA binding domain; or

(c) a first nucleic acid encodes the gag-pol polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA and the RNA binding domain.

[00620] Embodiment III- 10. The XDP system of Embodiment III- 1 , wherein the encoded components are selected from the gag-TFR-PR polyprotein, the CasX protein, the protease cleavage site, the gNA, the RNA binding domain, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00621] Embodiment III- 11. The XDP system of Embodiment III- 10, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the RNA binding domain; or

(c) a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA and the RNA binding domain.

[00622] Embodiment III- 12. The XDP system of any one of Embodiments III- 8-11, wherein the RNA binding domain is a retroviral Psi packaging element inserted into the gNA or is a protein selected from the group consisting of MS2, PP7 or Qbeta, U1A, phage replication loop, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, and pseudoknots.

[00623] Embodiment III- 13. The XDP system of Embodiment III- 1 , wherein the encoded components are selected from the gag-pol polyprotein, the CasX protein, the protease cleavage site, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gNA, wherein the components are encoded on one, two, or three individual nucleic acids.

[00624] Embodiment III- 14. The XDP system of Embodiment III- 13 , wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag-pol polyprotein, an intervening protease cleavage site, the CasX protein; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; or

(c) a first nucleic acid encodes the gag-pol polyprotein, an intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00625] Embodiment III- 15. The XDP system of Embodiment III- 1 , wherein the encoded components are selected from the MA, the CasX protein, the protease, the protease cleavage site, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, three, or four individual nucleic acids.

[00626] Embodiment III- 16. The XDP system of Embodiment III- 15, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the MA, the CasX protein, the protease, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA;

(c) a first nucleic acid encodes the MA, the CasX protein the protease, and intervening protease cleavage sites between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA; or

(d) a first nucleic acid encodes the MA, an intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; a third nucleic acid encodes the gNA; and a fourth nucleic acid encodes the protease.

[00627] Embodiment III- 17. The XDP system of Embodiment III- 15 or Embodiment III- 16, further comprising the CA component linked between the MA and the CasX protein components with intervening protease cleavage sites.

[00628] Embodiment III- 18. The XDP system of Embodiment III- 1 , wherein the encoded components are selected from the gag polyprotein, the CasX protein, the protease, the protease cleavage site, the gNA, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gag-pol polyprotein, wherein the components are encoded on two, three, or four individual nucleic acids.

[00629] Embodiment III- 19. The XDP system of Embodiment III- 18, wherein

(a) a first nucleic acid encodes the gag polyprotein, the CasX protein, the protease, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the gag-pol polyprotein, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gNA; or

(b) a first nucleic acid encodes the gag polyprotein, the intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the protease; and a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the gag-pol polyprotein; or (c) a first nucleic acid encodes the gag polyprotein, the intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the protease; a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a fourth nucleic acid encodes the gNA and the gag-pol polyprotein.

[00630] Embodiment III-20. The XDP system of Embodiment III-2 or Embodiment III-3, comprising all or a portion of any one of the nucleic acid sequences of Table 6.

[00631] Embodiment III-21. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the MA, the CA, the gag-TFR-PR polyprotein, the gag polyprotein, and the gag-pol polyprotein are derived from a retrovirus.

[00632] Embodiment III-22. The XDP system of Embodiment III-21, wherein the retrovirus is selected from the group consisting of an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, an epsilonretrovirus, and a lentivirus.

[00633] Embodiment III-23. The XDP system of Embodiment III-22, wherein the lentivirus is selected from the group consisting of human immunodeficiency- 1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[00634] Embodiment III-24. The XDP system of Embodiment III-23, wherein the lentivirus is HIV-1 or SIV.

[00635] Embodiment III-25. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the gag polypeptide further comprises a p6 polypeptide.

[00636] Embodiment III-26. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the gag polypeptide comprises a MA polypeptide, a CA polypeptide, a p2 polypeptide, an NC polypeptide, a pi polypeptide, and a p6 polypeptide, and wherein the gag polyprotein comprises one or more protease cleavage sites located between one or more of:

(a) the MA polypeptide and the CA polypeptide;

(b) the CA polypeptide and the p2 polypeptide;

(c) the p2 polypeptide and the NC polypeptide;

(d) the NC polypeptide and the pi polypeptide; and

(e) the pi polypeptide and the p6 polypeptide.

[00637] Embodiment III-27. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the protease capable of cleaving the protease cleavage site is selected from the group of proteases consisting of HIV-1 protease, tobacco etch virus protease (TEV), potyvirus HC protease, potyvirus PI protease, PreScission, b virus NIa protease, B virus RNA-2- encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C4ike protease, PYVF (parsnip yellow fleck virus) 3C-like protease, cathepsin, thrombin, factor Xa, metalloproteinase-2 (MMP-2), MMP -3, MMP-7, MMP-9, MMP-10, MMP-11, and enterokinase.

[00638] Embodiment III-28. The XDP system of Embodiment III-27, wherein the protease capable of cleaving the protease cleavage site is HIV-1 protease.

[00639] Embodiment III-29. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the pseudotyping viral envelope glycoprotein is derived from an enveloped virus selected from the group consisting of Argentine hemorrhagic fever virus, Australian bat virus, Autographa californica multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Borna disease virus, Breda virus, Bunyamwera virus, Chandipura virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue 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 virus 2, 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-lymphotro pic 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 virus, 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 virus, rubella virus, Sabia-associated hemorrhagic fever virus, SARS-associated coronavirus (SARS-CoV), Sendai virus, Tacaribe virus, Thogotovirus, tick-borne encephalitis causing virus, varicella zoster virus (HHV3), varicella zoster virus (HHV3), variola major virus, variola minor virus, Venezuelan equine encephalitis virus, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus (VSV), Vesiculovirus, West Nile virus, western equine encephalitis virus, and Zika Virus.

[00640] Embodiment III-30. The XDP system of Embodiment III-29, wherein the pseudotyping viral envelope glycoprotein is derived from vesicular stomatitis virus (VSV).

[00641] Embodiment III-31. The XDP system of any one of Embodiments III- 1-29, wherein the pseudotyping viral envelope glycoprotein comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 4.

[00642] Embodiment III-32. The XDP system of any one of Embodiments Ill-Embodiments III- 1-28, wherein the antibody fragment has binding affinity for a cell surface marker or receptor of a target cell.

[00643] Embodiment III-33. The XDP system of Embodiment III-32, wherein the antibody fragment is a scFv.

[00644] Embodiment III-34. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the gNA is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[00645] Embodiment III-35. The XDP system of Embodiment III-29, wherein the guide RNA scaffold sequence has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group of sequences consisting of SEQ ID NOS: 4, 5, and 2101-2241.

[00646] Embodiment III-36. The XDP system of Embodiment III-29 or Embodiment III-35, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[00647] Embodiment III-37. The XDP system of Embodiment III-36, wherein the targeting sequence of the guide RNA consists of 20 nucleotides.

[00648] Embodiment III-38. The XDP system of Embodiment III-36, wherein the targeting sequence of the guide RNA consists of 19 nucleotides. [00649] Embodiment III-39. The XDP system of Embodiment III-36, wherein the targeting sequence of the guide RNA consists of 18 nucleotides.

[00650] Embodiment III-40. The XDP system of Embodiment III-36, wherein the targeting sequence of the guide RNA consists of 17 nucleotides.

[00651] Embodiment III-41. The XDP system of Embodiment III-36, wherein the targeting sequence of the guide RNA consists of 16 nucleotides.

[00652] Embodiment III-42. The XDP system of Embodiment III-36, wherein the targeting sequence of the guide RNA consists of 15 nucleotides.

[00653] Embodiment III-43. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the guide RNA further comprises one or more ribozymes.

[00654] Embodiment III-44. The XDP system of Embodiment III-43, wherein the one or more ribozymes are independently fused to a terminus of the guide RNA.

[00655] Embodiment III-45. The XDP system of Embodiment III-43 or Embodiment III-44, wherein at least one of the one or more ribozymes is a hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

[00656] Embodiment III-46. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the guide RNA is chemically modified.

[00657] Embodiment III-47. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the CasX protein comprises a sequence having at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, 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%, or at least 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 1.

[00658] Embodiment III-48. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the CasX protein has binding affinity for a protospacer adjacent motif (PAM) sequence selected from the group consisting of TTC, ATC, GTC, and CTC.

[00659] Embodiment III-49. The XDP system of Embodiment III-48, wherein the binding affinity of the CasX protein for the PAM sequence is at least 1.5-fold greater compared to the binding affinity of any one of the CasX proteins of SEQ ID NOS: 1-3 for the PAM sequences. [00660] Embodiment III-50. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the CasX protein further comprises one or more nuclear localization signals (NLS).

[00661] Embodiment III-51. The XDP system of Embodiment III-50, wherein the one or more NLS are selected from the group of sequences consisting of PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP,

NQ S SNF GPMKGGNF GGRS S GP Y GGGGQ YF AKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEV S VELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDE VDGVDE V AKKK SKK, RKCLQAGMNLEARKTKK, PRPRKIPR, PPRKKRTVV, NLSKKKKRKREK, RRPSRPFRKP, KRPRSPSS, KRGINDRNFWRGENERKTR, PRPPKMARYDN, KRSFSKAF, KLKIKRPVK, PKTRRRPRRSQRKRPPT, RRKKRRPRRKKRR, PKKK SRKPKKK SRK, HKKKHPDASVNFSEFSK, QRPGPYDRPQRPGPYDRP, LSPSLSPLLSPSLSPL, RGKGGKGLGKGGAKRHRK, PKRGRGRPKRGRGR, and MSRRRKANPTKLSENAKKLAKEVEN.

[00662] Embodiment III-52. The CasX variant of Embodiment III-50 or Embodiment III-51, wherein the one or more NLS are fused to the C-terminus of the CasX protein.

[00663] Embodiment III-53. The CasX variant of Embodiment III-50 or Embodiment III-51, wherein the one or more NLS are fused to the N-terminus of the CasX protein.

[00664] Embodiment III-54. The CasX variant of Embodiment III-50 or Embodiment III-51, wherein the one or more NLS are fused to the N-terminus and C-terminus of the CasX protein. [00665] Embodiment III-55. The XDP system of any one of the preceding embodiments of Set I of Set III, wherein the CasX protein comprises a nuclease domain having nickase activity. [00666] Embodiment III-56. The XDP system of any one of Embodiments Ill-Embodiments III- 1-54, wherein the CasX protein comprises a nuclease domain having double-stranded cleavage activity.

[00667] Embodiment III-57. The XDP system of any one of the preceding embodiments of Set I of Set III, further comprising a nucleic acid encoding a retroviral packaging signal.

[00668] Embodiment III-58. The XDP system of any one of the preceding embodiments of Set I of Set III, further comprising a donor template nucleic acid complementary to a target nucleic acid. [00669] Embodiment III-59. The XDP system of Embodiment III-58, wherein the donor template comprises two homologous arms complementary to sequences flanking a cleavage site in the target nucleic acid.

[00670] Embodiment III-60. The XDP system of Embodiment III-58 or Embodiment III-59, wherein the donor template nucleic acid sequence comprises a corrective sequence for a mutation in the target nucleic acid.

[00671] Embodiment III-61. The XDP system of Embodiment III-58 or Embodiment III-59, wherein the donor template nucleic acid sequence comprises a mutation compared to the target nucleic acid.

[00672] Embodiment III-62. The XDP system of Embodiment III-61, where the mutation is an insertion, a deletion, or a substitution of one or more nucleotides in the donor template nucleic acid sequence.

[00673] Embodiment III-63. The XDP system of any one of Embodiments Ill-Embodiments III- 1-54, wherein the CasX protein is a catalytically inactive CasX (dCasX) protein, and wherein the dCasX and the guide RNA retain the ability to bind to the target nucleic acid.

[00674] Embodiment III-64. The XDP system of Embodiment III-63, wherein the dCasX comprises a mutation at residues:

(a) D672, E769, and/or D935 corresponding to the CasX protein of SEQ ID NO: 1; or

(b) D659, E756 and/or D922 corresponding to the CasX protein of SEQ ID NO: 2. [00675] Embodiment III-65. The XDP system of Embodiment III-64, wherein the mutation is a substitution of alanine for the residue.

[00676] Embodiment III-66. A eukaryotic cell comprising the XDP system of any one of the preceding embodiments of Set I of Set III.

[00677] Embodiment III-67. The eukaryotic cell of Embodiment III-66, wherein the cell is a packaging cell.

[00678] Embodiment III-68. The eukaryotic cell of any one of Embodiments Ill-Embodiments III-66 or Embodiment III-67, wherein the eukaryotic cell is selected from the group consisting of HEK293 cells, Lenti-X 293T cells, BHK cells, HepG2, Saos-2, HuH7, NS0 cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO, NIH3T3 cells, COS, WI38, MRC5, A549, HeLa cells, CHO cells, and HT1080 cells. [00679] Embodiment III-69. The eukaryotic cell of Embodiment III-67 or Embodiment III-68, wherein the packaging cell comprises one or more mutations to reduce expression of a cell surface marker.

[00680] Embodiment III-70. The eukaryotic cell of any one of Embodiments Ill-Embodiments III-66-69, wherein all or a portion of the nucleic acids encoding the XDP system of any one of Embodiments III-1-56 are integrated into the genome of the eukaryotic cell.

[00681] Embodiment III-71. A method of making an XDP comprising a CasX protein and a gNA, the method comprising:

(a) propagating the packaging cell of any one of Embodiments III-67-70 under conditions such that XDPs are produced; and

(b) harvesting the XDPs produced by the packaging cell.

[00682] Embodiment III-72. An XDP produced by the method of Embodiment TTT-71 [00683] Embodiment III-73. An XDP comprising one or more components selected from:

(a) a matrix polypeptide (MA);

(b) a capsid polypeptide (CA);

(c) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide

(CA), and a nucleocapsid polypeptide (NC);

(d) a CasX protein;

(e) a guide nucleic acid (gNA);

(f) a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell; and

(g) an RNA binding domain;

[00684] Embodiment III-74. The XDP of Embodiment III-73, wherein the XDP comprises

(a) the matrix polypeptide (MA);

(b) the pseudotyping viral envelope glycoprotein or antibody fragment; and

(c) the CasX and the gNA contained within the XDP.

[00685] Embodiment III-75. The XDP of Embodiment III-74, further comprising the capsid polypeptide (CA).

[00686] Embodiment III-76. The XDP of Embodiment III-74 or Embodiment III-75, further comprising the nucleocapsid polypeptide (NC).

[00687] Embodiment III-77. The XDP of any one of Embodiments III-74-76, further comprising an RNA binding domain. [00688] Embodiment III-78. The XDP of Embodiment III-77, wherein the RNA binding domain is a retroviral Psi packaging element inserted into the gNA or is a protein selected from the group consisting of MS2, PP7 or Qbeta, U 1 A, phage replication loop, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, and pseudoknots.

[00689] Embodiment III-79. The XDP of any one of Embodiments III-74-78, wherein the CasX and the gNA are associated together in a ribonuclear protein complex (RNP) within the XDP. [00690] Embodiment III-80. The XDP of any one of Embodiments III-74-79, comprising the CasX of any one of Embodiments III-47-65 and the guide RNA of any one of Embodiments III- 34-46.

[00691] Embodiment III-81. The XDP of any one of Embodiments III-74-80, wherein the pseudotyping viral envelope glycoprotein comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 4.

[00692] Embodiment III-82. The XDP of any one of Embodiments III-73-80, wherein the pseudotyping viral envelope glycoprotein is derived from an enveloped virus selected from the group consisting of Argentine hemorrhagic fever virus, Australian bat virus, Autographa californica multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Borna disease virus, Breda virus, Bunyamwera virus, Chandipura virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue 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 virus 2, 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-lymphotro pic 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 virus, 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 virus, rubella virus, Sabia-associated hemorrhagic fever virus, SARS-associated coronavirus (SARS-CoV), Sendai virus, Tacaribe virus, Thogotovirus, tick-borne encephalitis causing virus, varicella zoster virus (HHV3), varicella zoster virus (HHV3), variola major virus, variola minor virus, Venezuelan equine encephalitis virus, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus (VSV), Vesiculovirus, West Nile virus, western equine encephalitis virus, and Zika Virus.

[00693] Embodiment III-83. The XDP of any one of Embodiments III-73-82, further comprising the donor template nucleic acid sequence of any one of Embodiments III-58-62. [00694] Embodiment III-84. A method of method of modifying a target nucleic acid sequence in a cell, the method comprising contacting the cell with the XDP of any one of Embodiments III-73-83, wherein said contacting comprises introducing into the cell the CasX protein, the guide RNA, and, optionally, the donor template nucleic acid sequence, resulting in modification of the target nucleic acid sequence.

[00695] Embodiment III-85. The method of Embodiment III-84, wherein the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence. [00696] Embodiment III-86. The method of Embodiment III-84, wherein the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence. [00697] Embodiment III-87. The method of any one of Embodiments III-84-86, wherein the modification comprises insertion of the donor template into the target nucleic acid sequence. [00698] Embodiment III-88. The method of any one of Embodiments III-84-87, wherein the cell is modified in vitro.

[00699] Embodiment III-89. The method of any one of Embodiments III-84-87, wherein the cell is modified in vivo.

[00700] Embodiment III-90. The method of Embodiment III-89, wherein the XDP is administered to a subject.

[00701] Embodiment III-91. The method of Embodiment III-90, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human. [00702] Embodiment III-92. The method of Embodiment III-90 or Embodiment III-91, wherein the XDP is 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 routes and sub-retinal routes.

[00703] Embodiment III-93. The method of any one of Embodiments III-90-92, wherein the XDP is administered to the subject using a therapeutically effective dose.

[00704] Embodiment III-94. The method of Embodiment III-93, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles, or at least about 1 x 10⁶ particles, or at least about 1 x 10⁷ particles, or at least about 1 x 10⁸ particles, or at least about 1 x 10⁹ particles, or at least about 1 x 10¹⁰ particles, or at least about 1 x 10¹¹ particles, or at least about 1 x 10¹² particles, or at least about 1 x 10¹³ particles, or at least about 1 x 10¹⁴ particles, or at least about 1 x 10¹⁵ particles, or at least about 1 x 10¹⁶ particles.

[00705] Embodiment III-95. A method for introducing a CasX and gNA RNP into a cell having a target nucleic acid, comprising contacting the cell with the XDP of any one of Embodiments III-79-83, such that the RNP enters the cell.

[00706] Embodiment III-96. The method of Embodiment III-95, wherein the RNP binds to the target nucleic acid.

[00707] Embodiment III-97. The method of Embodiment III-96, wherein the target nucleic acid is cleaved by the CasX.

[00708] Embodiment III-98. The method of any one of Embodiments III-95-97, wherein the cell is modified in vitro.

[00709] Embodiment III-99. The method of any one of Embodiments III-95-97, wherein the cell is modified in vivo.

[00710] Embodiment III- 100. The method of Embodiment III-99, wherein the XDP is administered to a subject.

[00711] Embodiment III- 101. The method of Embodiment III- 100, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

[00712] Embodiment III- 102. The method of any one of Embodiments III-99- 101, wherein the XDP is administered to the subject using a therapeutically effective dose. [00713] Embodiment III- 103. The method of Embodiment III- 102, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles, or at least about 1 x 10⁶ particles, or at least about 1 x 10⁷ particles, or at least about 1 x 10⁸ particles, or at least about 1 x 10⁹ particles, or at least about 1 x 10¹⁰ particles, or at least about 1 x 10¹¹ particles, or at least about 1 x 10¹² particles, or at least about 1 x 10¹³ particles, or at least about 1 x 10¹⁴ particles, or at least about 1 x 10¹⁵ particles, or at least about 1 x 10¹⁶ particles.

Set IV

[00714] Embodiment IV-1. A delivery particle (XDP) system for CasX and one or more nucleic acids comprising sequences encoding one or more components selected from (a) to (o) or encoding one or more portions of the components selected from (a) to (o):

(a) a matrix polypeptide (MA);

(b) a capsid polypeptide (CA);

(c) a nucelocapsid polypeptide (NC);

(d) a pi spacer peptide;

(e) a p2 spacer peptide;

(f) p6 spacer peptide;

(g) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a pi spacer, and a p6 spacer;

(h) a CasX protein;

(i) a guide nucleic acid (gNA);

(j) a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell;

(k) an RNA binding domain;

(l) a protease cleavage site;

(m) a gag-transframe region-pol protease polyprotein (gag-TFR-PR);

(n) a gag-pol polyprotein; and

(o) a protease capable of cleaving the protease cleavage sites.

[00715] Embodiment IV-2. The XDP system of Embodiment IV-1, wherein the encoded components comprise the gag polyprotein, the protease cleavage site, the CasX protein, the gag- pol polyprotein, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on two, three, or four individual nucleic acids. [00716] Embodiment IV-3. The XDP system of Embodiment IV-2, wherein

(a) a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the gag-pol polyprotein, the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA;

(b) a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the gag-pol polyprotein; and a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; or

(c) a first nucleic acid encodes the gag polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; a third nucleic acid encodes the gag-pol polyprotein; and a fourth nucleic acid encodes the gNA.

[00717] Embodiment IV-4. The XDP system of Embodiment IV- 1, wherein the encoded components are selected from the gag-TFR-PR polyprotein, the protease cleavage site, the CasX protein, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00718] Embodiment IV-5. The XDP system of Embodiment IV-4, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA;

(c) a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00719] Embodiment IV-6. The XDP system of Embodiment IV-1, wherein the encoded components are selected from the gag polyprotein, the protease cleavage site, the protease, the CasX protein, the gNA and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00720] Embodiment IV-7. The XDP system of Embodiment IV-6, wherein

(a) the components are encoded on a single nucleic acid; (b) a first nucleic acid encodes the gag polyprotein, the protease, the CasX protein, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA;

(c) a first nucleic acid encodes the gag polyprotein, the protease, the CasX protein and intervening protease cleavage sites between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00721] Embodiment IV-8. The XDP system of Embodiment IV- 1, wherein the encoded components are selected from the gag-pol polyprotein, the CasX protein, the protease cleavage site, the gNA, the RNA binding domain, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00722] Embodiment IV-9. The XDP system of Embodiment IV-8, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag-pol polyprotein, the CasX protein, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the RNA binding domain; or

(c) a first nucleic acid encodes the gag-pol polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA and the RNA binding domain.

[00723] Embodiment IV-10. The XDP system of Embodiment IV-1, wherein the encoded components are selected from the gag-TFR-PR polyprotein, the CasX protein, the protease cleavage site, the gNA, the RNA binding domain, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.

[00724] Embodiment IV-11. The XDP system of Embodiment IV-10, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the RNA binding domain; or

(c) a first nucleic acid encodes the gag-TFR-PR polyprotein, the CasX protein, and an intervening protease cleavage site between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA and the RNA binding domain.

[00725] Embodiment IV-12. The XDP system of any one of Embodiments IV-8-11, wherein the RNA binding domain is a retroviral Psi packaging element inserted into the gNA or is a protein selected from the group consisting of MS2, PP7 or Qbeta, U 1 A, phage replication loop, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, and pseudoknots.

[00726] Embodiment IV-13. The XDP system of Embodiment IV-1, wherein the encoded components are selected from the gag-pol polyprotein, the CasX protein, the protease cleavage site, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gNA, wherein the components are encoded on one, two, or three individual nucleic acids.

[00727] Embodiment IV-14. The XDP system of Embodiment IV-13, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the gag-pol polyprotein, an intervening protease cleavage site, the CasX protein; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; or

(c) a first nucleic acid encodes the gag-pol polyprotein, an intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA.

[00728] Embodiment IV-15. The XDP system of Embodiment IV-1, wherein the encoded components are selected from the MA, the CasX protein, the protease, the protease cleavage site, the gNA, and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, three, or four individual nucleic acids.

[00729] Embodiment IV-16. The XDP system of Embodiment IV-15, wherein

(a) the components are encoded on a single nucleic acid;

(b) a first nucleic acid encodes the MA, the CasX protein, the protease, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment and the gNA; (c) a first nucleic acid encodes the MA, the CasX protein the protease, and intervening protease cleavage sites between the components; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a third nucleic acid encodes the gNA; or

(d) a first nucleic acid encodes the MA, an intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; a third nucleic acid encodes the gNA; and a fourth nucleic acid encodes the protease.

[00730] Embodiment IV-17. The XDP system of Embodiment IV-15 or Embodiment IV-16, further comprising the CA component linked between the MA and the CasX protein components with intervening protease cleavage sites.

[00731] Embodiment IV-18. The XDP system of Embodiment IV- 1, wherein the encoded components are selected from the gag polyprotein, the CasX protein, the protease, the protease cleavage site, the gNA, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gag-pol polyprotein, wherein the components are encoded on two, three, or four individual nucleic acids.

[00732] Embodiment IV-19. The XDP system of Embodiment IV-18, wherein

(a) a first nucleic acid encodes the gag polyprotein, the CasX protein, the protease, and intervening protease cleavage sites between the components; and a second nucleic acid encodes the gag-pol polyprotein, the pseudotyping viral envelope glycoprotein or antibody fragment, and the gNA; or

(b) a first nucleic acid encodes the gag polyprotein, the intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the protease; and a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment, the gNA and the gag-pol polyprotein; or

(c) a first nucleic acid encodes the gag polyprotein, the intervening protease cleavage site, and the CasX protein; a second nucleic acid encodes the protease; a third nucleic acid encodes the pseudotyping viral envelope glycoprotein or antibody fragment; and a fourth nucleic acid encodes the gNA and the gag-pol polyprotein.

[00733] Embodiment IV-20. The XDP system of Embodiment IV-2 or Embodiment IV-3, comprising all or a portion of any one of the nucleic acid sequences of Tables 6-8. [00734] Embodiment IV-21. The XDP system of any one of the preceding embodiments of Set I of Set IV, wherein the MA, the CA, the gag-TFR-PR polyprotein, the gag polyprotein, and the gag-pol polyprotein are derived from a retrovirus.

[00735] Embodiment IV-22. The XDP system of Embodiment IV-21, wherein the retrovirus is selected from the group consisting of an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, an epsilonretrovirus, and a lentivirus.

[00736] Embodiment IV-23. The XDP system of Embodiment IV-22, wherein the lentivirus is selected from the group consisting of human immunodeficiency- 1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[00737] Embodiment IV-24. The XDP system of Embodiment IV-23, wherein the lentivirus is HIV-1 or SIV.

[00738] Embodiment IV-25. The XDP system of any one of the preceding embodiments of Set I of Set IV, wherein the gag polypeptide further comprises a p6 polypeptide.

[00739] Embodiment IV-26. The XDP system of any one Embodiments IV- 1 to 25, wherein the gag polypeptide comprises a MA polypeptide, a CA polypeptide, a p2 polypeptide, an NC polypeptide, a pi polypeptide, and a p6 polypeptide, and wherein the gag polyprotein comprises one or more protease cleavage sites located between one or more of:

(a) the MA polypeptide and the CA polypeptide;

(b) the CA polypeptide and the p2 polypeptide;

(c) the p2 polypeptide and the NC polypeptide;

(d) the NC polypeptide and the pi polypeptide; and

(e) the pi polypeptide and the p6 polypeptide.

[00740] Embodiment IV-27. The XDP system of any one Embodiments IV- 1 to 26, wherein the protease capable of cleaving the protease cleavage site is selected from the group of proteases consisting of HIV- 1 protease, tobacco etch virus protease (TEV), potyvirus HC protease, potyvirus PI protease, PreScission, b virus NIa protease, B virus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2 A protease, rhinovirus 2 A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, cathepsin, thrombin, factor Xa, metalloproteinase-2 (MMP-2), MMP -3, MMP-7, MMP-9, MMP-10, MMP-11, and enterokinase. [00741] Embodiment IV-28. The XDP system of Embodiment IV-27, wherein the protease capable of cleaving the protease cleavage site is HIV-1 protease.

[00742] Embodiment IV-29. The XDP system of any one of Embodiments IV-1 to 28, wherein the pseudotyping viral envelope glycoprotein is derived from an enveloped virus selected from the group consisting of Argentine hemorrhagic fever virus, Australian bat virus, Autographa californica multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Borna disease virus, Breda virus, Bunyamwera virus, Chandipura virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue 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 virus 2, 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-lymphotro pic 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 virus, 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 virus, rubella virus, Sabia-associated hemorrhagic fever virus, SARS-associated coronavirus (SARS-CoV), Sendai virus, Tacaribe virus, Thogotovirus, tick-borne encephalitis causing virus, varicella zoster virus (HHV3), varicella zoster virus (HHV3), variola major virus, variola minor virus, Venezuelan equine encephalitis virus, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus (VSV), Vesiculovirus, West Nile virus, western equine encephalitis virus, and Zika Virus.

[00743] Embodiment IV-30. The XDP system of Embodiment IV-29, wherein the pseudotyping viral envelope glycoprotein is derived from vesicular stomatitis virus (VSV). [00744] Embodiment IV-31. The XDP system of any one of Embodiments IV- 1-29, wherein the pseudotyping viral envelope glycoprotein comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 4.

[00745] Embodiment IV-32. The XDP system of any one of Embodiments IV-1-28, wherein the antibody fragment has binding affinity for a cell surface marker or receptor of a target cell. [00746] Embodiment IV-33. The XDP system of Embodiment IV-32, wherein the antibody fragment is a scFv.

[00747] Embodiment IV-34. The XDP system of any one of the preceding embodiments of Set I of Set IV, wherein the gNA is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[00748] Embodiment IV-35. The XDP system of Embodiment IV-29, wherein the guide RNA scaffold sequence has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group of sequences consisting of SEQ ID NOS: 4, 5, and 2101-2241.

[00749] Embodiment IV-36. The XDP system of Embodiment IV-29 or Embodiment IV-35, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[00750] Embodiment IV-37. The XDP system of Embodiment IV-36, wherein the targeting sequence of the guide RNA consists of 20 nucleotides.

[00751] Embodiment IV-38. The XDP system of Embodiment IV-36, wherein the targeting sequence of the guide RNA consists of 19 nucleotides.

[00752] Embodiment IV-39. The XDP system of Embodiment IV-36, wherein the targeting sequence of the guide RNA consists of 18 nucleotides.

[00753] Embodiment IV-40. The XDP system of Embodiment IV-36, wherein the targeting sequence of the guide RNA consists of 17 nucleotides.

[00754] Embodiment IV-41. The XDP system of Embodiment IV-36, wherein the targeting sequence of the guide RNA consists of 16 nucleotides.

[00755] Embodiment IV-42. The XDP system of Embodiment IV-36, wherein the targeting sequence of the guide RNA consists of 15 nucleotides. [00756] Embodiment IV-43. The XDP system of any one of the preceding embodiments of Set I of Set IV, wherein the guide RNA further comprises one or more ribozymes.

[00757] Embodiment IV-44. The XDP system of Embodiment IV-43, wherein the one or more ribozymes are independently fused to a terminus of the guide RNA.

[00758] Embodiment IV-45. The XDP system of Embodiment IV-43 or Embodiment IV-44, wherein at least one of the one or more ribozymes is a hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

[00759] Embodiment IV-46. The XDP system of any one of the preceding embodiments of Set I of Set IV, wherein the guide RNA is chemically modified.

[00760] Embodiment IV-47. The XDP system of any one of the preceding embodiments of Set I of Set IV, wherein the CasX protein comprises a sequence having at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, 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%, or at least 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 1.

[00761] Embodiment IV-48. The XDP system of any one of the preceding embodiments of Set I of Set IV, wherein the CasX protein has binding affinity for a protospacer adjacent motif (PAM) sequence selected from the group consisting of TTC, ATC, GTC, and CTC.

[00762] Embodiment IV-49. The XDP system of Embodiment IV-48, wherein the binding affinity of the CasX protein for the PAM sequence is at least 1.5-fold greater compared to the binding affinity of any one of the CasX proteins of SEQ ID NOS: 1-3 for the PAM sequences. [00763] Embodiment IV-50. The XDP system of any one Embodiments IV- 1 to 49, wherein, wherein the CasX protein further comprises one or more nuclear localization signals (NLS). [00764] Embodiment IV-51. The XDP system of Embodiment IV-50, wherein the one or more NLS are selected from the group of sequences consisting of PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP,

NQ S SNF GPMKGGNF GGRS S GP Y GGGGQ YF AKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEV S VELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDE VDGVDE V AKKK SKK, RKCLQAGMNLEARKTKK,

PRPRKIPR, PPRKKRTVV, NLSKKKKRKREK, RRPSRPFRKP, KRPRSPSS, KRGINDRNFWRGENERKTR, PRPPKMARYDN, KRSFSKAF, KLKIKRPVK, PKTRRRPRRSQRKRPPT, RRKKRRPRRKKRR, PKKK SRKPKKK SRK, HKKKHPDASVNFSEFSK, QRPGPYDRPQRPGPYDRP, LSPSLSPLLSPSLSPL, RGKGGKGLGKGGAKRHRK, PKRGRGRPKRGRGR, and MSRRRKANPTKLSENAKKLAKEVEN.

[00765] Embodiment IV-52. The CasX variant of Embodiment IV-50 or Embodiment IV- 51, wherein the one or more NLS are fused to the C-terminus of the CasX protein.

[00766] Embodiment IV-53. The CasX variant of Embodiment IV-50 or Embodiment IV-51, wherein the one or more NLS are fused to the N-terminus of the CasX protein.

[00767] Embodiment IV-54. The CasX variant of Embodiment IV-50 or Embodiment IV-51, wherein the one or more NLS are fused to the N-terminus and C-terminus of the CasX protein. [00768] Embodiment IV-55. The XDP system of any one of the preceding embodiments of Set I of Set IV, wherein the CasX protein comprises a nuclease domain having nickase activity. [00769] Embodiment IV-56. The XDP system of any one of Embodiments IV-1-54, wherein the CasX protein comprises a nuclease domain having double-stranded cleavage activity.

[00770] Embodiment IV-57. The XDP system of any one Embodiments IV- 1 to 56, further comprising a nucleic acid encoding a retroviral packaging signal.

[00771] Embodiment IV-58. The XDP system of any one of the preceding embodiments of Set I of Set IV, further comprising a donor template nucleic acid complementary to a target nucleic acid.

[00772] Embodiment IV-59. The XDP system of Embodiment IV-58, wherein the donor template comprises two homologous arms complementary to sequences flanking a cleavage site in the target nucleic acid.

[00773] Embodiment IV-60. The XDP system of Embodiment IV-58 or Embodiment IV-59, wherein the donor template nucleic acid sequence comprises a corrective sequence for a mutation in the target nucleic acid.

[00774] Embodiment IV-61. The XDP system of Embodiment IV-58 or Embodiment IV-59, wherein the donor template nucleic acid sequence comprises a mutation compared to the target nucleic acid.

[00775] Embodiment IV-62. The XDP system of Embodiment IV-61, where the mutation is an insertion, a deletion, or a substitution of one or more nucleotides in the donor template nucleic acid sequence. [00776] Embodiment IV-63. The XDP system of any one of Embodiments IV-1-54, wherein the CasX protein is a catalytically inactive CasX (dCasX) protein, and wherein the dCasX and the guide RNA retain the ability to bind to the target nucleic acid.

[00777] Embodiment IV-64. The XDP system of Embodiment IV-63, wherein the dCasX comprises a mutation at residues:

(a) D672, E769, and/or D935 corresponding to the CasX protein of SEQ ID NO: 1; or

(b) D659, E756 and/or D922 corresponding to the CasX protein of SEQ ID NO: 2. [00778] Embodiment IV-65. The XDP system of Embodiment IV-64, wherein the mutation is a substitution of alanine for the residue.

[00779] Embodiment IV-66. A eukaryotic cell comprising the XDP system of any one of the preceding embodiments of Set I of Set IV.

[00780] Embodiment IV-67. The eukaryotic cell of Embodiment IV-66, wherein the cell is a packaging cell.

[00781] Embodiment IV-68. The eukaryotic cell of any one of Embodiments IV-66 or Embodiment IV-67, wherein the eukaryotic cell is selected from the group consisting of HEK293 cells, Lenti-X 293T cells, BHK cells, HepG2, Saos-2, HuH7, NS0 cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO, NIH3T3 cells, COS, WI38, MRC5, A549, HeLa cells, CHO cells, and HT1080 cells.

[00782] Embodiment IV-69. The eukaryotic cell of Embodiment IV-67 or Embodiment IV-68, wherein the packaging cell comprises one or more mutations to reduce expression of a cell surface marker.

[00783] Embodiment IV-70. The eukaryotic cell of any one of Embodiments IV-66-69, wherein all or a portion of the nucleic acids encoding the XDP system of any one of Embodiments IV- 1- 56 are integrated into the genome of the eukaryotic cell.

[00784] Embodiment IV-71. A method of making an XDP comprising a CasX protein and a gNA, the method comprising:

(a) propagating the packaging cell of any one of Embodiments IV-67-70 under conditions such that XDPs are produced; and

(b) harvesting the XDPs produced by the packaging cell.

[00785] Embodiment IV-72. An XDP produced by the method of Embodiment IV-71. [00786] Embodiment IV-73. An XDP comprising one or more components selected from:

(a) a matrix polypeptide (MA);

(b) a capsid polypeptide (CA);

(c) a gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC);

(d) a CasX protein;

(e) a guide nucleic acid (gNA);

(f) a pseudotyping viral envelope glycoprotein or antibody fragment that provides for binding and fusion of the XDP to a target cell; and

(g) an RNA binding domain;

[00787] Embodiment IV-74. The XDP of Embodiment IV-73, wherein the XDP comprises

(a) the matrix polypeptide (MA);

(b) the pseudotyping viral envelope glycoprotein or antibody fragment; and

(c) the CasX and the gNA contained within the XDP.

[00788] Embodiment IV-75. The XDP of Embodiment IV-74, further comprising the capsid polypeptide (CA).

[00789] Embodiment IV-76. The XDP of Embodiment IV-74 or Embodiment IV-75, further comprising the nucleocapsid polypeptide (NC).

[00790] Embodiment TV-77. The XDP of any one of Embodiments IV-74-76, further comprising an RNA binding domain.

[00791] Embodiment IV-78. The XDP of Embodiment TV-77, wherein the RNA binding domain is a retroviral Psi packaging element inserted into the gNA or is a protein selected from the group consisting of MS2, PP7 or Qbeta, U1A, phage replication loop, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, and pseudoknots.

[00792] Embodiment IV-79. The XDP of any one of Embodiments IV-74-78, wherein the CasX and the gNA are associated together in a ribonuclear protein complex (RNP) within the XDP. [00793] Embodiment IV-80. The XDP of any one of Embodiments IV-74-79, comprising the CasX of any one of Embodiments IV-47-65 and the guide RNA of any one of Embodiments IV- 34-46.

[00794] Embodiment IV-81. The XDP of any one of Embodiments IV-74-80, wherein the pseudotyping viral envelope glycoprotein comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the sequences set forth in Table 4.

[00795] Embodiment IV-82. The XDP of any one of Embodiments IV-73-80, wherein the pseudotyping viral envelope glycoprotein is derived from an enveloped virus selected from the group consisting of Argentine hemorrhagic fever virus, Australian bat virus, Autographa californica multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Borna disease virus, Breda virus, Bunyamwera virus, Chandipura virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue 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 virus 2, 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-lymphotro pic 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 virus, 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 virus, rubella virus, Sabia-associated hemorrhagic fever virus, SARS-associated coronavirus (SARS-CoV), Sendai virus, Tacaribe virus, Thogotovirus, tick-borne encephalitis causing virus, varicella zoster virus (HHV3), varicella zoster virus (HHV3), variola major virus, variola minor virus, Venezuelan equine encephalitis virus, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus (VSV), Vesiculovirus, West Nile virus, western equine encephalitis virus, and Zika Virus.

[00796] Embodiment IV-83. The XDP of any one of Embodiments IV-73-82, further comprising the donor template nucleic acid sequence of any one of Embodiments IV-58-62. [00797] Embodiment IV-84. A method of method of modifying a target nucleic acid sequence in a cell, the method comprising contacting the cell with the XDP of any one of Embodiments IV-73-83, wherein said contacting comprises introducing into the cell the CasX protein, the guide RNA, and, optionally, the donor template nucleic acid sequence, resulting in modification of the target nucleic acid sequence.

[00798] Embodiment IV-85. The method of Embodiment IV-84, wherein the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence. [00799] Embodiment IV-86. The method of Embodiment IV-84, wherein the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence. [00800] Embodiment IV-87. The method of any one of Embodiments IV-84-86, wherein the modification comprises insertion of the donor template into the target nucleic acid sequence. [00801] Embodiment IV-88. The method of any one of Embodiments IV-84-87, wherein the cell is modified in vitro.

[00802] Embodiment IV-89. The method of any one of Embodiments IV-84-87, wherein the cell is modified in vivo.

[00803] Embodiment IV-90. The method of Embodiment IV-89, wherein the XDP is administered to a subject.

[00804] Embodiment IV-91. The method of Embodiment IV-90, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human. [00805] Embodiment IV-92. The method of Embodiment IV-90 or Embodiment IV-91, wherein the XDP is 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 routes and sub-retinal routes.

[00806] Embodiment IV-93. The method of any one of Embodiments IV-90-92, wherein the XDP is administered to the subject using a therapeutically effective dose.

[00807] Embodiment IV-94. The method of Embodiment IV-93, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles, or at least about 1 x 10⁶ particles, or at least about 1 x 10⁷ particles, or at least about 1 x 10⁸ particles, or at least about 1 x 10⁹ particles, or at least about 1 x 10¹⁰ particles, or at least about 1 x 10¹¹ particles, or at least about 1 x 10¹² particles, or at least about 1 x 10¹³ particles, or at least about 1 x 10¹⁴ particles, or at least about 1 x 10¹⁵ particles, or at least about 1 x 10¹⁶ particles.

[00808] Embodiment IV-95. A method for introducing a CasX and gNA RNP into a cell having a target nucleic acid, comprising contacting the cell with the XDP of any one of Embodiments IV-79-83, such that the RNP enters the cell.

[00809] Embodiment IV-96. The method of Embodiment IV-95, wherein the RNP binds to the target nucleic acid.

[00810] Embodiment IV-97. The method of Embodiment IV-96, wherein the target nucleic acid is cleaved by the CasX.

[00811] Embodiment IV-98. The method of any one of Embodiments IV-95-97, wherein the cell is modified in vitro.

[00812] Embodiment IV-99. The method of any one of Embodiments IV-95-97, wherein the cell is modified in vivo.

[00813] Embodiment IV-100. The method of Embodiment IV-99, wherein the XDP is administered to a subject.

[00814] Embodiment IV-101. The method of Embodiment IV-100, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

[00815] Embodiment IV-102. The method of any one of Embodiments IV-99-101, wherein the XDP is administered to the subject using a therapeutically effective dose.

[00816] Embodiment IV-103. The method of Embodiment IV-102, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles, or at least about 1 x 10⁶ particles, or at least about 1 x 10⁷ particles, or at least about 1 x 10⁸ particles, or at least about 1 x 10⁹ particles, or at least about 1 x 10¹⁰ particles, or at least about 1 x 10¹¹ particles, or at least about 1 x 10¹² particles, or at least about 1 x 10¹³ particles, or at least about 1 x 10¹⁴ particles, or at least about 1 x 10¹⁵ particles, or at least about 1 x 10¹⁶ particles.

Set V

[00817] Embodiment V-l. A delivery particle (XDP) system comprising one or more nucleic acids encoding:

(a) one or more retroviral components;

(b) a therapeutic payload; and (c) a tropism factor

[00818] Embodiment V-2. The XDP system of Embodiment V-l, 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.

[00819] Embodiment V-3. The XDP system of Embodiment V-2, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509,

511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,

549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,

587, 589, 591, 593 and 595 as set forth in Table 4, 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.

[00820] Embodiment V-4. The XDP system of Embodiment V-2, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,

549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,

587, 589, 591, 593 and 595.

[00821] Embodiment V-5. The XDP system of any one of the preceding embodiments of Set V, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

[00822] Embodiment V-6. The XDP system of Embodiment V-5, wherein the protein payload 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, and an anti-cancer modality.

[00823] Embodiment V-7. The XDP system of Embodiment V-6, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein. [00824] Embodiment V-8. The XDP system of Embodiment V-7, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of a Type II, a Type V, or a Type VI protein.

[00825] Embodiment V-9. The XDP system of Embodiment V-8, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[00826] Embodiment V-10. The XDP system of Embodiment V-9, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

[00827] Embodiment V-l 1. The XDP system of Embodiment V-5, wherein the therapeutic payload comprises a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[00828] Embodiment V-12. The XDP system of Embodiment V-l 1, wherein the CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 14 and 30 nucleotides and is complementary to a target nucleic acid sequence.

[00829] Embodiment V-13. The XDP system of Embodiment V-12, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781 as set forth in Table 3, 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.

[00830] Embodiment V-14. The XDP system of Embodiment V-13, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781.

[00831] Embodiment V-l 5. The XDP system of any one of the preceding embodiments of Set V, wherein the nucleic acids further encode one or more components selected from:

(a) all or a portion of a retroviral gag polyprotein;

(b) one or more protease cleavage sites;

(c) a gag-transframe region-pol protease polyprotein (gag-TFR-PR);

(d) a retroviral gag-pol polyprotein; and (e) a non-retroviral protease capable of cleaving the protease cleavage sites.

[00832] Embodiment V-16. The XDP system of any one of the preceding embodiments of Set V, wherein one or more of the retroviral components are derived from an Orthoretrovirinae virus or a Spumaretrovirinae virus.

[00833] Embodiment V- 17. The XDP system of Embodiment V-16, wherein the Orthoretrovirinae virus is selected from the group consisting of an Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus.

[00834] Embodiment V-18. The XDP system of Embodiment V-16, wherein the Spumaretrovirinae virus is selected from the group consisting of Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, or Spumavirus.

[00835] Embodiment V-19. The XDP system of any one of the preceding embodiments of Set V, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoded on two nucleic acids;

(c) the components are encoded on three nucleic acids;

(d) the components are encoded on four nucleic acids; or

(e) the components are encoded on five nucleic acids.

[00836] Embodiment V-20. The XDP system of Embodiment V-19, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

[00837] Embodiment V-21. The XDP system of Embodiment V-19 or Embodiment V-20, wherein the one or more of the retroviral components are encoded by a nucleic acid selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, and 234-339 as set forth in Table 5.

[00838] Embodiment V-22. The XDP system of any one of the preceding embodiments of Set V, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

[00839] Embodiment V-23. The XDP of Embodiment V-22, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

[00840] Embodiment V-24. The XDP system of Embodiment V-23, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template. [00841] Embodiment V-25. The XDP of Embodiment V-22, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

[00842] Embodiment V-26. The XDP system of Embodiment V-25, wherein the tropism factor confers preferential interaction of the XDP with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

[00843] Embodiment V-27. An XDP system comprising one or more nucleic acids encoding components:

(a) all or a portion of an Alpharetrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

[00844] Embodiment V-28. The XDP system of Embodiment V-27, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a P2A peptide, a P2B peptide, a P10 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00845] Embodiment V-29. The XDP system of Embodiment V-28, wherein the gag polyprotein comprises, from N-terminus to C-terminus, a matrix polypeptide (MA), a P2A peptide, a P2B peptide, a P10 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00846] Embodiment V-30. The XDP system of any one of Embodiments V-27-29, wherein the one or more nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

[00847] Embodiment V-31. The XDP system of any one of Embodiments V-27-30, 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.

[00848] Embodiment V-32. The XDP system of Embodiment V-31, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,

549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,

587, 589, 591, 593 and 595, 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.

[00849] Embodiment V-33. The XDP system of Embodiment V-31, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group of sequences consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505,

507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543,

545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581,

583, 585, 587, 589, 591, 593 and 595 as set forth in Table 4.

[00850] Embodiment V-34. The XDP system of Embodiment V-33, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G), optionally wherein the VSV-G glycoprotein comprises a sequence of SEQ ID NO: 438.

[00851] Embodiment V-35. The XDP system of any one of Embodiments V-27-34, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

[00852] Embodiment V-36. The XDP system of Embodiment V-35, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality.

[00853] Embodiment V-37. The XDP system of Embodiment V-36, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

[00854] Embodiment V-38. The XDP system of Embodiment V-37, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein. [00855] Embodiment V-39. The XDP system of Embodiment V-38, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[00856] Embodiment V-40. The XDP system of Embodiment V-39, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

[00857] Embodiment V-41. The XDP system of Embodiment V-39, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

[00858] Embodiment V-42. The XDP system of any one of Embodiments V-39-41, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of PKKKRKV (SEQ ID NO: 130), KRPAATKKAGQAKKKK (SEQ ID NO: 131),

PAAKRVKLD (SEQ ID NO: 132), RQRRNELKRSP (SEQ ID NO: 133),

NQ S SNF GPMKGGNF GGRS S GP Y GGGGQ YF AKPRNQGGY (SEQ ID NO: 134), RMRIZFKNKGKDTAELRRRRVEV S VELRKAKKDEQILKRRNV (SEQ ID NO: 135), VSRKRPRP (SEQ ID NO: 136), PPKKARED (SEQ ID NO: 137), PQPKKKPL (SEQ ID NO: 138), SALIKKKKKMAP (SEQ ID NO: 139), DRLRR (SEQ ID NO: 140), PKQKKRK (SEQ ID NO: 141), RKLKKKIKKL (SEQ ID NO: 142), REKKKFLKRR (SEQ ID NO: 143), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 144), RKCLQAGMNLEARKTKK (SEQ ID NO: 145), PRPRKIPR (SEQ ID NO: 146), PPRKKRTVV (SEQ ID NO: 147),

NL SKKKKRKREK (SEQ ID NO: 148), RRPSRPFRKP (SEQ ID NO: 149), KRPRSPSS (SEQ ID NO: 150), KRGINDRNFWRGENERKTR (SEQ ID NO: 151), PRPPKMARYDN (SEQ ID NO: 152), KRSFSKAF (SEQ ID NO: 153), KLKIKRPVK (SEQ ID NO: 154), PKTRRRPRRSQRKRPPT (SEQ ID NO: 156), RRKKRRPRRKKRR (SEQ ID NO: 159),

PKKK SRKPKKK SRK (SEQ ID NO: 160), HKKKHPD AS VNF SEF SK (SEQ ID NO: 161), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 162), LSPSLSPLLSPSLSPL (SEQ ID NO: 163), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 164), PKRGRGRPKRGRGR (SEQ ID NO: 165), M SRRRK ANPTKL SENAKKL AKEVEN (SEQ ID NO: 157), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 155), and PKKKRKVPPPPKKKRKV (SEQ ID NO: 166), wherein the NLS are located at or near the N-terminus and/or the C-terminus. [00859] Embodiment V-43. The XDP system of Embodiment V-35, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[00860] Embodiment V-44. The XDP system of Embodiment V-43, wherein the CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[00861] Embodiment V-45. The XDP system of Embodiment V-44, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

[00862] Embodiment V-46. The XDP system of Embodiment V-45, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

[00863] Embodiment V-47. The XDP system of any one of Embodiments V-44-46, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[00864] Embodiment V-48. The XDP system of any one of Embodiments V-27-47, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

[00865] Embodiment V-49. The XDP system of Embodiment V-48, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

[00866] Embodiment V-50. The XDP system of Embodiment V-48 or Embodiment V-49, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

[00867] Embodiment V-51. The XDP system of any one of Embodiments V-27-50, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

[00868] Embodiment V-52. The XDP of Embodiment V-51, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

[00869] Embodiment V-53. The XDP system of Embodiment V-52, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

[00870] Embodiment V-54. The XDP of Embodiment V-51, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

[00871] Embodiment V-55. The XDP system of Embodiment V-54, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

[00872] Embodiment V-56. An XDP system comprising one or more nucleic acids encoding components:

(a) all or a portion of an Betaretrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

[00873] Embodiment V-57. The XDP system of Embodiment V-56, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a PP21/24 peptide, a P12/P3/P8 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00874] Embodiment V-58. The XDP system of Embodiment V-56, wherein the gag polyprotein comprises, from N-terminus to C-terminus, a matrix polypeptide (MA), a PP21/24 peptide, a P12/P3/P8 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC). [00875] Embodiment V-59. The XDP system of any one of Embodiments V-56-58, wherein the nucleic acids further encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein; (d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

[00876] Embodiment V-60. The XDP system of any one of Embodiments V-56-59, 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.

[00877] Embodiment V-61. The XDP system of Embodiment V-60, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,

549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,

587, 589, 591, 593 and 595, 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.

[00878] Embodiment V-62. The XDP system of Embodiment V-61, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551,

553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589,

591, 593 and 595.

[00879] Embodiment V-63. The XDP system of Embodiment V-62, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

[00880] Embodiment V-64. The XDP system of any one of Embodiments V-56-63, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

[00881] Embodiment V-65. The XDP system of Embodiment V-64, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality.

[00882] Embodiment V-66. The XDP system of Embodiment V-65, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

[00883] Embodiment V-67. The XDP system of Embodiment V-66, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

[00884] Embodiment V-68. The XDP system of Embodiment V-67, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[00885] Embodiment V-69. The XDP system of Embodiment V-68, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, or 11, 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.

[00886] Embodiment V-70. The XDP system of Embodiment V-68, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

[00887] Embodiment V-71. The XDP system of any one of Embodiments V-68-70, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C- terminus.

[00888] Embodiment V-72. The XDP system of Embodiment V-64, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[00889] Embodiment V-73. The XDP system of Embodiment V-72, wherein the CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence. [00890] Embodiment V-74. The XDP system of Embodiment V-73, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

[00891] Embodiment V-75. The XDP system of Embodiment V-73, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

[00892] Embodiment V-76. The XDP system of any one of Embodiments V-73-75, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[00893] Embodiment V-77. The XDP system of any one of Embodiments V-56-76, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

[00894] Embodiment V-78. The XDP system of Embodiment V-77, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

[00895] Embodiment V-79. The XDP system of Embodiment V-77 or Embodiment V-78, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

[00896] Embodiment V-80. The XDP system of any one of Embodiments V-56-79, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

[00897] Embodiment V-81. The XDP of Embodiment V-80, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP. [00898] Embodiment V-82. The XDP system of Embodiment V-81, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

[00899] Embodiment V-83. The XDP of Embodiment V-80, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

[00900] Embodiment V-84. The XDP system of Embodiment V-83, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

[00901] Embodiment V-85. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Deltaretrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

[00902] Embodiment V-86. The XDP system of Embodiment V-85, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00903] Embodiment V-87. The XDP system of Embodiment V-86, wherein the gag polyprotein comprises, from N-terminus to C-terminus, matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00904] Embodiment V-88. The XDP system of any one of Embodiments V-85-87, wherein the nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

[00905] Embodiment V-89. The XDP system of any one of Embodiments V-85-88, 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.

[00906] Embodiment V-90. The XDP system of Embodiment V-89, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,

549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,

587, 589, 591, 593 and 595, 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.

[00907] Embodiment V-91. The XDP system of Embodiment V-89, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551,

553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589,

591, 593 and 595.

[00908] Embodiment V-92. The XDP system of Embodiment V-91, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

[00909] Embodiment V-93. The XDP system of any one of Embodiments V-85-92, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

[00910] Embodiment V-94. The XDP system of Embodiment V-93, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality.

[00911] Embodiment V-95. The XDP system of Embodiment V-94, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

[00912] Embodiment V-96. The XDP system of Embodiment V-95, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein. [00913] Embodiment V-97. The XDP system of Embodiment V-96, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[00914] Embodiment V-98. The XDP system of Embodiment V-97, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

[00915] Embodiment V-99. The XDP system of Embodiment V-97, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

[00916] Embodiment V-100. The XDP system of any one of Embodiments V-97-99, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

[00917] Embodiment V-101. The XDP system of Embodiment V-93, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[00918] Embodiment V-102. The XDP system of Embodiment V-101, wherein the

CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[00919] Embodiment V-103. The XDP system of Embodiment V-102, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

[00920] Embodiment V-104. The XDP system of Embodiment V-102, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781. [00921] Embodiment V-105. The XDP system of any one of Embodiments V-102-104, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[00922] Embodiment V-106. The XDP system of any one of Embodiments V-85-105, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

[00923] Embodiment V-107. The XDP system of Embodiment V-106, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

[00924] Embodiment V-108. The XDP system of Embodiment V-106 or Embodiment

V-107, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

[00925] Embodiment V-109. The XDP system of any one of Embodiments V-85-108, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

[00926] Embodiment V-l 10. The XDP of Embodiment V-109, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

[00927] Embodiment V-l 11. The XDP system of Embodiment V-l 10, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

[00928] Embodiment V-l 12. The XDP of Embodiment V-109, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP. [00929] Embodiment V-l 13. The XDP system of Embodiment V-l 12, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

[00930] Embodiment V-l 14. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Epsilonretrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

[00931] Embodiment V-l 15. The XDP system of Embodiment V-l 14, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a p20 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00932] Embodiment V-l 16. The XDP system of Embodiment V-l 14, wherein the gag polyprotein comprises, from N-terminus to C-terminus, matrix polypeptide (MA), a p20 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00933] Embodiment V-l 17. The XDP system of any one of Embodiments V-l 14-116, wherein the nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

[00934] Embodiment V-l 18. The XDP system of any one of Embodiments V-l 14-117, 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.

[00935] Embodiment V-l 19. The XDP system of Embodiment V-l 18, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505,

507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543,

545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595, 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.

[00936] Embodiment V-120. The XDP system of Embodiment V-l 18, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509,

511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,

549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,

587, 589, 591, 593 and 595.

[00937] Embodiment V-121. The XDP system of Embodiment V-120, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

[00938] Embodiment V-122. The XDP system of any one of Embodiments V-l 14-121, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

[00939] Embodiment V-123. The XDP system of Embodiment V-122, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality.

[00940] Embodiment V-124. The XDP system of Embodiment V-123, wherein the

CRISPR protein is a Class 1 or Class 2 CRISPR protein.

[00941] Embodiment V-125. The XDP system of Embodiment V-124, wherein the

CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II,

Type V, or Type VI protein.

[00942] Embodiment V-126. The XDP system of Embodiment V-125, wherein the

CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[00943] Embodiment V-127. The XDP system of Embodiment V-126, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

[00944] Embodiment V-128. The XDP system of Embodiment V-126, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

[00945] Embodiment V-129. The XDP system of any one of Embodiments V-126-128, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

[00946] Embodiment V-130. The XDP system of Embodiment V-122, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[00947] Embodiment V-131. The XDP system of Embodiment V-130, wherein the

CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[00948] Embodiment V-132. The XDP system of Embodiment V-131, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-78 lor 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.

[00949] Embodiment V-133. The XDP system of Embodiment V-131, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

[00950] Embodiment V-134. The XDP system of any one of Embodiments V-131-133, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[00951] Embodiment V-135. The XDP system of any one of Embodiments V-l 14-134, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids; (c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

[00952] Embodiment V-136. The XDP system of Embodiment V-135, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

[00953] Embodiment V-137. The XDP system of Embodiment V-135 or Embodiment

V-136, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

[00954] Embodiment V-138. The XDP system of any one of Embodiments V-l 14-137, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

[00955] Embodiment V-139. The XDP of Embodiment V-138, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

[00956] Embodiment V-140. The XDP system of Embodiment V-139, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

[00957] Embodiment V-141. The XDP of Embodiment V-139, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

[00958] Embodiment V-142. The XDP system of Embodiment V-141, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

[00959] Embodiment V-143. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Gammaretrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor. [00960] Embodiment V-144. The XDP system of Embodiment V-143, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a pl2 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00961] Embodiment V-145. The XDP system of Embodiment V-144, wherein the gag polyprotein comprises, from N-terminus to C-terminus, matrix polypeptide (MA), a pl2 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

[00962] Embodiment V-146. The XDP system of any one of Embodiments V-143-145, wherein the nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

[00963] Embodiment V-147. The XDP system of any one of Embodiments V-143-146, 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.

[00964] Embodiment V-148. The XDP system of Embodiment V-147, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505,

507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543,

545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581,

583, 585, 587, 589, 591, 593 and 595, 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.

[00965] Embodiment V-149. The XDP system of Embodiment V-147, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595.

[00966] Embodiment V-150. The XDP system of Embodiment V-149, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

[00967] Embodiment V-151. The XDP system of any one of Embodiments V-143-150, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

[00968] Embodiment V-152. The XDP system of Embodiment V-151, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality.

[00969] Embodiment V-153. The XDP system of Embodiment V-152, wherein the

CRISPR protein is a Class 1 or Class 2 CRISPR protein.

[00970] Embodiment V-154. The XDP system of Embodiment V-153, wherein the

CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

[00971] Embodiment V-155. The XDP system of Embodiment V-154, wherein the

CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[00972] Embodiment V-156. The XDP system of Embodiment V-155, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

[00973] Embodiment V-157. The XDP system of Embodiment V-155, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

[00974] Embodiment V-158. The XDP system of any one of Embodiments V-155-157, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

[00975] Embodiment V-159. The XDP system of Embodiment V-151, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[00976] Embodiment V-160. The XDP system of Embodiment V-159, wherein the

CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[00977] Embodiment V-161. The XDP system of Embodiment V-160, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

[00978] Embodiment V-162. The XDP system of Embodiment V-160, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

[00979] Embodiment V-163. The XDP system of any one of Embodiments V-160-162, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[00980] Embodiment V-164. The XDP system of any one of Embodiments V-143-163, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

[00981] Embodiment V-165. The XDP system of Embodiment V-164, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

[00982] Embodiment V-166. The XDP system of Embodiment V-164 or Embodiment

V-165, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

[00983] Embodiment V-167. The XDP system of any one of Embodiments V-164-166, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

[00984] Embodiment V-168. The XDP of Embodiment V-167, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

[00985] Embodiment V-169. The XDP system of Embodiment V-168, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

[00986] Embodiment V-170. The XDP of Embodiment V-167, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

[00987] Embodiment V-171. The XDP system of Embodiment V-170, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

[00988] Embodiment V-172. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Lentivirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

[00989] Embodiment V-173. The XDP system of Embodiment V-172, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a capsid polypeptide (CA), a p2 peptide, a nucleocapsid polypeptide (NC), a pi peptide, and a p6 peptide.

[00990] Embodiment V-174. The XDP system of Embodiment V-173, wherein the gag polyprotein comprises, from N-terminus to C-terminus, matrix polypeptide (MA), a capsid polypeptide (CA), a p2 peptide, a nucleocapsid polypeptide (NC), a pi peptide, and a p6 peptide. [00991] Embodiment V-175. The XDP system of any one of Embodiments V-172-173, wherein the nucleic acids encode one or more components selected from (a) a Gag-Pol polyprotein;

(b) one or more protease cleavage sites;

(c) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(d) a gag-transframe region-pol protease polyprotein.

[00992] Embodiment V-176. The XDP system of any one of Embodiments V-172-175, wherein the lentivirus is selected from the group consisting of human immunodeficiency- 1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[00993] Embodiment V-177. The XDP system of Embodiment V-176, wherein the lentivirus is HIV-1

[00994] Embodiment V-178. The XDP system of any one of Embodiments V-172-177, 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.

[00995] Embodiment V-179. The XDP system of Embodiment V-178, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543,

545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581,

583, 585, 587, 589, 591, 593 and 595, 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.

[00996] Embodiment V-180. The XDP system of Embodiment V-178, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509,

511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,

549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,

587, 589, 591, 593 and 595.

[00997] Embodiment V-181. The XDP system of Embodiment V-180, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G). [00998] Embodiment V-182. The XDP system of any one of Embodiments V-172-181, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

[00999] Embodiment V-183. The XDP system of Embodiment V-182, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality.

[001000] Embodiment V-184. The XDP system of Embodiment V-183, wherein the

CRISPR protein is a Class 1 or Class 2 CRISPR protein.

[001001] Embodiment V-185. The XDP system of Embodiment V-184, wherein the

CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

[001002] Embodiment V-186. The XDP system of Embodiment V-185, wherein the

CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[001003] Embodiment V-187. The XDP system of Embodiment V-186, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

[001004] Embodiment V-188. The XDP system of Embodiment V-186, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

[001005] Embodiment V-189. The XDP system of any one of Embodiments V-186-188, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

[001006] Embodiment V-190. The XDP system of Embodiment V-182, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[001007] Embodiment V-191. The XDP system of Embodiment V-190, wherein the

CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[001008] Embodiment V-192. The XDP system of Embodiment V-191, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-78 lor 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.

[001009] Embodiment V-193. The XDP system of Embodiment V-191, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

[001010] Embodiment V-194. The XDP system of any one of Embodiments V-191-193, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[001011] Embodiment V-195. The XDP system of any one of Embodiments V-172-194, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

[001012] Embodiment V-196. The XDP system of Embodiment V-195, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

[001013] Embodiment V-197. The XDP system of Embodiment V-195 or Embodiment

V-196, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339,

880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

[001014] Embodiment V-198. The XDP system of any one of Embodiments V-195-197, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

[001015] Embodiment V-199. The XDP of Embodiment V-198, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

[001016] Embodiment V-200. The XDP system of Embodiment V-198, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

[001017] Embodiment V-201. The XDP of Embodiment V-198, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

[001018] Embodiment V-202. The XDP system of Embodiment V-201, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

[001019] Embodiment V-203. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Spumaretrovirinae gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

[001020] Embodiment V-204. The XDP system of Embodiment V-203, wherein the gag polyprotein comprises one or more components selected from the group consisting of a p68 Gag polypeptide and a p3 Gag polypeptide.

[001021] Embodiment V-205. The XDP system of Embodiment V-204, wherein the gag polyprotein comprises, from N-terminus to C-terminus, p68 Gag polypeptide and a p3 Gag polypeptide.

[001022] Embodiment V-206. The XDP system of any one of Embodiments V-203-205, wherein the nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites; (e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

[001023] Embodiment V-207. The XDP system of any one of Embodiments V-203-206, 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.

[001024] Embodiment V-208. The XDP system of Embodiment V-207, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543,

545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581,

583, 585, 587, 589, 591, 593 and 595, 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.

[001025] Embodiment V-209. The XDP system of Embodiment V-207, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,

549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,

587, 589, 591, 593 and 595.

[001026] Embodiment V-210. The XDP system of Embodiment V-209, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

[001027] Embodiment V-211. The XDP system of any one of Embodiments V-203-210, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

[001028] Embodiment V-212. The XDP system of Embodiment V-211, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality. [001029] Embodiment V-213. The XDP system of Embodiment V-212, wherein the

CRISPR protein is a Class 1 or Class 2 CRISPR protein.

[001030] Embodiment V-214. The XDP system of Embodiment V-213, wherein the

CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II,

Type V, or Type VI protein.

[001031] Embodiment V-215. The XDP system of Embodiment V-214, wherein the

CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[001032] Embodiment V-216. The XDP system of Embodiment V-215, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

[001033] Embodiment V-217. The XDP system of Embodiment V-216, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

[001034] Embodiment V-218. The XDP system of any one of Embodiments V-203-217, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

[001035] Embodiment V-219. The XDP system of Embodiment V-211, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[001036] Embodiment V-220. The XDP system of Embodiment V-219, wherein the

CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

[001037] Embodiment V-221. The XDP system of Embodiment V-220, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

[001038] Embodiment V-222. The XDP system of Embodiment V-221, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

[001039] Embodiment V-223. The XDP system of any one of Embodiments V-220-222, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

[001040] Embodiment V-224. The XDP system of any one of Embodiments V-203-223, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

[001041] Embodiment V-225. The XDP system of Embodiment V-224, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

[001042] Embodiment V-226. The XDP system of Embodiment V-224 or Embodiment

V-225, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

[001043] Embodiment V-221. The XDP system of any one of Embodiments V-224-226, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

[001044] Embodiment V-228. The XDP of Embodiment V-221 , wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

[001045] Embodiment V-229. The XDP system of Embodiment V-228, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template. [001046] Embodiment V-230. The XDP of Embodiment V-227, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

[001047] Embodiment V-231. The XDP system of Embodiment V-230, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

[001048] Embodiment V-232. The XDP system of any one of the preceding embodiments of Set V, wherein the gag polyprotein and the therapeutic payload is expressed as a fusion protein.

[001049] Embodiment V-233. The XDP system of Embodiment V-232, wherein the fusion protein does not comprise a protease cleavage site between the gag polyprotein and the therapeutic payload.

[001050] Embodiment V-234. The XDP system of Embodiment V-232, wherein the fusion protein comprises a protease cleavage site between the gag polyprotein and the therapeutic payload.

[001051] Embodiment V-235. The XDP system of any one of Embodiments V-232-234, wherein the fusion protein comprises protease cleavage sites between the components of the gag polyprotein.

[001052] Embodiment V-236. The XDP system of Embodiment V-234 and/or

Embodiment V-235, wherein the cleavage sites are capable of being cleaved by the protease of the Gag-Pol polyprotein, the protease of the gag-transframe region-pol protease polyprotein, or the non-retroviral, heterologous protease.

[001053] Embodiment V-237. The XDP system of Embodiment V-236, wherein the cleavage sites are capable of being cleaved by the protease of the gag-transframe region-pol protease polyprotein.

[001054] Embodiment V-238. The XDP system of Embodiment V-236, wherein the cleavage sites are capable of being cleaved by the protease of the Gag-Pol polyprotein [001055] Embodiment V-239. The XDP system of Embodiment V-236, wherein the non- retroviral, heterologous protease is selected from the group consisting of tobacco etch virus protease (TEV), potyvirus HC protease, potyvirus PI protease, PreScission (HRV3C protease), b virus NIa protease, B virus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2 A protease, picoma 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, parsnip yellow fleck virus protease, 3C-like protease, heparin, cathepsin, thrombin, factor Xa, metalloproteinase, and enterokinase.

[001056] Embodiment V-240. The XDP system of Embodiment V-239, wherein the non- retroviral, heterologous protease is PreScission (HRV3C protease).

[001057] Embodiment V-241. The XDP system of Embodiment V-239, wherein the non- retroviral, heterologous protease is tobacco etch virus protease (TEV).

[001058] Embodiment V-242. The XDP system of any one of Embodiments V-12-13, 44-

47, 73-76, 96-99, 103-106, 132-135, 161-164, 192-195 or 221-224, wherein the guide RNA further comprises one or more ribozymes.

[001059] Embodiment V-243. The XDP system of Embodiment V-242, wherein the one or more ribozymes are independently fused to a terminus of the guide RNA.

[001060] Embodiment V-244. The XDP system of Embodiment V-242 or Embodiment

V-243, wherein at least one of the one or more ribozymes is a hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

[001061] Embodiment V-245. The XDP system of any one of Embodiments V-12-13, 44-

47, 73-76, 96-99, 103-106, 132-135, 161-164, 192-195 or 221-224, wherein the guide RNA is chemically modified.

[001062] Embodiment V-246. The XDP system of any one of Embodiments V-12-13, 44-

47, 73-76, 96-99, 103-106, 132-135, 161-164, 192-195 or 221-224, wherein the guide RNA comprises an element selected from the group consisting of a Psi packaging element, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, or pseudoknot, wherein the element has affinity to a protein incorporated into the CasX selected from the group consisting of MS2, PP7, Qbeta, U 1 A, and phage R-loop. [001063] Embodiment V-247. A eukaryotic cell comprising the XDP system of any one of the preceding embodiments of Set V.

[001064] Embodiment V-248. The eukaryotic cell of Embodiment V-247, wherein the cell is a packaging cell.

[001065] Embodiment V-249. The eukaryotic cell of Embodiment V-247 or Embodiment

V-248, wherein the eukaryotic cell is selected from the group consisting of HEK293 cells, Lenti- X 293T cells, BHK cells, HepG2, Saos-2, HuH7, NS0 cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO, NIH3T3 cells, COS, WI38, MRC5, A549, HeLa cells, CHO cells, and HT1080 cells.

[001066] Embodiment V-250. The eukaryotic cell of Embodiment V-248 or Embodiment

V-249, wherein the packaging cell comprises one or more mutations to reduce expression of a cell surface marker.

[001067] Embodiment V-251. The eukaryotic cell of any one of Embodiments V-247-

250, wherein all or a portion of the nucleic acids encoding the XDP system are integrated into the genome of the eukaryotic cell.

[001068] Embodiment V-252. A method of making an XDP comprising a therapeutic payload, the method comprising:

(a) propagating the packaging cell of any one of Embodiments V-248-251 under conditions such that XDPs are produced; and

(b) harvesting the XDPs produced by the packaging cell.

[001069] Embodiment V-253. An XDP produced by the method of Embodiment V-252.

[001070] Embodiment V-254. The XDP of Embodiment V-253, comprising a therapeutic payload of an RNP of a CasX and guide RNA and, optionally, a donor template.

[001071] Embodiment V-255. A method of method of modifying a target nucleic acid sequence in a cell, the method comprising contacting the cell with the XDP of Embodiment V- 254, wherein said contacting comprises introducing into the cell the RNP and, optionally, the donor template nucleic acid sequence, wherein the target nucleic acid targeted by the guide RNA is modified by the CasX.

[001072] Embodiment V-256. The method of Embodiment V-255, wherein the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence.

[001073] Embodiment V-257. The method of Embodiment V-255, wherein the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence.

[001074] Embodiment V-258. The method of any one of Embodiments V-255-257, wherein the modification comprises insertion of the donor template into the target nucleic acid sequence.

[001075] Embodiment V-259. The method of any one of Embodiments V-255-258, wherein the cell is modified in vitro or ex vivo. [001076] Embodiment V-260. The method of any one of Embodiments V-255-258, wherein the cell is modified in vivo.

[001077] Embodiment V-261. The method of Embodiment V-260, wherein the XDP is administered to a subject.

[001078] Embodiment V-262. The method of Embodiment V-261, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

[001079] Embodiment V-263. The method of Embodiment V-261 or Embodiment V-262, wherein the XDP is 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 routes and sub-retinal routes.

[001080] Embodiment V-264. The method of any one of Embodiments V-261-263, wherein the XDP is administered to the subject using a therapeutically effective dose.

[001081] Embodiment V-265. The method of Embodiment V-264, wherein the XDP is administered at a dose of at least about 1 x 10^L5 particles/kg, or at least about 1 x 10^L6 particles/kg, or at least about 1 x 10^L7 particles/kg, or at least about 1 x 10^L8 particles/kg, or at least about 1 x 10^L9 particles/kg, or at least about 1 x 10^L10 particles/kg, or at least about 1 x 10^L11 particles/kg, or at least about 1 x 10^L12 particles/kg, or at least about 1 x 10^L13 particles/kg, or at least about 1 x 10^L14 particles/kg, or at least about 1 x 10^L15 particles/kg, or at least about 1 x 10^L16 particles/kg.

[001082] Embodiment V-266. The method of any one of Embodiments V-261 -265, wherein the XDP is administered to the subject according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose of the XDP.

[001083] Embodiment V-267. The method of Embodiment V-266, 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. [001084] Embodiment V-268. A method for introducing a CasX and gNA RNP into a cell having a target nucleic acid, comprising contacting the cell with the XDP of Embodiment V-253 or Embodiment V-254, such that the RNP enters the cell.

[001085] Embodiment V-269. The method of Embodiment V-268, wherein the RNP binds to the target nucleic acid.

[001086] Embodiment V-270. The method of Embodiment V-269, wherein the target nucleic acid is cleaved by the CasX.

[001087] Embodiment V-271. The method of any one of Embodiments V-268-270, wherein the cell is modified in vitro.

[001088] Embodiment V-272. The method of any one of Embodiments V-268-270, wherein the cell is modified in vivo.

[001089] Embodiment V-273. The method of Embodiment V-272, wherein the XDP is administered to a subject.

[001090] Embodiment V-274. The method of Embodiment V-273, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

[001091] Embodiment V-275. The method of any one of Embodiments V-272-274, wherein the XDP is administered to the subject using a therapeutically effective dose.

[001092] Embodiment V-276. The method of Embodiment V-275, wherein the XDP is administered at a dose of at least about 1 x 10^L5 particles/kg, or at least about 1 x 10^L6 particles/kg, or at least about 1 x 10^L7 particles/kg, or at least about 1 x 10^L8 particles/kg, or at least about 1 x 10^L9 particles/kg, or at least about 1 x 10^L10 particles/kg, or at least about 1 x 10^L11 particles/kg, or at least about 1 x 10^L12 particles/kg, or at least about 1 x 10^L13 particles/kg, or at least about 1 x 10^L14 particles/kg, or at least about 1 x 10^L15 particles/kg, or at least about 1 x 10^L16 particles/kg.

[001093] Embodiment V-277. A XDP particle comprising:

(a) a retroviral matrix (MA) polypeptide;

(b) a therapeutic payload encapsidated within the XDP; and

(c) a tropism factor incorporated on the XDP surface.

[001094] Embodiment V-278. The XDP particle of Embodiment V-277, further comprising one or more retroviral components selected from:

(a) a capsid polypeptide (CA); (b) a nucleocapsid polypeptide (NC);

(c) a P2A peptide, a P2B peptide;

(d) a P10 peptide;

(e) a p 12 peptide

(f) a PP21/24 peptide;

(g) a P12/P3/P8 peptide;

(h) a P20 peptide;

(i) A pi peptide; and

(j) a p6 peptide.

[001095] Embodiment V-279. The XDP particle of Embodiment V-277 or Embodiment

V-278, 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.

[001096] Embodiment V-280. The XDP particle of Embodiment V-279, wherein the tropism factor is a glycoprotein having an sequence selected from the group consisting of SEQ ID NOS: 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470,

472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508,

510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546,

548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584,

586, 588, 590, 592, 594 and 596, 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.

[001097] Embodiment V-281. The XDP particle of Embodiment V-279, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466,

468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504,

506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542,

544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580,

582, 584, 586, 588, 590, 592, 594 and 596.

[001098] Embodiment V-282. The XDP particle of any one of Embodiments V-277-281, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid. [001099] Embodiment V-283. The XDP particle of Embodiment V-282, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti-cancer modality.

[001100] Embodiment V-284. The XDP particle of Embodiment V-283, wherein the

CRISPR protein is a Class 1 or Class 2 CRISPR protein.

[001101] Embodiment V-285. The XDP particle of Embodiment V-284, wherein the

CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II,

Type V, or Type VI protein.

[001102] Embodiment V-286. The XDP particle of Embodiment V-285, wherein the

CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

[001103] Embodiment V-287. The XDP particle of Embodiment V-286, wherein the

CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

[001104] Embodiment V-288. The XDP particle of Embodiment V-282, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

[001105] Embodiment V-289. The XDP particle of Embodiment V-288, wherein the

CRISPR guide nucleic acid is a single-molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 14 and 30 nucleotides and is complementary to a target nucleic acid sequence.

[001106] Embodiment V-290. The XDP particle of Embodiment V-289, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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. [001107] Embodiment V-291. The XDP particle of Embodiment V-290, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781.

[001108] Embodiment V-292. The XDP particle of any one of Embodiments V-286-291, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

[001109] Embodiment V-293. The XDP particle of any one of Embodiments V-277-292, wherein the retroviral components are derived from a Orthoretrovirinae virus or a Spumaretrovirinae virus.

[001110] Embodiment V-294. The XDP particle of Embodiment V-293, wherein the

Orthoretrovirinae virus is selected from the group consisting of Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus.

[001111] Embodiment V-295. The XDP particle of Embodiment V-293, wherein the

Spumaretrovirinae virus is selected from the group consisting of Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, and Spumavirus. [001112] Embodiment V-296. The XDP particles, or the XDP systems of any one of the preceding embodiments, for use as a medicament for the treatment of a subject having a disease. [001113] 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.

EXAMPLES

Example 1: Creation, Expression and Purification of CasX Constructs

1. Growth and Expression

[001114] An expression construct for CasX Stx2 (also referred to herein as CasX2), derived from Planctomycetes (having the amino acid sequence of SEQ ID NO: 2 and encoded by the sequence of the Table 6, below), was constructed from gene fragments (Twist Biosciences) that were codon optimized for E.coli. The assembled construct contains a TEV-cleavable, C- terminal, TwinStrep tag and was cloned into a pBR322-derivative plasmid backbone containing an ampicillin resistance gene. The expression construct was transformed into chemically competent BL21* (DE3) E. coli and a starter culture was grown overnight in LB broth supplemented with carbenicillin at 37°C, 200 RPM, in UltraYield Flasks (Thomson Instrument Company). The following day, this culture was used to seed expression cultures at a 1 : 100 ratio (starter culture:expression culture). Expression cultures were Terrific Broth (Novagen) supplemented with carbenicillin and grown in UltraYield flasks at 37°C, 200 RPM. Once the cultures reached an OD of 2, they were chilled to 16°C and IPTG (isopropyl b-D-l- thiogalactopyranoside) was added to a final concentration of 1 mM, from a 1 M stock. The cultures were induced at 16°C, 200 RPM for 20 hours before being harvested by centrifugation at 4,000xg for 15 minutes, 4°C. The cell paste was weighed and resuspended in lysis buffer (50 mM HEPES-NaOH, 250 mM NaCl, 5 mM MgCk, 1 mM TCEP, 1 mM benzamidine-HCL, 1 mM PMSF, 0.5% CHAPS, 10% glycerol, pH 8) at a ratio of 5 mL of lysis buffer per gram of cell paste. Once resuspended, the sample was frozen at -80°C until purification.

Table 6: DNA sequence of CasX Stx2 construct

2. Purification

[001115] Frozen samples were thawed overnight at 4°C with magnetic stirring. The viscosity of the resulting lysate was reduced by sonication and lysis was completed by homogenization in three passes at 17k PSI using an Emulsiflex C3 (Avestin). Lysate was clarified by centrifugation at 50,000x g, 4°C, for 30 minutes and the supernatant was collected. The clarified supernatant was applied to a Heparin 6 Fast Flow column (GE Life Sciences) by gravity flow. The column was washed with 5 CV of Heparin Buffer A (50 mM HEPES-NaOH, 250 mM NaCl, 5 mM MgCb, 1 mM TCEP, 10% glycerol, pH 8), then with 5 CV of Heparin Buffer B (Buffer A with the NaCl concentration adjusted to 500 mM). Protein was eluted with 5 CV of Heparin Buffer C (Buffer A with the NaCl concentration adjusted to 1 M), collected in fractions. Fractions were assayed for protein by Bradford Assay and protein-containing fractions were pooled. The pooled heparin eluate was applied to a Strep-Tactin XT Superflow column (IBA Life Sciences) by gravity flow. The column was washed with 5 CV of Strep Buffer (50 mM HEPES-NaOH, 500 mM NaCl, 5 mM MgCh, 1 mM TCEP, 10% glycerol, pH 8). Protein was eluted from the column using 5 CV of Strep Buffer with 50 mM D-Biotin added and collected in fractions. CasX-containing fractions were pooled, concentrated at 4°C using a 30 kDa cut-off spin concentrator, and purified by size exclusion chromatography on a Superdex 200 pg column (GE Life Sciences). The column was equilibrated with SEC Buffer (25 mM sodium phosphate, 300 mM NaCl, 1 mM TCEP, 10% glycerol, pH 7.25) operated by an ART A Pure FPLC system (GE Life Sciences). CasX-containing fractions that eluted at the appropriate molecular weight were pooled, concentrated at 4°C using a 30 kDa cut-off spin concentrator, aliquoted, and snap-frozen in liquid nitrogen before being stored at -80°C.

3. Results

[001116] Samples from throughout the purification were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as shown in FIG. 1 and FIG. 3. In FIG. 1, the lanes, from left to right, are: molecular weight standards, Pellet: insoluble portion following cell lysis, Lysate: soluble portion following cell lysis, Flow Thru: protein that did not bind the Heparin column, Wash: protein that eluted from the column in wash buffer, Elution: protein eluted from the heparin column with elution buffer, Flow Thru: Protein that did not bind the StrepTactinXT column, Elution: protein eluted from the StrepTactin XT column with elution buffer, Injection: concentrated protein injected onto the s200 gel filtration column, Frozen: pooled fractions from the s200 elution that have been concentrated and frozen. In FIG. 3, the lanes from right to left, are the injection (sample of protein injected onto the gel filtration column) molecular weight markers, lanes 3 -9 are samples from the indicated elution volumes. Results from the gel filtration are shown in FIG. 2. The 68.36 mL peak corresponds to the apparent molecular weight of CasX and contained the majority of CasX protein. The average yield was 0.75 mg of purified CasX protein per liter of culture, with 75% purity, as evaluated by colloidal Coomassie staining.

Example 2: CasX construct CasX 119, 438 and 457

[001117] In order to generate the CasX 119, 438, and 457 constructs (sequences in Table 7), the codon-optimized CasX 37 construct (based on the CasX Stx2 construct of Example 1, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) was cloned into a mammalian expression plasmid (pStX; see FIG. 4) using standard cloning methods. To build CasX 119, the CasX 37 construct DNA was PCR amplified in two reactions using Q5 DNA polymerase (New England BioLabs Cat# M0491L) according to the manufacturer’s protocol, using primers oIC539 and 0IC88 as well as oIC87 and oIC540 respectively (see FIG. 5). To build CasX 457, the CasX 365 construct DNA was PCR amplified in four reactions using Q5 DNA polymerase (New England BioLabs Cat# M0491L) according to the manufacturer’s protocol, using primers OIC539 and oIC212, oIC211 and oIC376, oIC375 and oIC551, and oIC550 and oIC540 respectively. To build CasX 438, the CasX 119 construct DNA was PCR amplified in four reactions using Q5 DNA polymerase according to the manufacturer’s protocol, using primers OIC539 and oIC689, 0IC688 and oIC376, oIC375 and oIC551, and oIC550 and oIC540 respectively. The resulting PCR amplification products were then purified using Zymoclean DNA clean and concentrator (Zymo Research Cat# 4014) according to the manufacturer’s protocol. The pStX backbone was digested using Xbal and Spel in order to remove the 2931 base pair fragment of DNA between the two sites in plasmid pStx34. The digested backbone fragment was purified by gel extraction from a 1% agarose gel (Gold Bio Cat# A-201-500) using Zymoclean Gel DNA Recovery Kit (Zymo Research Cat#D4002) according to the manufacturer’s protocol. The three fragments were then pieced together using Gibson assembly (New England BioLabs Cat# E2621S) following the manufacturer’s protocol. Assembled products in the pStx34 were transformed into chemically-competent or electro-competent Turbo Competent A. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing carbenicillin. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit (Qiagen Cat# 27104) following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. pStX34 includes an EF-la promoter for the protein as well as a selection marker for both puromycin and carbenicillin. Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates containing carbenicillin. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit and following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. SaCas9 and SpyCas9 control plasmids were prepared similarly to pStX plasmids described above, with the protein and guide regions of pStX exchanged for the respective protein and guide. Targeting sequences for SaCas9 and SpyCas9 were either obtained from the literature or were rationally designed according to established methods. The expression and recovery of the CasX 119, 438 and 457 proteins was performed using the general methodologies of Example 1 (however the DNA sequences were codon optimized for expression in E. coli).

[001118] CasX Variant 119: following the same expression and purification scheme for WT CasX, the following results were obtained for CasX variant 119. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as shown in FIG. 6 and FIG. 8. Results from the gel filtration are shown in FIG. 7. The average yield was 11.7 mg of purified CasX protein per liter of culture at 95% purity, as evaluated by colloidal Coomassie staining.

[001119] CasX Variant 438: Following the same expression and purification scheme for WT CasX, the following results were obtained for CasX variant 438. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as shown in FIGS. 9 and 11. Results from the gel filtration are shown in FIG. 10. The average yield was 13.1 mg of purified CasX protein per liter of culture at 97.5% purity, as evaluated by colloidal Coomassie staining.

[001120] CasX Variant 457: Following the same expression and purification scheme for WT CasX, the following results were obtained for CasX variant 457. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as shown in FIGS. 12 and 14 and gel filtration, as shown in FIG. 13. The average yield was 9.76 mg of purified CasX protein per liter of culture at 91.6% purity, as evaluated by colloidal Coomassie staining.

[001121] Overall, the results support that CasX variants can be produced and recovered at high levels of purity sufficient for experimental assays and evaluation.

Table 7: Sequences of CasX 119, 438 and 457

Example 3: CasX construct 488, 491, 515 and 527

[001122] In order to generate the CasX 488 construct (sequences in Table 8), the codon- optimized CasX 119 construct (based on the CasX Stx2 construct of Example 1, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution, a L379R substitution, and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) was cloned into a destination plasmid (pStX; see FIG. 4) using standard cloning methods. In order to generate the CasX 491 construct (sequences in Table 8), the codon-optimized CasX 484 construct (based on the CasX Stx2 construct of Example 1, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution, aL379R substitution, a [P793] deletion, a I658V substitution, and a F399L substitution with fused NLS, and linked guide and non-targeting sequences) was cloned into a destination plasmid (pStX; see FIG. 4) using standard cloning methods. Construct CasX 1 (CasX SEQ ID NO: 1) was cloned into a destination vector using standard cloning methods. To build CasX 488, the CasX 119 construct DNA was PCR amplified using Q5 DNA polymerase according to the manufacturer’s protocol, using primers oIC765 and oIC762 (see FIG. 5). To build CasX 491, the codon optimized CasX 484 construct DNA was PCR amplified using Q5 DNA polymerase according to the manufacturer’s protocol, using primers oIC765 and oIC762 (see FIG. 5). The CasX 1 construct was PCR amplified using Q5 DNA polymerase according to the manufacturer’s protocol, using primers oIC766 and oIC784. Each of the PCR products were purified by gel extraction from a 1% agarose gel (Gold Bio Cat# A-201-500) using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. The corresponding fragments were then pieced together using Gibson assembly (New England BioLabs Cat# E2621S) following the manufacturer’s protocol. Assembled products in pStxl were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing kanamycin. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. The correct clones were then subcloned into the mammalian expression vector pStx34 using restriction enzyme cloning. The pStx34 backbone and the CasX 488 and 491 clones in pStxl were digested with Xbal and BamHI respectively. The digested backbone and respective insert fragments were purified by gel extraction from a 1% agarose gel (Gold Bio Cat# A-201-500) using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. The clean backbone and insert were then ligated together using T4 Ligase (New England Biolabs Cat# M0202L) according to the manufacturer’s protocol. The ligated products were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing carbenicillin. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.

[001123] To build CasX 515 (sequences in Table 8), the CasX 491 construct DNA was PCR amplified in two reactions using Q5 DNA polymerase according to the manufacturer’s protocol, using primers oIC539 and oSH556 as well as oSH555 and oIC540 respectively (see FIG. 5). To build CasX 527 (sequences in Table 8), the CasX 491 construct DNA was PCR amplified in two reactions using Q5 DNA polymerase according to the manufacturer’s protocol, using primers oIC539 and oSH584 as well as oSH583 and oIC540 respectively. The PCR products were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. The pStX backbone was digested using Xbal and Spel in order to remove the 2931 base pair fragment of DNA between the two sites in plasmid pStx56. The digested backbone fragment was purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. The insert and backbone fragments were then pieced together using Gibson assembly (New England BioLabs Cat# E2621S) following the manufacturer’s protocol. Assembled products in the pStx56 were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing kanamycin. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. pStX34 includes an EF-la promoter for the protein as well as a selection marker for both puromycin and carbenicillin. pStX56 includes an EF-la promoter for the protein as well as a selection marker for both puromycin and kanamycin Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates containing the appropriate antibiotic. Individual colonies were picked and miniprepped using Qiaprep spin Miniprep Kit and following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. SaCas9 and SpyCas9 control plasmids were prepared similarly to pStX plasmids described above, with the protein and guide regions of pStX exchanged for the respective protein and guide. Targeting sequences for SaCas9 and SpyCas9 were either obtained from the literature or were rationally designed according to established methods. The expression and recovery of the CasX constructs was performed using the general methodologies of Example 1 and are summarized as follows:

[001124] CasX variant 488: following the same expression and purification scheme for WT CasX SEQ ID NO: 2, the following results were obtained for CasX variant 488. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as well as resolved by gel filtration. The average yield was 2.7 mg of purified CasX protein per liter of culture at 98.8% purity, as evaluated by colloidal Coomassie staining.

[001125] CasX Variant 491 : following the same expression and purification scheme for WT CasX SEQ ID NO: 2, the following results were obtained for CasX variant 488. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as well as resolved by gel filtration. The average yield was 12.4 mg of purified CasX protein per liter of culture at 99.4% purity, as evaluated by colloidal Coomassie staining.

[001126] CasX variant 515: following the same expression and purification scheme for WT CasX SEQ ID NO: 2, the following results were obtained for CasX variant 488. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as well as resolved by gel filtration. The average yield was 7.8 mg of purified CasX protein per liter of culture at 87.2% purity, as evaluated by colloidal Coomassie staining.

Table 8: Sequences of CasX 488, 491, 515 and 527

Example 4: Design and Generation of CasX Constructs 278-280, 285-288, 290, 291, 293, 300, 492, and 493

[001127] In order to generate the CasX 278-280, 285-288, 290, 291, 293, 300, 492, and 493 constructs (sequences in Table 9), the N- and C-termini of the codon-optimized CasX 119 construct (based on the CasX Stx37 construct of Example 2, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) in a mammalian expression vector were manipulated to delete or add NLS sequences (sequences in Table 10). Constructs 278, 279, and 280 were manipulations of the N- and C-termini using only an SV40 NLS sequence. Construct 280 had no NLS on the N-terminus and added two SV40 NLS’ on the C-terminus with a triple proline linker in between the two SV40 NLS sequences. Constructs 278, 279, and 280 were made by amplifying pStx34.119.174.NT with Q5 DNA polymerase according to the manufacturer’s protocol, using primers oIC527 and oIC528, oIC730 and oIC522, and oIC730 and oIC530 for the first fragments each and using oIC529 and oIC520, oIC519 and oIC731, and oIC529 and oIC731 to create the second fragments each. These fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. The respective fragments were cloned together using Gibson assembly (New England BioLabs Cat# E2621S) following the manufacturer’s protocol. Assembled products in the pStx34 were transformed into chemically-competent Turbo Competent A. coli bacterial cells, plated on LB- Agar plates containing carbenicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single- stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically- or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates containing carbenicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit and following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

[001128] In order to generate constructs 285-288, 290, 291, 293, and 300, a nested PCR method was used for cloning. The backbone vector and PCR template used was construct pStx34 279.119.174.NT, having the CasX 119, guide 174, and non-targeting spacer (see Examples 8 and 9 and Tables therein for sequences). Construct 278 has the configuration SV40NLS-CasXl 19. Construct 279 has the configuration CasXl 19-SV40NLS. Construct 280 has the configuration CasXl 19-SV40NLS-PPP linker-SV40NLS. Construct 285 has the configuration CasXl 19- SV40NLS-PPP linker-SynthNLS3. Construct 286 has the configuration CasXl 19-SV40NLS- PPP linker-SynthNLS4. Construct 287 has the configuration CasXl 19-SV40NLS-PPP linker- SynthNLS5. Construct 288 has the configuration CasXl 19-SV40NLS-PPP linker- SynthNLS6. Constrict 290 has the configuration CasXl 19-SV40NLS-PPP linker-EGL-13 NLS. Construct 291 has the configuration CasXl 19-SV40NLS-PPP linker-c-Myc NLS. Construct 293 has the configuration CasXl 19-SV40NLS-PPP linker-Nucleolar RNA Helicase II NLS. Construct 300 has the configuration CasXl 19-SV40NLS-PPP linker-influenza A protein NLS. Construct 492 has the configuration SV40NLS-CasXl 19- SV40NLS-PPP linker-SV40NLS. Construct 493 has the configuration SV40NLS-CasXl 19- SV40NLS-PPP linker-c-Myc NLS. Each variant had a set of three PCRs; two of which were nested, were purified by gel extraction, digested, and then ligated into the digested and purified backbone. Assembled products in the pStx34 were transformed into chemically-competent Turbo Competent A. coli bacterial cells, plated on LB- Agar plates containing carbenicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into the resulting pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically- or electro-competent cells such as NEB Turbo competent A coli (NEB Cat #C2984I), plated on LB-Agar plates containing carbenicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit and following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

[001129] In order to generate constructs 492 and 493, constructs 280 and 291 were digested using Xbal and BamHI (NEB# R0145S and NEB# R3136S) according to the manufacturer’s protocol. Next, they were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. Finally, they were ligated using T4 DNA ligase (NEB# M0202S) according to the manufacturer’s protocol into the digested and purified pStx34.119.174.NT using Xbal and BamHI and Zymoclean Gel DNA Recovery Kit. Assembled products in the pStx34 were transformed into chemically-competent Turbo Competent A. coli bacterial cells, plated on LB-Agar plates containing carbenicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting spacer sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting spacer sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into each pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and an appropriate restriction enzyme for the respective plasmids. Golden Gate products were transformed into chemically- or electro-competent cells such as NEB Turbo competent A. coli (NEB Cat #C2984I), plated on LB-Agar plates containing carbenicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit and following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. The plasmids would be used to produce and recover CasX protein utilizing the general methodologies of Examples 1 and 2.

Table 9: CasX 278-280, 285-288, 290, 291, 293, 300, 492, and 493 sequences

Table 10: Nuclear localization sequence list

Example 5: Design and Generation of CasX Constructs 387, 395, 485-491, and 494 [001130] In order to generate CasX 395, CasX 485, CasX 486, CasX 487, the codon optimized CasX 119 (based on the CasX 37 construct of Example 2, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences), CasX 435, CasX 438, and CasX 484 (each based on CasX 119 construct of Example 2 encoding Planctomycetes CasX SEQ ID NO: 2, with a L379R substitution, a A708K substitution, and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) were cloned respectively into a 4kb staging vector comprising a KanR marker, colEl ori, and CasX with fused NLS (pStxl) using standard cloning methods. Gibson primers were designed to amplify the CasX SEQ ID NO: 1 Helical I domain from amino acid 192-331 in its own vector to replace this corresponding region (aa 193-332) on CasX 119, CasX 435, CasX 438, and CasX 484 in pStxl respectively. The Helical I domain from CasX SEQ ID NO: 1 was amplified with primers oIC768 and oIC784 using Q5 DNA polymerase according to the manufacturer’s protocol. The destination vector containing the desired CasX variant was amplified with primers oIC765 and oIC764 using Q5 DNA polymerase according to the manufacturer’s protocol. The two fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. The insert and backbone fragments were then pieced together using Gibson assembly (New England BioLabs Cat# E2621S) following the manufacturer’s protocol. Assembled products in the pStxl staging vector were transformed into chemically-competent Turbo Competent E. colt bacterial cells, plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing kanamycin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Correct clones were then cut and pasted into a mammalian expression plasmid (see FIG. 5) using standard cloning methods. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.

[001131] Sequences encoding the targeting spacer sequences that target the gene of interest were designed based on CasX PAM locations. Targeting spacer sequence DNA was ordered as single- stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB- Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing carbenicillin and incubated at 37oC. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

[001132] In order to generate CasX 488, CasX 489, CasX 490, and CasX 491 (sequences in Table 11), the codon optimized CasX 119) CasX 435, CasX 438, and CasX 484 (each based on CasXl 19 construct of Example 2) were cloned respectively into a 4kb staging vector that was made up of a KanR marker, colEl ori, and STX with fused NLS (pStxl) using standard cloning methods. Gibson primers were designed to amplify the CasX Stxl NTSB domain from amino acid 101-191 and Helical I domain from amino acid 192-331 in its own vector to replace this similar region (aa 103-332) on CasX 119, CasX 435, CasX 438, and CasX 484 in pStxl respectively. The NTSB and Helical I domain from CasX SEQ ID NO: 1 were amplified with primers oIC766 and oIC784 using Q5 DNA polymerase according to the manufacturer’s protocol. The destination vector containing the desired CasX variant was amplified with primers oIC762 and oIC765 using Q5 DNA polymerase according to the manufacturer’s protocol. The two fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. The insert and backbone fragments were then pieced together using Gibson assembly (New England BioLabs Cat# E2621S) following the manufacturer’s protocol. Assembled products in the pStxl staging vector were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing kanamycin and incubated at 37oC. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Correct clones were then cut and pasted into a mammalian expression plasmid (see FIG. 5) using standard cloning methods. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting spacer sequences that target the gene of interest were designed based on CasX PAM locations. Targeting spacer sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing carbenicillin and incubated at 37oC. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit and following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

[001133] In order to generate CasX 387 and CasX 494 (sequences in Table 11), the codon optimized CasX 119 and CasX 484 were cloned respectively into a 4kb staging vector that was made up of a KanR marker, colEl ori, and STX with fused NLS (pStxl) using standard cloning methods. Gibson primers were designed to amplify the CasX Stxl NTSB domain from amino acid 101-191 in its own vector to replace this similar region (aa 103-192) on CasX 119 and CasX 484 in pStxl respectively. The NTSB domain from CasX Stxl was amplified with primers oIC766 and oIC767 using Q5 DNA polymerase according to the manufacturer’s protocol. The destination vector containing the desired CasX variant was amplified with primers oIC763 and oIC762 using Q5 DNA polymerase according to the manufacturer’s protocol. The two fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. The insert and backbone fragments were then pieced together using Gibson assembly (New England BioLabs Cat# E2621S) following the manufacturer’s protocol. Assembled products in the pStxl staging vector were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing kanamycin and incubated at 37oC. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Correct clones were then cut and pasted into a mammalian expression plasmid ( see FIG. 5) using standard cloning methods. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing carbenicillin and incubated at 37oC. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit and following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

Sequences of the resulting constructs are listed in Table 11.

Table 11: Sequences of CasX 395 and 485-491

Example 6: Generation of RNA guides

[001134] For the generation of RNA single guides and spacers, templates for in vitro transcription were generated by performing PCR with Q5 polymerase (NEB M0491) according to the recommended protocol, with template oligos for each backbone and amplification primers with the T7 promoter and the spacer sequence. The DNA primer sequences for the T7 promoter, guide and spacer for guides and spacers are presented in Table 12, below. The template oligos, labeled “backbone fwd” and “backbone rev” for each scaffold, were included at a final concentration of 20 nM each, and the amplification primers (T7 promoter and the unique spacer primer) were included at a final concentration of 1 mM each. The sg2, sg32, sg64, and sgl74 guides correspond to SEQ ID NOS: 5, 600, 602, and 734, respectively, with the exception that sg2, sg32, and sg64 were modified with an additional 5’ G to increase transcription efficiency (compare sequences in Table 12 to Table 2). The 7.37 spacer targets beta2-microglobulin (B2M). Following PCR amplification, templates were cleaned and isolated by phenol- chloroform-isoamyl alcohol extraction followed by ethanol precipitation.

[001135]//? vitro transcriptions were carried out in buffer containing 50 mM Tris pH 8.0, 30 mM MgC12, 0.01% Triton X-100, 2 mM spermidine, 20 mM DTT, 5 mM NTPs, 0.5 mM template, and 100 pg/mL T7 RNA polymerase. Reactions were incubated at 37°C overnight. 20 units of DNase I (Promega #M6101)) were added per 1 mL of transcription volume and incubated for one hour. RNA products were purified via denaturing PAGE, ethanol precipitated, and resuspended in IX phosphate buffered saline. To fold the sgRNAs, samples were heated to 70°

C for 5 min and then cooled to room temperature. The reactions were supplemented to 1 mM final MgCb concentration, heated to 50°C for 5 min and then cooled to room temperature. Final RNA guide products were stored at -80°C.

Table 12: Sequences for generation of guide RNA

Example 7: Assessing binding affinity to the guide RNA

[001136] Purified wild-type and improved CasX will be incubated with synthetic single-guide RNA containing a 3’ Cy7.5 moiety in low-salt buffer containing magnesium chloride as well as heparin to prevent non-specific binding and aggregation. The sgRNA will be maintained at a concentration of 10 pM, while the protein will be titrated from 1 pM to 100 mM in separate binding reactions. After allowing the reaction to come to equilibrium, the samples will be run through a vacuum manifold filter-binding assay with a nitrocellulose membrane and a positively charged nylon membrane, which bind protein and nucleic acid, respectively. The membranes will be imaged to identify guide RNA, and the fraction of bound vs unbound RNA will be determined by the amount of fluorescence on the nitrocellulose vs nylon membrane for each protein concentration to calculate the dissociation constant of the protein-sgRNA complex. The experiment will also be carried out with improved variants of the sgRNA to determine if these mutations also affect the affinity of the guide for the wild-type and mutant proteins. We will also perform electromobility shift assays to qualitatively compare to the filter-binding assay and confirm that soluble binding, rather than aggregation, is the primary contributor to protein-RNA association.

Example 8: Assessing binding affinity to the target DNA

[001137] Purified wild-type and improved CasX will be complexed with single-guide RNA bearing a targeting sequence complementary to the target nucleic acid. The RNP complex will be incubated with double-stranded target DNA containing a PAM and the appropriate target nucleic acid sequence with a 5’ Cy7.5 label on the target strand in low-salt buffer containing magnesium chloride as well as heparin to prevent non-specific binding and aggregation. The target DNA will be maintained at a concentration of 1 nM, while the RNP will be titrated from 1 pM to 100 mM in separate binding reactions. After allowing the reaction to come to equilibrium, the samples will be run on a native 5% polyacrylamide gel to separate bound and unbound target DNA. The gel will be imaged to identify mobility shifts of the target DNA, and the fraction of bound vs unbound DNA will be calculated for each protein concentration to determine the dissociation constant of the RNP -target DNA ternary complex.

Example 9: CasXrgNA In Vitro Cleavage Assays

1. Determining cleavage-competent fractions for protein variants compared to wild-type reference CasX

[001138] 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) 7.37 target for the cleavage assay was created as follows. DNA oligos with the sequence

TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC GCT (non-target strand, NTS (SEQ ID NO: 415)) and

TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC GCT (target strand, TS (SEQ ID NO: 416)) were purchased with 5’ fluorescent labels (LI-COR IRDye 700 and 800, respectively). dsDNA targets were formed by mixing the oligos in a 1:1 ratio in lx cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCb), heating to 95° C for 10 minutes, and allowing the solution to cool to room temperature.

[001139] CasX RNPs were reconstituted with the indicated CasX and guides (see graphs) at a final concentration of 1 mM with 1.5-fold excess of the indicated guide unless otherwise specified in 1 x cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCb) at 37° C for 10 min before being moved to ice until ready to use. The 7.37 target was used, along with sgRNAs having spacers complementary to the 7.37 target. [001140] Cleavage reactions were prepared with final RNP concentrations of 100 nM and a final target concentration of 100 nM. Reactions were carried out at 37° C and initiated by the addition of the 7.37 target DNA. Aliquots were taken at 5, 10, 30, 60, and 120 minutes and quenched by adding to 95% formamide, 20 mM EDTA. Samples were denatured by heating at 95° C for 10 minutes and run on a 10% urea-PAGE gel. The gels were either imaged with a LI-COR Odyssey CLx and quantified using the LI-COR Image Studio software or imaged with a Cytiva Typhoon and quantified using the Cytiva IQTL software. The resulting data were plotted and analyzed using Prism. We 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- stoichiometric 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. The cleavage traces were fit with a biphasic rate model, as the cleavage reaction clearly deviates from monophasic under this concentration regime, and the plateau was determined for each of three independent replicates. The mean and standard deviation were calculated to determine the active fraction (Table 13).

The graph is shown in FIG. 15.

[001141] Apparent active (competent) fractions were determined for RNPs formed for CasX2 + guide 174 + 7.37 spacer, CasX119 + guide 174 + 7.37 spacer, CasX457 + guide 174 +7.37 spacer, CasX488 + guide 174 + 7.37 spacer, and CasX491 + guide 174 + 7.37 spacer. The determined active fractions are shown in Table 13. All CasX variants had higher active fractions than the wild-type CasX2, indicating that the engineered CasX variants form significantly more active and stable RNP with the identical guide under tested conditions compared to wild-type CasX. This may be due to an increased affinity for the sgRNA, increased stability or solubility in the presence of sgRNA, or greater stability of a cleavage-competent conformation of the engineered CasX:sgRNA complex. An increase in solubility of the RNP was indicated by a notable decrease in the observed precipitate formed when CasX457, CasX488, or CasX491 was added to the sgRNA compared to CasX2.

2. In vitro Cleavage Assays - Determining kdeave for CasX variants compared to wild-type reference CasX

[001142] Cleavage-competent fractions were also determined using the same protocol for CasX2.2.7.37, CasX2.32.7.37, CasX2.64.7.37, and CasX2.174.7.37 to be 16 ± 3%, 13 ± 3%, 5 ± 2%, and 22 ± 5%, as shown in FIG. 16 and Table 13.

[001143] A second set of guides were tested under different conditions to better isolate the contribution of the guide to RNP formation. 174, 175, 185, 186, 196, 214, and 215 guides with 7.37 spacer were mixed with CasX491 at final concentrations of 1 mM for the guide and 1.5 mM for the protein, rather than with excess guide as before. Results are shown in FIG. 17 and Table 13. Many of these guides exhibited additional improvement over 174, with 185 and 196 achieving 91 ± 4% and 91 ± 1% competent fractions, respectively, compared with 80 ± 9% for 174 under these guide-limiting conditions.

[001144] The data indicate that both CasX variants and sgRNA variants are able to form a higher degree of active RNP with guide RNA compare to wild-type CasX and wild-type sgRNA. [001145] The apparent cleavage rates of CasX variants 119, 457, 488, and 491 compared to wild-type reference CasX were determined using an in vitro fluorescent assay for cleavage of the target 7.37.

[001146] CasX RNPs were reconstituted with the indicated CasX (see FIG. 18) at a final concentration of 1 pM with 1.5-fold excess of the indicated guide in lx cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCh) at 37° C for 10 min before being moved to ice until ready to use. 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 37° C except where otherwise noted and initiated by the addition of the target DNA. Aliquots were taken at 0.25, 0.5, 1, 2, 5, and 10 minutes and quenched by adding to 95% formamide, 20 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 LI-COR Odyssey CLx and quantified using the LI- COR Image Studio software or imaged with a Cytiva Typhoon and quantified using the Cytiva IQTL software. The resulting data were plotted and analyzed using Prism, and the apparent first- order rate constant of non-target strand cleavage (kdeave) was determined for each CasX: sgRNA combination replicate individually. The mean and standard deviation of three replicates with independent fits are presented in Table 13, and the cleavage traces are shown in FIG 18.

[001147] Apparent cleavage rate constants were determined for wild-type CasX2, and CasX variants 119, 457, 488, and 491 with guide 174 and spacer 7.37 utilized in each assay (see Table 13 and FIG. 18). All CasX variants had improved cleavage rates relative to the wild-type CasX2. CasX457 cleaved more slowly than 119, despite having a higher competent fraction as determined above. CasX488 and CasX491 had the highest cleavage rates by a large margin; as the target was almost entirely cleaved in the first timepoint, the true cleavage rate exceeds the resolution of this assay, and the reported kcieave should be taken as a lower bound.

[001148] The data indicate that the CasX variants have a higher level of activity, with kcieave rates reaching at least 30-fold higher compared to wild-type CasX2.

3. In vitro Cleavage Assays: Comparison of guide variants to wild-type guides [001149] Cleavage assays were also performed with wild-type reference CasX2 and reference guide 2 compared to guide variants 32, 64, and 174 to determine whether the variants improved cleavage. The experiments were performed as described above. As many of the resulting RNPs did not approach full cleavage of the target in the time tested, we determined initial reaction velocities (Vo) rather than first-order rate constants. The first two timepoints (15 and 30 seconds) were fit with a line for each CasX:sgRNA combination and replicate. The mean and standard deviation of the slope for three replicates were determined.

[001150] Under the assayed conditions, the Vo for CasX2 with guides 2, 32, 64, and 174 were 20.4 ± 1.4 nM/min, 18.4 ± 2.4 nM/min, 7.8 ± 1.8 nM/min, and 49.3 ± 1.4 nM/min (see Table 13 and FIG. 19 and FIG. 20). Guide 174 showed substantial improvement in the cleavage rate of the resulting RNP (~2.5-fold relative to 2, see FIG. 20), while guides 32 and 64 performed similar to or worse than guide 2. Notably, guide 64 supports a cleavage rate lower than that of guide 2 but performs much better in vivo (data not shown). Some of the sequence alterations to generate guide 64 likely improve in vivo transcription at the cost of a nucleotide involved in triplex formation. Improved expression of guide 64 likely explains its improved activity in vivo , while its reduced stability may lead to improper folding in vitro.

[001151] Additional experiments were carried out with guides 174, 175, 185, 186, 196, 214, and 215 with spacer 7.37 and CasX491 to determine relative cleavage rates. To reduce cleavage kinetics to a range measurable with our assay, the cleavage reactions were incubated at 10° C. Results are in FIG. 21 and Table 13. Under these conditions, 215 was the only guide that supported a faster cleavage rate than 174. 196, which exhibited the highest active fraction of RNP under guide-limiting conditions, had kinetics essentially the same as 174, again highlighting that different variants result in improvements of distinct characteristics.

[001152] The data support that, under the conditions of the assay, use of the majority of the guide variants with CasX results in RNP with a higher level of activity than one with the wild- type guide, with improvements in initial cleavage velocity ranging from ~2-fold to >6-fold. Numbers in Table 13 indicate, from left to right, CasX variant, sgRNA scaffold, and spacer sequence of the RNP construct. In the RNP construct names in the table below, CasX protein variant, guide scaffold and spacer are indicated from left to right.

Table 13: Results of cleavage and RNP formation assays

*Mean and standard deviation Example 10: Assessing differential PAM recognition in vitro

[001153] In vitro cleavage assays were performed essentially as described in Example 9, using CasX2, CasX119, and CasX438 complexed with sgl74.7.37. Fluorescently labeled dsDNA targets with a 7.37 spacer and either a TTC, CTC, GTC, or ATC PAM were used (sequences are in Table 14). Time points were taken at 0.25, 0.5, 1, 2, 5, 10, 30, and 60 minutes. Gels were imaged with an Cytiva Typhoon and quantified using the IQTL 8.2 software. Apparent first- order rate constants for non-target strand cleavage (kcieave) were determined for each Casx:sgRNA complex on each target. Rate constants for targets with non-TTC PAM were compared to the TTC PAM target to determine whether the relative preference for each PAM was altered in a given protein variant.

[001154] For all variants, the TTC target supported the highest cleavage rate, followed by the ATC, then the CTC, and finally the GTC target (FIG. 22A-D, Table 15). For each combination of CasX variant and NTC PAM, the cleavage rate kcieave is shown. For all non-NTC PAMs, the relative cleavage rate as compared to the TTC rate for that variant is shown in parentheses. All non-TTC PAMs exhibited substantially decreased cleavage rates (>10-fold for all). The ratio between the cleavage rate of a given non-TTC PAM and the TTC PAM for a specific variant remained generally consistent across all variants. The CTC target supported cleavage 3.5-4.3% as fast as the TTC target; the GTC target supported cleavage 1.0-1.4% as fast; and the ATC target supported cleavage 6.5-8.3% as fast. The exception is for 491, where the kinetics of cleavage at TTC PAMs are too fast to allow accurate measurement, which artificially decreases the apparent difference between TTC and non-TTC PAMs. Comparing the relative rates of 491 on GTC, CTC, and ATC PAMs, which fall within the measurable range, results in ratios comparable to those for other variants when comparing across non-TTC PAMs, consistent with the rates increasing in tandem. Overall, differences between the variants are not substantial enough to suggest that the relative preference for the various NTC PAMs have been altered. However, the higher basal cleavage rates of the variants allow targets with ATC or CTC PAMs to be cleaved nearly completely within 10 minutes, and the apparent kdeaves are comparable to or greater than the kcieave of CasX2 on a TTC PAM (Table 14). This increased cleavage rate may cross the threshold necessary for effective genome editing in a human cell, explaining the apparent increase in PAM flexibility for these variants. Table 14. Sequences of DNA substrates used in in vitro PAM cleavage assay.

*The PAM sequences for each are bolded. TS - target strand. NTS - Non-target strand.

Table 15. Apparent cleavage rates of CasX variants against NTC PAMs.

Example 11: Identification of nicking variants

[001155] Purified modified CasX variants will be complexed with single-guide RNA bearing a fixed targeting sequence. The RNP complexes will be added to buffer containing MgC12 at a final concentration of 100 nM and incubated with double-stranded target DNA with a 5’ fluorescein label on the target strand and a 5’ Cy5 label on the non-target strand at a concentration of 10 nM. Aliquots of the reactions will be taken at fixed time points and quenched by the addition of an equal volume of 50 mM EDTA and 95% formamide. The samples will be run on a denaturing polyacrylamide gel to separate cleaved and uncleaved DNA substrates. Efficient cleavage of one strand but not the other would be indicative that the variant possessed single-strand nickase activity.

Example 12: Assessing improved expression and solubility characteristics of CasX variants for RNP production

[001156] Wild-type and modified CasX variants will be expressed in BL21 (DE3) E. coli under identical conditions. All proteins will be under the control of an IPTG-inducible T7 promoter. Cells will be grown to an OD of 0.6 in TB media at 37°C, at which point the growth temperature will be reduced to 16°C and expression will be induced by the addition of 0.5 mM IPTG. Cells will be harvested following 18 hours of expression. Soluble protein fractions will be extracted and analyzed on an SDS-PAGE gel. The relative levels of soluble CasX expression will be identified by Coomassie staining. The proteins will be purified in parallel according to the protocol above, and final yields of pure protein will be compared. To determine the solubility of the purified protein, the constructs will be concentrated in storage buffer until the protein begins to precipitate. Precipitated protein will be removed by centrifugation and the final concentration of soluble protein will be measured to determine the maximum solubility for each variant. Finally, the CasX variants will be complexed with single guide RNA and concentrated until precipitation begins. Precipitated RNP will be removed by centrifugation and the final concentration of soluble RNP will be measured to determine the maximum solubility of each variant when bound to guide RNA.

Example 13: XDP construct, transfection and recovery.

Plasmids and Cell lines

[001157] CasX delivery particles (XDPs) containing RNP of CasX, CasX 119, CasX 438, or CasX 457 protein and single guide RNA 174 with spacer sequence 12.7 (encoded by CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825) targeting tdTomato were produced by transient transfection of LentiX HEK293T cells (Takara Biosciences) using the four plasmids portrayed in FIG. 23 and listed in Table 16 (with different plasmids depending on which CasX was utilized). The pStx43 plasmid contains the Gag polyprotein sequence followed by a CasX protein fused at the C-terminus (pXDlO encodes CasX 119; pXDl 1 encodes CasX 438; pXD12 encodes CasX 457). A SQNYPIVQ (SEQ ID NO: 20) HIV-1 cleavage site separated the Gag protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold and spacer components (targeted to tdTomato) in a single guide format. Another pStx42 plasmid was utilized to make a CasX guide cassette having scaffold and non-targeting spacer components, used as a control in the editing assay. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP and Gag-Pol (psPax2) proteins were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 16.

Table 16: Plasmid Encoding Sequences

Transfection

[001158] The steps for creation of the XDP are depicted graphically in FIG. 24. HEK293T Lenti-X^® cells were maintained in 10% FBS supplemented DMEM with HEPES, penicillin/streptomycin (Pen/Step), sodium pyruvate, and 2-mercaptoethanol. Cells were seeded in 10 cm dishes at 8e6 cells per dish in 10 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection cells were 70-90% confluent. For transfection, the following plasmid amounts were used: 19.8 pg of pXDPlO, pXDPl 1, or pXDP12.5 pg of pStx42.174.12.7, 3.3 pg of psPax2, and 1 pg of pGP2 in 680 pi of Opti-MEM media. 87.5 pi of 1 mg/ml linear polyethylenimine (PEI, MW=25,000 Da) was then added to the plasmid mixture, mixed, and allowed to incubate at room temperature before being added to the cell culture.

Collection and concentration

[001159] Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 pM filter using a 10 mL syringe. 1 ml of the approximately 8 mL remaining after filtration was stored at 4°C for titering and subsequent assays. The remaining filtered supernatant was used directly for cell editing or was concentrated by centrifugation at 10,000 x g at 4°C for 4h using a 10% sucrose buffer in NTE, as described below.

Example 14: Purification of XDP

[001160] As described in the various Examples for production of XDP, production cells were maintained in DMEM supplemented with 10% fetal bovine system at 37°C in a humidified 5% CO2 atmosphere. Cells were plated in 15 cm plates 24 hours before transfection. Transfections were carried out using PEI with the appropriate plasmids. The media was removed and replaced with Opti-MEM containing 6.25 U/mL of Benzonase 24 hours after transfection. XDP- containing supernatant was collected 72 hours after transfection and filtered through 0.45 mM PES filters before being stored at 4°C until purification.

Centrifugation Protocol

[001161] Filtered supernatant was divided evenly into an appropriate number of centrifuge tubes or bottles and 1/5*¹¹ of the supernatant volume of Sucrose Buffer (50mM Tris-HCL, lOOmM NaCl, 10% Sucrose, pH 7.4) was underlaid using serological pipettes. The samples were centrifuged at 10,000xg, 4°C, in a swinging-bucket rotor for 4 hours with no brake. The supernatant was carefully removed and the pellet briefly dried by inverting the centrifuge vessels. Pellets were then resuspended in Storage Buffer (PBS + 113 mM NaCl, 15% Trehalose dihydrate, pH 8) or an appropriate media by gentle trituration and vortexing.

Column Protocol

[001162] Filtered supernatant was purified by anion exchange chromatography (AEX) using an FPLC instrument, at 4°C. The AEX column was equilibrated with buffer A, the supernatant was applied, and the column was washed with 10 CV of Buffer A (100 mM Tris-HCl, pH 7.5).

Bound material was eluted using a gradient elution from 0% - 100% Buffer B (100 mM Tris- HCl, 1M NaCl, pH 7.5) over 40 column volumes. XDP-containing fractions were pooled and further purified using a CaptoCore 700 column (Cytiva), equilibrated with buffer C (100 mM Tris-HCl, 300 mM NaCl, pH 7.5). The XDP-containing flow-through was then concentrated using 100 kDa cutoff spin concentrators at room temperature. The resulting concentrated sample was diafiltered into Storage buffer, aliquoted, and snap-frozen in liquid nitrogen before being stored at -80°C.

Quantification

[001163] Samples were quickly thawed at 37°C in a heat bath, vortexed, and diluted in 2xPBS supplemented with 0.1% Tween 20. Particle titer and size was evaluated using the qNano Gold TRPS system (Izon Science) on anNP150 nanopore.

[001164] FIG. 34 shows representative SDS-PAGE and Western blot images of samples taken from throughout the centrifugation purification process. Lanes from left to right: Cells: producer cells, Pre: Supernatant pre-filtration, Post: 0.45 mM filtered supernatant, Supe: Supernatant remaining after centrifugation, Pellet: resuspended XDP pellet. Total protein was visualized with StainFree technology (BioRad), Western blotting was performed with the indicated antibodies. These figures show that XDPs can be purified and concentrated from mammalian producer cell supernatant either by centrifugation or by the column chromatography. In FIG. 34, the total protein staining shows that certain proteins are concentrated in the supernatant, that are not over represented in the whole cell lysate (Cells lane). The pre, post, and supe lanes are indistinguishable, demonstrating that the bulk proteins are not being concentrated into the XDP pellet. This is further shown by the change in makeup of the pellet lane, which has unique bands consistent with the molecular weight of gag-CasX-HA, VSV-G, and gag. Western blotting confirmed these results, showing that, despite the same amount of protein being loaded in each lane, the most significant staining is in the pellet lane. The second darkest staining can be seen in the input lane, showing that the particles are concentrated by this process. The lack of staining in the other lanes indicates that only an insignificant amount of particles are lost at each step. [001165] On average, this purifications process yields 4.13 x 10¹² particles per liter of filtered supernatant, at a concentration of 2.48 x 10¹¹ particles per milliliter, averaging 113 nm in diameter, as measured by TRPS. The average activity of particles purified in this way was 4.27 x 10⁷ editing units (EU) per mL, once purified. This works out to 1.42 x 10⁷ EU/L of culture, which is a feasible yield for production of vectors for therapeutic use.

Example 15: XDP construct, transfection and recovery

[001166] Alternative configuration versions of the CasX delivery particles (XDPs) named Versions 1-24 (see Table 17) were designed to contain RNP of four different CasX variants proteins; CasXl 19, CasX438, CasX 457, or CasX 491, complexed with single guide RNA variant 174 having spacer sequence 12.7 targeted to tdTomato (encoded by CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825). The XDP were produced by transient transfection of LentiX HEK293T cells (Takara Biosciences) using one or more structural plasmids (derived from one or more components of the Gag-Pol HIV-1 system, a plasmid encoding a pseudotyping glycoprotein, and a plasmid encoding a single guide RNA (see FIG.

17, representing Version 1), using the methods described below. Table 17, grouped by version number, lists the plasmids (and their sequences) that were used to produce each version of the XDP containing the components indicated in the column “Design”, and FIG. 24 shows schematics of the organization of the various plasmids in the versions. The plasmids were constructed utilizing the methods outlined in Example 13. For the plasmid encoding the guide RNA, the pStx42 plasmid was created with a human U6 promoter upstream of a guide RNA cassette having scaffold and spacer components targeted to tdTomato in a single-guide format, as described in Example 13. Another pStx42 plasmid was utilized to make a guide RNA cassette having scaffold and non-targeting spacer components, used as a control in the editing assay. Plasmids encoding VSV-G (pGP2) for pseudotyping the XDP and Gag-Pol (psPax2) proteins was also used (representative sequence in Table 16). All plasmids contained either an ampicillin or kanamycin resistance gene.

Table 17: Plasmid Encoding Sequences Transfection

[001167] The steps for creation of the XDP are depicted graphically in FIG. 24. HEK293T Lenti-X cells were maintained in 10% FBS supplemented DMEM with HEPES, penicillin/streptomycin (Pen/Step), sodium pyruvate, and 2-mercaptoethanol. Cells were seeded in 10 cm dishes at 8e6 cells per dish in 10 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection cells were 70-90% confluent. For transfection, the plasmids of Table 17, together with the 5 pg of the guide plasmid, and 0.1 pg of pMD2.G in 680 pi of Opti-MEM media. 87.5 pi of 1 mg/ml linear polyethylenimine (PEI, MW=40,000 Da) was then added to the plasmid mixture, mixed, and allowed to incubate at room temperature before being added to the cell culture.

Collection and concentration

[001168] Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 pM filter using a 10 mL syringe.

1 ml of the approximately 8 mL remaining after filtration was stored at 4°C for titering and subsequent assays. The remaining filtered supernatant was used directly for cell editing or was concentrated by centrifugation at 10,000 x g at 4°C for 4h using a 10% sucrose buffer in NTE.

Example 16: Editing of tdTomato neural progenitor cells using XDP

[001169]tdTomato neural progenitor cells (tdT NPCs) were grown in DMEM F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using a Takara Biosciences Neuron Dissociation Kit and seeded on PLF coated 96 well plates. Cells were allowed to grow at 37°C for 48 hours before being treated with targeting XDPs (having spacer 12.7 for tdTomato) and non-targeting XDPs (having a non-targeting spacer) as a lOx concentrate from the sucrose buffer concentrates using half-log dilutions, as well as a Opti-MEM negative control. NPCs were grown for 96 hours before analysis of fluorescence as a marker of editing of tdTomato.

[001170] Results: The results of the editing assay are shown in FIG. 25 and in Table 18, below. FIG. 25 shows results of a single experiment (Targeting XLP is XDP CasXl 19 with VSV-g; Bald VLP is XDP CasXl 19 with no GP; and Negative Control is Buffer Control as labelled in Table 18, while the table represents the mean results of 3 experiments showing 20% editing of the dtTomato target sequence was achieved with the XDP comprising the CasX 119 construct. Table 18: Results of Editing Assay

Example 17: Construction of XDP with incorporated glycoproteins to evaluate tropism and editing capabilities

[001171] Viral vectors including lentiviral and retroviral vectors are most often pseudotyped with the envelope protein of vesicular stomatitis virus (VSV-G); a glycoprotein that endows both a broad host cell range and high vector particle stability. Experiments were performed in which XDPs with incorporated RNP of CasX and gNA specific for editing tdTomato in mouse neural progenitor cells (tdT NPCs) were created with varying concentrations of incorporated VSV-G to determine the corresponding effects on editing in tdT NPCs via the enhanced delivery of the editing moiety by the VSV-G.

[001172] Experiments shown in FIGS. 26-28 follow the XDP production methods (for the CasX 119 and single guide RNA 174 with spacer sequence 12.7 targeted to tdT) and, where applicable, testing procedures detailed in Examples 13 and 15. Sequences are shown in Table 19. For the experiments resulting in the data in FIGS. 26A and 26B, the effects of varying concentrations of the pseudotyping (VSV-G) plasmid incorporated into the XDP were evaluated as follows: 1 pg of the VSV-G plasmid was used for the 100% VSV-G group, 0.3 pg was used for the 30% VSV- G group, 0.1 pg was used for the 10% VSV-G group, 0.03 pg was used for the 3% VSV-G group, 0.01 pg was used for the 1% VSV-G group, and 0.003 pg was used for the 0.3% VSV-G group. Titering of the XDPs produced was done using the Takara p24 rapid titer kit. Editing was assessed in the tdTomato NPC cells as detailed in Example 16.

[001173] The results for the 10% and 30% VSV-G groups trend towards a better editing outcome as compared to the 100% VSV-G group, as shown in FIG. 26A, without affecting viral titer or stability, as shown in FIG. 26B.

[001174] As the results indicate that one can achieve, under the experimental conditions, the same, if not higher editing with 10-30% VSV-G compared to the 100% VSV-G group, this opens up the possibility of pseudotyping the XDP particle with other encoded glycoproteins, either with or without VSV-G, to confer differential or enhanced cellular tropism to the resulting XDP, including the viral glycoproteins disclosed herein, examples of which were produced and evaluated as follows. Utilizing the XDP production and editing methods of Example 13 and 15, each XDP transfection used 3.3 pg (0.467 pM) of psPax2 plasmid, 19.8 pg (3.24 pM) of pStx43.119 plasmid, 5 pg (3.13 pM) of pStx42 plasmid (with guide 174) targeting the tdTomato locus using spacer 12.7 and 0.262 pM of the respective glycoprotein(s) plasmid which varied in molecular weight. Glycoprotein plasmids contained the same backbone pGP2 and only varied by expressing different viral envelope proteins which they expressed. The following plasmids were used for transfections: rabies used 0.94 pg of pGP29; FUG E used 0.95 pg of pGP60; HSV-1 used 0.28 pg of pGPM.l, 0.22 pg of pGP14.2, 0.27 pg of pGP14.3, and 0.20 pg of pGP14.4; RD114 used 0.96 pg of pGP8; HCV used 0.97 ug of pGP23; EBOVused 1.02 pg of pGP41; Mokola used 1.02 pg of pGP30. Canonical HSV-1 pseudotyping requires four glycoproteins which were used in equimolar amounts in this assay (Polpitiya Arachchige, S., Henke, W., Kalamvoki, M. et al. Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles. Retrovirology 16:9 (2019)). Glycoprotein amino acid sequences come from wild type viral sequences. Nucleic acid sequences also came from wild type viral sequences though some were codon optimized for synthesis and expression in human cell lines.

[001175] The editing efficiencies in mouse tdTomato NPCs were tested with an initial panel of pseudotyped XDPs having glycoproteins from VSV-G, rabies, FUG E, HSV-1, RD114, hepatitis C virus (HCV), and Ebola virus (EBOV), produced as described above. The results are shown in FIG. 27. While constructs with FUG E, Mokola and herpes simplex virus-1 (HSV-1) incorporated glycoproteins were expected to achieve some level of cell entry in NPCs, rabies was the only glycoprotein other than VSV-G resulting in an observable level of editing under the conditions of the assay, which is a readout for cell entry into mouse neural progenitor cells. Conversely, XDPs pseudotyped with HCV, EBOV and RD114 did not achieve any editing in mouse NPCs, which indicates the potential cell specificity requirements for this cell type. [001176] We also assessed whether pseudotyping with different viral glycoproteins could have an impact on overall size distributions, which could have an impact on in vivo editing efficiencies in different tissues of interest. For this experiment, the rabies pseudotyped XDP 10X and VSV-G pseudotyped XDP lx were produced using the protocol described above scaled to a 6 well format and using pGP29 in place of the pGP2 plasmid. All plasmid quantities and cells used were scaled down 8-fold. The VSV-G pseudotyped XDP IX were generated as described above. These preparations were then concentrated at 20,000 x g at 4°C for 90 minutes without a sucrose buffer. LV was transfected with the following plasmid weights: 5.4 pg of psPax2, 1.8 pg of pGP2, and 7.2 pg of pStx34.119.174.12.7, generating lentivirus designed to induce production and incorporation of RNP with the same enzymatic capabilities as VSV-G pseudotyped XDP IX. Samples were diluted appropriately for analysis. The size and number of particles were assessed using a Tunable Resistive Pulse Sensor (Izon Biosciences qNano Gold). While both rabies and VSV-G XDPs ranged in size from 75-140 nm, lentiviruses (LVs) tend to be a bit larger, ranging in size from 85-160 nm, as shown in FIG. 28 A. FIG. 28B shows that rabies pseudotyped XDPs trend towards a smaller mode as compared to VSV-G pseudotyped XDPs.

Table 19. Plasmid encoding sequences for glycoproteins. Example 18: Construction and evaluation of XDP with RNP comprising CasX with enhanced editing capabilities

[001177] In addition to improving the targeting capability and specificity within the XDP platform, the ability to concurrently improve the editing capability of XDPs incorporating improved RNP variants having CasX 438 and CasX 457 (compared to CasX 119) was examined (with guide 174 and spacer 12.7). The RNP variants were constructed by exchanging the CasX encoding sequences within the pStx43 plasmid. RNP 457 was transfected using 19.8 pg of pStx43.119, RNP 438 was transfected using 19.8 pg of pStx43.438, and RNP 119 was transfected using 19.8 pg of pStx43.119 (sequences in Table 20). Percent editing in mouse NPCs was assessed using the tdTomato assay described above and read-out was performed using an Attune NxT Flow Cytometer. Titers were assessed using a Takara p24 Rapid Titer Kit. The results, shown in FIG. 29, demonstrate enhanced editing of the tdTomato NPCs by the XDP with RNP comprising the CasX 438 and CasX 457 compared to RNP comprising CasX 119.

Example 19: Construction and evaluation of XDP with non-essential lentiviral components removed

[001178] The ability to improve XDP editing by optimizing RNP packaging into the viral vectors was evaluated by stripping away non-essential components such as the viral genome (Gag-Pol) from the Gag-CasX construct. Moreover , the removal of these components would alleviate some of the safety concerns with these platforms by taking away the reverse transcriptase (RT), integrase (IN) components that have been a source of concern for their use in humans. Furthermore, it offers the possibility of increased packaging of the RNP complex into an XDP molecule, as every Gag molecule packaged would have a CasX molecule attached to it. [001179] The XDP were created using the same approach as described above (i.e., 8 x 10⁶ LentiX cells were plated in a 10 cm dish, 24 hours later cells were transfected with DNA, media was changed 16 hours after transfection , XDPs were collected 72 hours post-transfection and concentrated). Here, we introduced a new plasmid having the components Gag, CasX, and protease, referred to as Gag-CasX-PR (or pMRG103; sequence in Table 20). This plasmid contains a gag polyprotein followed by a CasX molecule linked by a SQNYPIVQ (SEQ ID NO: 20) HIV-1 cleavage site. The CasX molecule is followed by an HA tag and another SQNYPIVQ (SEQ ID NO: 20) HIV-1 cleavage site linked to a component of the Pol protein from HIV-1.

This component contains the HIV-1 protease (PR) and lacks the HIV-1 reverse transcriptase (RT), pi 5, and integrase (INT) components. Upon budding of the XDP from the cell membrane, the protease functions identically to the protease found in the native Gag-Pol complex; it dimerizes and facilitates cleavage of the SQNYPIVQ (SEQ ID NO: 20) HIV-1 cleavage sites, freeing CasX from Gag and PR. To generate XDPs with this new construct, the following plasmid amounts were used: 0.3 pg of pGP2, 5 pg of pStx42 (guide 174) with spacer 12.7, and 19.8 pg of pStx43.119 (CasX 119). Additional constructs used the following plasmid amounts: 100% Gag-Pol used 3.3 pg of psPax2; the 50% Gag-Pol + 50% Gag-CasX construct used 1.65 pg of psPax2 and 1.48 pg of Gag-CasX-PR; the 30% Gag-Pol + 70% Gag-CasX construct used 0.99 pg of psPax2 and 1.47 pg of Gag-CasX-PR; the 15% Gag-Pol + 85% Gag-CasX construct used 0.50 pg of psPax2 and 2.51 pg of Gag-CasX-PR; and the 100% Gag-CasX construct used 3.00 pg of Gag-CasX-PR. Sequences are provided in Table 20.

[001180] Editing of tdTomato NPCs was assessed as described above, and the titer of the XDP preparations was assessed using the Takara p24 Rapid Titer Kit. The results, shown in FIG. 30, demonstrated that XDP created with Gag-CasX-PR and no inclusion of Gag-Pol were able to achieve the same amount of editing at ~10⁶ particles as compared to ~10⁸ particles with XDPs that have 100% Gag-Pol. The other constructs showed editing in proportion to the titer of the particles. The titer data for the various constructs that were produced is shown in FIG. 31. We believe that this observed enhancement in editing efficiency is due to enhanced packaging of RNP molecules per XDP, as shown by guide RNA quantification for the different XDP constructs as depicted in FIG. 32.

Table 20: Plasmid encoding sequences

Example 20: Construction and evaluation of XDP targeting human cells

[001181] The tdTomato mouse neural progenitor cell model is a powerful tool to assess the potency of XDPs. However, in view of the intended clinical application of XDP, the potency of these particles must be assessed on human cells using easily accessible, quantifiable and therapeutically relevant cell lines. As the human HLA locus for MHC I beta 2 microglobulin (B2M) fits these criteria, XDP were generated using the methodology described in Examples 13 and 15 above, with RNP comprising CasX 119 and gNA 174 with spacer sequences targeting B2M for assessment in Jurkat cells, a human T- cell line. The spacers 7.9 (GT GT AGT AC A AG AG AT AG A A, SEQ ID NO: 824) and 7.37

(GGCCGAGATGTCTCGCTCCG, SEQ ID NO: 826) target the human B2M locus and spacer 12.7 (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825), which targets the artificial tdTomato locus in mice, was used as the non-targeting spacer. Jurkat cells were seeded in a 96 well plate in RPMI media supplemented with 10% FBS, sodium pyruvate, and GlutaMAX. XDPs, resuspended in Opti-MEM, were diluted in half-log serial dilutions in RPMI media before being put on Jurkat cells and were spinfected at 1000 x g for 15 minutes. Cells were incubated at 37°C for 120 hours before analysis. To stain HLA, we used DAPI to mark dead cells, and the PE-Cy7 Mouse Anti -Human HLA- ABC staining kit (BD Pharmingen) was used to stain major histocompatibility complex, class I. Expression of this complex at the cell surface is blocked by B2M knockout.

Results:

[001182] The results, shown in FIG. 33, depicts the relative HLA negative (edited) populations in Jurkat cells, after being treated with XDPs containing CasX molecules with spacer 7.9, spacer 7.37, or a non-targeting spacer. The results indicate that under the experimental conditions, the XDPs with spacer 7.9 are capable of knocking out B2M in -10% of Jurkat cells.

Example 21: The generation and assessment of potency of HIV-1 XDPs with alternative structures of HIV-1 Gag in various configurations.

[001183] The purpose of these experiments was to make various configurations of XDP constructs comprising CasX and guide RNA as RNP to demonstrate their utility in the editing of eukaryotic cells; either by in vitro or by in vivo delivery. To generate the most efficient and minimal HIV-1 capsid designed specifically for RNP delivery, we created thirty-five different versions of HIV-1 based XDPs with CasX 491 and guide RNA 174 and spacer 12.7 to tdTomato to 1) determine which components of HIV- 1 were and were not necessary for the successful delivery of RNP to cells capable of editing target nucleic acid; and 2) demonstrate that multiple configurations of XDP were able to successfully delivery RNP to cells and edit target nucleic acid. Methods

Method for the generation of XDPs

[001184] Alternative configuration versions of the XDPs, referred to as versions 1, 4, 5, 7-27, 32-40, and 122-124, 126 and 128 (see FIGS. 36-68), were designed to contain RNP of CasX 491 complexed with a single guide RNA variant having spacer sequence 12.7 targeted to tdTomato (encoded by CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825). Utilizing methods described in the sections below, the XDP versions were produced by transient transfection of LentiX HEK293T cells (Takara Biosciences) with one or more structural plasmids encoding components of the gag-pol HIV-1 system, a plasmid encoding a pseudotyping glycoprotein, and a plasmid encoding a single guide RNA (see FIGS. 36-68 for schematics of each version, the plasmids employed and the components the plasmid encoded)). Table 21, grouped by version number, lists the plasmids and their sequences that were used to produce each version of the XDP containing the components indicated in the Table and the corresponding version of the Figures. For the plasmid encoding the guide RNA, the pStx42 plasmid was created with a human U6 promoter upstream of a guide RNA cassette having scaffold and spacer components targeted to tdTomato in a single-guide format (p42.174.12.7). Another pStx42 plasmid was utilized to make a guide RNA cassette having scaffold and non-targeting spacer components (Stx42.174.NT), used as a control in the editing assays. A plasmid encoding VSV-G (pGP2) for pseudotyping the XDP was also used (Table 22). All plasmids contained either an ampicillin or kanamycin resistance gene.

Structural plasmid cloning

[001185] In order to generate pXDP3, pXDP17, pXDP23-32, pXDP98-100, pXDP102 and pXDP103, pXDPl (UC Berkeley) was digested using EcoRI to remove the gag-pol sequence. Between one and three fragments containing CasX and HIV-1 components were amplified using In Fusion primers with 15-20 base pair overlaps and Kapa HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. These fragments were cloned into plasmid backbones using In-Fusion HD Cloning Kit from Takara (Cat# 639650) according to the manufacturer’s protocols. Assembled products were transformed into chemically-competent Turbo Competent A. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing ampicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. The encoding sequences are presented in Table 23. The first column of the table describes the version number and CasX molecule included. The second is the configuration of the HIV components and CasX molecules. The plasmid number for those design plasmids are in the third column. The fourth column contains SEQ IDS for only the encoding sequences for HIV-1 gag, HIV-1 pol, and CasX molecules, as applicable.

Guide plasmid cloning

[001186] The p42.174.NT (NT sequence CGAGACGTAATTACGTCTCG, SEQ ID NO: 827) plasmid encoding the guide RNA 174 and the non-targeting spacer and the p42.174.12.7 targeting tdTomato were cloned using standard cloning methods. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. These fragments were amplified using Kapa HiFi DNA polymerase according to the manufacturer’s protocols and primers appropriate for In-Fusion cloning. The fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. These fragments were cloned into plasmid backbones using In-Fusion® HD Cloning Kit from Takara (Cat 639650) according to manufacturer protocols. Assembled products were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat# 214510) containing ampicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.

Cloning tdTomato spacer 12.7 into p42.174.NT

[001187] The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825) and the reverse complement of this sequence. These two oligos were annealed together and cloned into p42.174 NT or a p42 plasmids with an alternate scaffold. This was done by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and Esp3I restriction enzyme from NEB (New England BioLabs Cat# R0734L). Golden Gate products were transformed into chemically competent NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat# 214510) containing carbenicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning

[001188] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G (UC Berkeley) using Kapa HiFi DNA polymerase according to the manufacturer’s protocols and primers appropriate for In-Fusion cloning. The backbone was taken from a kanamycin resistant plasmid and amplified using the same methods. These were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. These fragments were cloned into plasmid backbones using In-Fusion® HD Cloning Kit from Takara (Cat 639650) according to manufacturer protocols. Assembled products were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing kanamycin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.

Cell culture and transfection

[001189] HEK293T Lenti-X cells were maintained in 10% FBS supplemented DMEM with HEPES and Glutamax (Thermo Fisher). Cells were seeded in 15 cm dishes at 20 x 10⁶ cells per dish in 20 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection, cells were 70-90% confluent. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 21 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 0.25 pg of pGP2. Polyethylenimine (PEI Max, Polyplus) was then added to the plasmid mixture, mixed, and allowed to incubate at room temperature before being added to the cell culture.

Collection and concentration

[001190] Media was aspirated from the plates 24 hours post-transfection and replaced with Opti- MEM (Thermo Fisher). XDP-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 at 10,000 x g at 4°C for 4h using a 10% sucrose buffer in NTE (50mM Tris-HCL, lOOmM NaCl, 10% Sucrose, pH 7.4). XDPs were resuspended in 300 pL of DMEM/ F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.

Resuspension and transduction

[001191]tdTomato neural progenitor cells (tdT NPCs) were grown in DMEM/ F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using StemPro Accutase Cell Dissociation Reagent and seeded on PLF coated 96 well plates. Cells were allowed to grow for 48 hours before being treated for targeting XDPs (having a spacer for tdTomato) starting with neat resuspended virus and proceeding through 5 half-log dilutions. Cells were then centrifuged for 15 minutes at 1000 g. NPCs were grown for 96 hours before analysis of fluorescence as a marker of editing of tdTomato. The assays were run 2-3 times for each sample with similar results. Editing results for a single assay are shown in Table 21.

Results

[001192] The editing results confirmed that, under the conditions of the assay, the majority of the 35 alternative configurations were able to edit the NPCs with at least 10% or greater editing, with 7 versions showing >80% editing. Additionally, it was confirmed that some of the HIV structural components of Gag were dispensable, with editing seen in one configuration in which only the matrix (MA) component was linked to the CasX. The pl/p6 component, which promotes budding from the host cell, was associated in all versions with high levels of editing (>= 70%, VI, V7, V8, V33, V34, V40, V123, V124) suggesting that this component is important for potency. Particles without NC, such as versions 34, 40 and 123, were able to achieve high levels of editing whereas particles without CA (such as version 17) had lower levels of editing (37%). The results also demonstrated that the protease component is not necessary for the XDP to retain high levels of editing potency, as demonstrated by versions 7, 8, 40, 123, and 124. Furthermore, p2, a component of NC, was also detrimental to potency as seen when comparing versions 122 and 128 on table XX where 122 (MA-CA-pl/p6) has no p2 and achieves 44.4% editing and versions 128 (MA-CA-p2-pl/p6) includes p2 and archives only 29.2% editing. In addition, constructs with multiple pl/p6 may contribute to enhance editing, as seen in FIG. 35 (version 122 versus 123), however, this did not prove to be the case for other configurations; e.g., version 7 (MA-CA-NC-pl/p6-X) versus version 124 (MA-CA-NC-pl/p6-pl/p6), where version 7 achieved 92.2% editing and version 124 achieved only 72.3% editing. [001193] Overall, the results support that, under the conditions of the assays, multiple configurations of XDP are able to successfully assemble particles able to deliver the CasX and guide RNA therapeutic payloads to eukaryotic cells, resulting in editing of the target nucleic acid.

Table 21: Editing of NPCs by XDP constructs, by version configuration.

*% Editing was calculated by taking the maximum editing percentage of the 5 dilutions’ averaged replicates. Table 22: Encoding sequences for guides and glycoproteins

Table 23: XDP Versions and Component Encoding Sequences

Example 22: Transfection and recovery of XDP constructs in the Gag-(-l)-protease-CasX configuration derived from Retroviruses.

[001194] Editing efficiency and specificity can be altered and enhanced with the method of CasX delivery that is employed. A wide variety of viral vector families, including those of retroviral origin, can be engineered for the transient delivery of CasX RNPs. In addition to potentially enhancing editing with altered cell and tissue tropism, use of RNPs also offers the unique advantage of negating the potential risks of insertional mutagenesis and long-term transgene expression. The purpose of the following experiment was to create and identify unique CasX delivery particles derived from different genera of the Retroviridae family. The genera investigated in the following experiments include Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses, Epsilonretroviruses, Non-primate lentiviruses and Spumaretroviruses.

Method for the generation of XDPs

[001195] XDPs derived from Alpharetroviruses (avian leukosis virus (ALV) and rous sarcoma virus (RSV)) in the Gag-protease-CasX variation (Version 44 and 45; see FIG. 52A) were produced by transient transfection of LentiX HEK293T cells (Takara Biosciences) using the three plasmids encoding the Gag-protease-CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 24. The pXDP40 and pXDP41 plasmid contains the Gag polyprotein sequence followed by a protease and a CasX 491 protein fused at the C-terminus. A TSCYHCGT (SEQ ID NO: 944) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide RNA cassette having scaffold 174 and spacer components (targeted to tdTomato: CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 24.

[001196] XDPs derived from Betaretroviruses (Enzootic Nasal Tumor Virus (ENTV), mouse mammary tumor virus (MMTV) and Mason-Pfizer monkey virus (MPMV)) in the Gag-(-l)- protease-CasX variation (Version 46, 47, 62 and 90; see FIG. 52B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids encoding the Gag-(-l)-protease- CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 24. The pXDP42, pXDP43, pXDP44 and pXDP61 plasmid contains the Gag polyprotein sequence followed by ribosomal frameshift, a protease and a CasX protein fused at the C-terminus. A DCLDFDND (SEQ ID NO: 934), DLVLLSAE (SEQ ID NO: 935), PQVMAAVA (SEQ ID NO: 936) and PQVMAAVA (SEQ ID NO: 936) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP42, pXDP43, pXDP44 and pXDP61 plasmids, respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 24.

[001197] XDPs derived from Deltaretroviruses (bovine leukemia virus (BLV) and human T lymphotropic virus (HTLV1)) in the Gag-(-l)-protease-CasX variation (Version 48, 49 and 63) were produced by transient transfection of LentiX HEK293T cells using three plasmids encoding the Gag-(-l)-protease-CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 24. The pXDP45, pXDP46, and pXDP62 plasmid contains the Gag polyprotein sequence followed by ribosomal frameshift, a protease and a CasX protein fused at the C-terminus. A PAILPIIS (SEQ ID NO: 945), PQVLPVMH (SEQ ID NO: 946) and PQVLPVMH (SEQ ID NO: 946) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP45, pXDP46, and pXDP62 plasmid respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 24. [001198] XDPs derived from Epsilonretroviruses (walleye dermal sarcoma virus (WDSV)) in the Gag-protease-CasX variation (Version 50) were produced by transient transfection of LentiX HEK293T cells using three plasmids encoding the Gag-protease-CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 24. The pXDP47 plasmid contains the Gag polyprotein sequence followed by a protease and a CasX protein fused at the C-terminus. A ARQMTAHT (SEQ ID NO: 937) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP47 plasmid. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 24. [001199] XDPs derived from Gammaretroviruses (feline leukemia virus (FLV) and murine leukemia virus (MMLV)) in the Gag-protease-CasX variation (Version 51 and 52) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 54A and listed in Table 24. The pXDP48, and pXDP49 plasmid contains the Gag polyprotein sequence followed by a protease and a CasX protein fused at the C-terminus. A SSLYPVLP (SEQ ID NO: 938), and SSLYPALT (SEQ ID NO: 939) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP48, and pXDP49 plasmid respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 24.

[001200] XDPs derived from Non-primate Lentiviruses (caprine arthritis encephalitis (CAEV), equine infectious anaemia virus (EIAV), simian immunodeficiency virus (SIV) and visna maedi virus (VMV)) in the Gag-(-l)-protease-CasX variation (Version 53, 54, 55 and 91) were produced by transient transfection of LentiX HEK293T cells using three plasmids encoding the Gag-(-l)-protease-CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 24. The pXDP50, pXDP51, pXDP52, pXDP53 plasmid contains the Gag polyprotein sequence followed by a ribosomal frameshift, a protease and a CasX protein fused at the C-terminus. A AGGRSWKA (SEQ ID NO: 940), SEEYPIMI (SEQ ID NO: 941), GGNYPVQQ (SEQ ID NO: 942) and REVYPIVN (SEQ ID NO: 943) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP50, pXDP51, pXDP52, pXDP53 plasmid respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudo-typing the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 24.

[001201] XDPs derived Spumaretrovirinae family (bovine foamy virus (BFV), equine foamy virus (EFV), feline foamy virus (FFV), Brown greater galago prosimian foamy virus (BGPFV), Rhesus macaque simian foamy virus (RHSFV) and Simian foamy virus (SFV)) in the Gag-(-l)- protease-CasX variation (Version 56, 57, 58, 59, 60, 61 and 92) were produced by transient transfection of LentiX HEK293T cells using the three plasmids encoding the Gag-(-l)-protease- CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 24. The pXDP54, pXDP55, pXDP56, pXDP57, pXDP58, pXDP59 and pXDP60 plasmid contains the Gag polyprotein sequence followed by a ribosomal frameshift, a protease and a CasX protein fused at the C-terminus. A SAVHSVRL (SEQ ID NO: 784), RTVNTVRV (SEQ ID NO: 785), NTVHTVRQVES (SEQ ID NO: 786), AAVHTVKA (SEQ ID NO: 787), RTVNTVTT (SEQ ID NO: 788) and RSVNTVTA (SEQ ID NO: 789) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP54, pXDP55, pXDP56, pXDP57, pXDP58, pXDP59 and pXDP60 plasmid respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 24.

Table 24: Plasmid Encoding Sequences for XDP Versions

Transfection

[001202] The steps for creation of the XDP are depicted graphically in FIG. 24. HEK293T Lenti-X^® cells were maintained in 10% FBS supplemented DMEM with HEPES, penicillin/streptomycin (Pen/Step), sodium pyruvate, and 2-mercaptoethanol. Cells were seeded in TWO 15 cm dishes at 8e6 cells per dish in 10 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection cells were 70-90% confluent. For transfection, the following plasmid amounts were used for the structural plasmid individually: pXDP40 (151 pg), pXDP41(151 pg), pXDP42 (157 pg), pXDP43 (157 pg), pXDP44 (159 pg), pXDP45 (145 pg), pXDP46 (149 pg), pXDP47 (152 pg), pXDP48 (148 pg), pXDP49 (149 pg), pXDP50 (145 pg), pXDP51 (146 pg), pXDP52 (147 pg), pXDP53 (144 pg), pXDP54 (149 pg), pXDP55 (153 pg), pXDP56 (154 pg), pXDP57 (150 pg), pXDP58 (146 pg), pXDP59 (154 pg), pXDP60 (154 pg), pXDP61 (159 pg), pXDP62 (149 pg), pXDP63 (147 pg), pXDP88 (146 pg). Along with the structural plasmid, each transfection also received 26.3 pg of pStx42.174.12.7, and the 5 pg of pGP2 in 3800 pi of Opti-MEM media. 1 mg/ml linear polyethylenimine (PEI, MW=25,000 Da) was then added to the plasmid mixture at 1:3 DNA:PEI concentration, mixed, and allowed to incubate at room temperature before being added to the cell culture.

Collection and concentration

[001203] Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 pM filter using a 60 mL syringe. The filtered supernatant was concentrated by centrifugation at 17,000 x g at 4°C for 4h using a 10% sucrose buffer in NTE. The concentrated XDPs were held at -20°C until use.

Editing of tdTomato neural progenitor cells using XDP

[001204] tdTomato neural progenitor cells (tdT NPCs) were grown in DMEM F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using a Takara Biosciences Neuron Dissociation Kit and seeded on PLF coated 96 well plates. Cells were allowed to grow at 37°C for 48 hours before being treated with targeting XDPs (having spacer 12.7 for tdTomato) as a lOx concentrate from the sucrose buffer concentrates using half-log dilutions. NPCs were grown for 96 hours before analysis of fluorescence as a marker of editing of tdTomato. Version 29 XDP made with pXDP88 is the HIV lentivirus control for these experiments testing out Gag-Pro-Stx versions of the various retroviruses.

[001205] Results: The results of the editing assay are shown in FIGS. 69A and B, FIG. 70 and in Table 25 and Table 26 below. FIGS. 69A and B show the percentage editing efficacy for specific amounts of the various XDP versions in tdTomato NPCs. FIG. 70 shows specifically the editing efficacy of the various XDP versions when 16.6 pi of the concentrated XDP prep is used to treat tdTomato NPCs. Tables 25 and 26 represent the results showing % editing of the dtTomato target sequence when 50 mΐ and 16.6 mΐ of the concentrated XDP prep were used to treat NPCs. The results indicate that, under the conditions of the assay, XDPs constructed using members of the Retroviridae in several different configurations of the XDP, were able, for the majority of the genera, to result in significant editing of the target nucleic acid in the NPC cells, with several editing above 10%.

Table 25: Results of Editing Assay for the first dilution (50 mΐ)

Table 26: Results of Editing Assay for the second dilution (16.6ul)

Example 23: Transfection and recovery of XDP constructs in a MA-CA-CasX configuration derived from Retroviruses

[001206] Editing efficiency and specificity can be altered and enhanced with the method of CasX delivery that is employed. A wide variety of viral vector families, including those of retroviral origin, can be engineered for the transient delivery of CasX RNPs. In addition to potentially enhancing editing with altered cell and tissue tropism, use of RNPs packaged within these viral vectors also offers the unique advantage of negating the potential risks of insertional mutagenesis and long-term transgene expression. The purpose of the following experiment was to build upon the previous example and to create and identify unique CasX delivery particles derived from different genera of the Retroviridae family using different architectures. The genera investigated in the following experiments include Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses, Epsilonretroviruses and Non-primate lentiviruses in a MA-CA-CasX configuration, thereby eliminating the NC and protease domains.

Methods

Method for the generation of XDPs

[001207] XDPs derived from Alpharetroviruses (ALV and RSV) in the MA-CA-CasX variation (Version 66a and 67a; see FIG. 55B) were produced by transient transfection of LentiX HEK293T cells (Takara Biosciences) using the three plasmids encoding the MA-CA-CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 27. The pXDP64 and pXDP65 plasmid contains the Matrix sequence followed by the Capsid sequence and a CasX 491 protein fused at the C-terminus. The cleavage site between the Capsid and the Nucleocapsid protein was kept intact for each virus and immediately preceded the CasX protein sequences to mediate separation of the editing molecules during XDP maturation, when coupled with a plasmid that contained the respective viral protease. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide RNA cassette having scaffold 174 and spacer components (targeted to tdTomato: CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 27.

[001208] XDPs derived from Betaretroviruses (ENTV, MMTV and MPMV) in the MA-CA- CasX variation (Version 68A, 69A, 70A and 87A, FIG. 56B) were produced by transient transfection of LentiX HEK293T cells using three plasmids encoding the MA-CA-CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 27. The pXDP66, pXDP67, pXDP68 and pXDP85 plasmid contains the Matrix sequence followed by the Capsid sequence and a CasX protein fused at the C-terminus. The cleavage site between the Capsid and the Nucleocapsid protein was kept intact for each virus and immediately preceded the CasX protein sequences to mediate separation of the editing molecules during XDP maturation, when coupled with a plasmid that contained the respective viral protease. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 27.

[001209] XDPs derived from Deltaretroviruses (BLV and HTLV1) in the MA-CA-CasX variation (Version 71 A, 72A and 88A, FIG. 57B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 57B and listed in Table 27. The pXDP69, pXDP70, and pXDP86 plasmid contains the Matrix sequence followed by the Capsid sequence and a CasX protein fused at the C-terminus. The cleavage site between the Capsid and the Nucleocapsid protein was kept intact for each virus and immediately preceded the CasX protein sequences to mediate separation of the editing molecules during XDP maturation, when coupled with a plasmid that contained the respective viral protease. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 27.

[001210] XDPs derived from Epsilonretroviruses (WDSV) in the MA-CA-CasX variation (Version 73A, FIG. 58B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 58B and listed in Table 27. The pXDP71 plasmid contains the Matrix sequence followed by the Capsid sequence and a CasX protein fused at the C-terminus. The cleavage site between the Capsid and the Nucleocapsid protein was kept intact for each virus and immediately preceded the CasX protein sequences to mediate separation of the editing molecules during XDP maturation, when coupled with a plasmid that contained the respective viral protease. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 27.

[001211] XDPs derived from Gammaretroviruses (FLV and MMLV) in the MA-CA-CasX variation (Version 74A and 75A, FIG. 59B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 59B and listed in Table 27. The pXDP72, and pXDP73 plasmid contains the Matrix sequence followed by the Capsid sequence and a CasX protein fused at the C-terminus. The cleavage site between the Capsid and the Nucleocapsid protein was kept intact for each virus and immediately preceded the CasX protein sequences to mediate separation of the editing molecules during XDP maturation, when coupled with a plasmid that contained the respective viral protease. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 27. [001212] XDPs derived from Non-primate Lentiviruses (CAEV, EIAV, SIV and VMV) in the MA-CA-CasX variation (Version 76A, 77A, 78A, 79A and 89A, FIG. 60B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 60B and listed in Table 27. The pXDP74, pXDP75, pXDP76, pXDP77 and pXDP87 plasmid contains the Matrix sequence followed by the Capsid sequence and a CasX protein fused at the C-terminus. The cleavage site between the Capsid and the Nucleocapsid protein was kept intact for each virus and immediately preceded the CasX protein sequences to mediate separation of the editing molecules during XDP maturation, when coupled with a plasmid that contained the respective viral protease. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudo-typing the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 27.

Table 27: Plasmid Encoding Sequences for XDP Versions

Transfection

[001213] The steps for creation of the XDP are depicted graphically in FIG. 24. HEK293T Lenti-X^® cells were maintained in 10% FBS supplemented DMEM with HEPES, penicillin/streptomycin (Pen/Step), sodium pyruvate, and 2-mercaptoethanol. Cells were seeded in TWO 15 cm dishes at 8e6 cells per dish in 10 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection cells were 70-90% confluent. For transfection, the following plasmid amounts were used for the structural plasmid individually: pXDP64 (143 pg), pXDP65 (143 pg), pXDP66 (142 pg), pXDP67 (143 pg), pXDP68 (144 pg), pXDP69 (136 pg), pXDP70 (137 pg), pXDP71 (141 pg), pXDP72 (140 pg), pXDP73 (142 pg), pXDP74 (134 pg), pXDP75 (134 pg), pXDP76 (134 pg), pXDP85 (144 pg), pXDP86 (137 pg), pXDP87 (138 pg), pXDP32 (114 pg). Along with the structural plasmid, each transfection also received 26.3 pg of pStx42.174.12.7, and the 5 pg of pGP2 in 3800 pi of Opti-MEM media. 1 mg/ml linear polyethylenimine (PEI, MW=25,000 Da) was then added to the plasmid mixture at 1:3 DNA:PEI concentration, mixed, and allowed to incubate at room temperature before being added to the cell culture.

Collection and concentration

[001214] Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 pM filter using a 60 mL syringe. The filtered supernatant was concentrated by centrifugation at 17,000 x g at 4oC for 4h using a 10% sucrose buffer in NTE. The concentrated XDPs were held at -20°C until use.

Editing of tdTomato neural progenitor cells using XDP

[001215] tdTomato neural progenitor cells (tdT NPCs) were grown in DMEM F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using a Takara Biosciences Neuron Dissociation Kit and seeded on PLF coated 96 well plates. Cells were allowed to grow at 37°C for 48 hours before being treated with targeting XDPs (having spacer 12.7 for tdTomato) as a lOx concentrate from the sucrose buffer concentrates using half-log dilutions. NPCs were grown for 96 hours before analysis of fluorescence as a marker of editing of tdTomato. Version 18 with pXDP32 serves as the control for these experiments.

[001216] Results: The results of the editing assay are shown in FIGS. 71 A and B, FIG. 72 and in Tables 28 and 29 below. FIGS. 73A and B shows the percentage editing efficacy for specific amounts of the various XDP versions in tdTomato NPCs. FIG. 72 shows specifically the editing efficacy of the various XDP versions when 16.6 pi of the concentrated XDP prep is used to treat tdTomato NPCs. Tables 28 and 29 represent the results showing % editing of the dtTomato target sequence when 50 pi and 16.6 pi of the concentrated XDP prep were used to treat NPCs. The results indicate that, under the conditions of the assay, XDPs constructed using members of the Retroviridae in MA-CA-X configuration of the XDP, were able, for the majority of the genera, to result in significant editing of the target nucleic acid in the NPC cells, with several editing above 10%. Table 28: Results of Editing Assay for the first dilution (50 ul)

Table 29: Results of Editing Assay for the second dilution (16.6 mΐ)

Example 24: Transfection and recovery of XDP constructs in the Gag-(-l)-protease-CasX configuration derived from Retroviruses.

[001217] Editing efficiency and specificity can be altered and enhanced with the method of CasX delivery that is employed. A wide variety of viral vector families, including those of retroviral origin, can be engineered for the transient delivery of CasX RNPs. In addition to potentially enhancing editing with altered cell and tissue tropism, use of RNPs also offers the unique advantage of negating the potential risks of insertional mutagenesis and long-term transgene expression. The purpose of the following experiment was to create and identify unique CasX delivery particles derived from different genera of the Retroviridae family. The genera investigated in the following experiments include Alpharetroviruse, Betaretroviruse, Gammaretroviruse, Deltaretroviruse, Epsilonretroviruse, Non-primate lentiviruses and Spumare troviruse .

Method for the generation of XDPs

[001218] XDPs derived from Alpharetroviruses (avian leukosis virus (ALV) and rous sarcoma virus (RSV)) in the Gag-protease-CasX variation (Version 44 and 45; see FIG. 52A) were produced by transient transfection of LentiX HEK293T cells (Takara Biosciences) using the three plasmids portrayed in FIG. 52A and listed in Table 30. The pXDP40 and pXDP41 plasmid contains the Gag polyprotein sequence followed by a protease and a CasX 491 protein fused at the C-terminus. A TSCYHCGT (SEQ ID NO: 944) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide RNA cassette having scaffold 174 and spacer components (targeted to tdTomato: CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.

[001219] XDPs derived from Betaretroviruses (Enzootic Nasal Tumor Virus (ENTV), mouse mammary tumor virus (MMTV) and Mason-Pfizer monkey virus (MPMV)) in the Gag-(-l)- protease-CasX variation (Version 46, 47, 62 and 90; see FIG. 52B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 52B and listed in Table 30 . The pXDP42, pXDP43, pXDP44 and pXDP61 plasmid contains the Gag polyprotein sequence followed by ribosomal frameshift, a protease and a CasX protein fused at the C-terminus. A DCLDFDND (SEQ ID NO: 934), DLVLLSAE (SEQ ID NO: 935), PQVMAAVA (SEQ ID NO: 936) and PQVMAAVA (SEQ ID NO: 936) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP42, pXDP43, pXDP44 and pXDP61 plasmids, respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30. [001220] XDPs derived from Deltaretroviruses (bovine leukemia virus (BLV) and human T lymphotropic virus (HTLV1)) in the Gag-(-l)-protease-CasX variation (Version 48, 49 and 63; see FIG. 53A) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 53A and listed in Table 30 . The pXDP45, pXDP46, and pXDP62 plasmid contains the Gag polyprotein sequence followed by ribosomal frameshift, a protease and a CasX protein fused at the C-terminus. A PAILPIIS (SEQ ID NO: 945), PQVLPVMH (SEQ ID NO: 946) and PQVLPVMH (SEQ ID NO: 946) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP45, pXDP46, and pXDP62 plasmid respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.

[001221] XDPs derived from Epsilonretroviruses (walleye dermal sarcoma virus (WDSV)) in the Gag-protease-CasX variation (Version 50; see FIG. 53B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 53B and listed in Table 30 . The pXDP47 plasmid contains the Gag polyprotein sequence followed by a protease and a CasX protein fused at the C-terminus. A ARQMTAHT (SEQ ID NO: 937) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP47 plasmid. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.

[001222] XDPs derived from Gammaretroviruses (feline leukemia virus (FLV) and murine leukemia virus (MMLV)) in the Gag-protease-CasX variation (Version 51 and 52; see FIG. 54A) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 54A and listed in Table 30. The pXDP48, and pXDP49 plasmid contains the Gag polyprotein sequence followed by a protease and a CasX protein fused at the C-terminus. A SSLYPVLP (SEQ ID NO: 938), and SSLYPALT (SEQ ID NO: 939) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP48, and pXDP49 plasmid respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.

[001223] XDPs derived from Non-primate Lentiviruses (caprine arthritis encephalitis (CAEV), equine infectious anaemia virus (EIAV), simian immunodeficiency virus (SIV) and visna maedi virus (VMV)) in the Gag-(-l)-protease-CasX variation (Version 53, 54, 55 and 91; see FIG.

54B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 54B and listed in Table 30. The pXDP50, pXDP51, pXDP52, pXDP53 plasmid contains the Gag polyprotein sequence followed by a ribosomal frameshift, a protease and a CasX protein fused at the C-terminus. A AGGRSWKA (SEQ ID NO: 940), SEEYPIMI (SEQ ID NO: 941), GGNYPVQQ (SEQ ID NO: 942) and REVYPIVN (SEQ ID NO: 943) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP50, pXDP51, pXDP52, pXDP53 plasmid respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudo-typing the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.

[001224] XDPs derived Spumaretrovirinae family (bovine foamy virus (BFV), equine foamy virus (EFV), feline foamy virus (FFV), Brown greater galago prosimian foamy virus (BGPFV), Rhesus macaque simian foamy virus (RHSFV) and Simian foamy virus (SFV)) in the Gag-(-l)- protease-CasX variation (Version 56, 57, 58, 59, 60, 61 and 92; see FIG. 55A) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 55A and listed in Table 30. The pXDP54, pXDP55, pXDP56, pXDP57, pXDP58, pXDP59 and pXDP60 plasmid contains the Gag polyprotein sequence followed by a ribosomal frameshift, a protease and a CasX protein fused at the C-terminus. A SAVHSVRL (SEQ ID NO: 784), RTVNTVRV (SEQ ID NO: 785), NTVHTVRQVES (SEQ ID NO: 786), AAVHTVKA (SEQ ID NO: 787), RTVNTVTT (SEQ ID NO: 788) and RSVNTVTA (SEQ ID NO: 789) cleavage site separated the Protease protein and CasX protein sequences to mediate separation of the editing molecules during XDP maturation in the pXDP54, pXDP55, pXDP56, pXDP57, pXDP58, pXDP59 and pXDP60 plasmid respectively. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.

Table 30: Plasmid and XDP Encoding Sequences

Transfection

[001225] The steps for creation of the XDP are depicted graphically in FIG. 24. HEK293T Lenti-X^® cells were maintained in 10% FBS supplemented DMEM with HEPES, penicillin/streptomycin (Pen/Step), sodium pyruvate, and 2-mercaptoethanol. Cells were seeded in TWO 15 cm dishes at 8e6 cells per dish in 10 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection cells were 70-90% confluent. For transfection, the following plasmid amounts were used for the structural plasmid individually: pXDP40 (151 pg), pXDP41(151 pg), pXDP42 (157 pg), pXDP43 (157 pg), pXDP44 (159 pg), pXDP45 (145 pg), pXDP46 (149 pg), pXDP47 (152 pg), pXDP48 (148 pg), pXDP49 (149 pg), pXDP50 (145 pg), pXDP51 (146 pg), pXDP52 (147 pg), pXDP53 (144 pg), pXDP54 (149 pg), pXDP55 (153 pg), pXDP56 (154 pg), pXDP57 (150 pg), pXDP58 (146 pg), pXDP59 (154 pg), pXDP60 (154 pg), pXDP61 (159 pg), pXDP62 (149 pg), pXDP63 (147 pg), pXDP88 (146 pg). Along with the structural plasmid, each transfection also received 26.3 pg of pStx42.174.12.7, and the 5 pg of pGP2 in 3800 pi of Opti-MEM media. 1 mg/ml linear polyethylenimine (PEI, MW=25,000 Da) was then added to the plasmid mixture at 1:3 DNA:PEI concentration, mixed, and allowed to incubate at room temperature before being added to the cell culture.

Collection and concentration

[001226] Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 mM filter using a 60 mL syringe. The filtered supernatant was concentrated by centrifugation at 17,000 x g at 4°C for 4h using a 10% sucrose buffer in NTE. The concentrated XDPs were held at -20°C until use.

Editing of tdTomato neural progenitor cells using XDP

[001227] tdTomato neural progenitor cells (tdT NPCs) were grown in DMEM F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using a Takara Biosciences Neuron Dissociation Kit and seeded on PLF coated 96 well plates. Cells were allowed to grow at 37°C for 48 hours before being treated with targeting XDPs (having spacer 12.7 for tdTomato) as a lOx concentrate from the sucrose buffer concentrates using half-log dilutions. NPCs were grown for 96 hours before analysis of fluorescence as a marker of editing of tdTomato. Version 29 XDP made with pXDP88 is the HIV lentivirus control for these experiments testing out Gag-Pro-Stx versions of the various retroviruses.

[001228] Results: The results of the editing assay are shown in FIGS. 69A and B and in Table 31 and Table 32 below. FIGS. 69A and B show the percentage editing efficacy for specific amounts of the various XDP versions in tdTomato NPCs. Tables 31 and 32 represent the results showing % editing of the dtTomato target sequence when 50 mΐ and 16.6 mΐ of the concentrated XDP prep were used to treat NPCs. The results indicate that, under the conditions of the assay, XDPs constructed using members of the Retroviridae in several different configurations of the XDP, were able, for the majority of the genera, to result in significant editing of the target nucleic acid in the NPC cells, with several editing above 10%.

Table 31: Results of Editing Assay for the first dilution (50 mΐ)

Table 32: Results of Editing Assay for the second dilution (16.6 mΐ)

Example 25: Transfection and recovery of XDP constructs in the Gag-CasX configuration derived from Retroviruses.

[001229] Editing efficiency and specificity can be altered and enhanced with the method of CasX delivery that is employed. A wide variety of viral vector families, including those of retroviral origin, can be engineered for the transient delivery of CasX RNPs. In addition to potentially enhancing editing with altered cell and tissue tropism, use of RNPs packaged within these viral vectors also offers the unique advantage of negating the potential risks of insertional mutagenesis and long-term transgene expression. The purpose of the following experiment was to build upon the previous example and to create and identify unique CasX delivery particles derived from different genera of the Retroviridae family using different architectures. The genera investigated in the following experiments include Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses, Epsilonretroviruses and Non-primate lentiviruses in a Gag-CasX configuration. The experiments were meant to be a direct comparison with the HIV Lentivirus based V7 construct, with the Gag component being replaced with the corresponding Gag components of Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses, Epsilonretroviruses, Non-primate lentiviruses and Spumaretroviruses, with the protease domains eliminated in all constructs to test whether XDP capable of editing required active release from Gag.

Methods for the generation of XDPs

[001230] XDPs derived from Alpharetroviruses (avian leukosis virus (ALV) and rous sarcoma virus (RSV)) in the Gag-CasX variation (VI 02 and VI 14; see FIG. 62B) were produced by transient transfection of LentiX HEK293T cells (Takara Biosciences) using the three plasmids portrayed in FIG. 62B and listed in Table 33. The pXDP127 and pXDP139 plasmid contains the Gag polyprotein sequence followed by the CasX 491 protein fused at the C-terminus. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide RNA cassette having scaffold 174 and spacer components (targeted to tdTomato: CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also incorporated into the constructs. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33 .

[001231] XDPs derived from Betaretroviruses (Enzootic Nasal Tumor Virus (ENTV), mouse mammary tumor virus (MMTV) and Mason-Pfizer monkey virus (MPMV)) in the Gag-CasX variation (VI 06, VI 11, VI 12 and VI 13, FIG. 64 A) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 64A and listed in Table 33 . The pXDP131, pXDP136, pXDP137 and pXDP138 plasmid contains the Gag polyprotein sequence followed by the CasX 491 protein fused at the C-terminus. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also incorporated into the constructs. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33 .

[001232] XDPs derived from Deltaretroviruses (bovine leukemia virus (BLV) and human T lymphotropic virus (HTLV1)) in the Gag-CasX variation (Version V103, V108 and V109, FIG. 63 A) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 63A and listed in Table 33 . The pXDP128, pXDP133 and pXDP134 plasmid contains the Gag polyprotein sequence followed by the CasX 491 protein fused at the C-terminus. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also incorporated into the constructs. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33 . [001233] XDPs derived from Epsilonretroviruses (walleye dermal sarcoma virus (WDSV)) in the Gag-CasX variation (Version 73 A, FIG. 58B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 58B and listed in Table 33. The pXDP127 and pXDP139 plasmid contains the Gag polyprotein sequence followed by the CasX 491 protein fused at the C-terminus. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also incorporated into the constructs. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33.

[001234] XDPs derived from Gammaretroviruses (feline leukemia virus (FLV) and murine leukemia virus (MMLV)) in the Gag-CasX variation (VI 07 and VI 10, FIG. 64B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 64B and listed in Table 33 . The pXDP132, and pXDP135 plasmid contains the Gag polyprotein sequence followed by the CasX 491 protein fused at the C-terminus. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also incorporated into the constructs. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33 .

[001235] XDPs derived from Non-primate Lentiviruses (caprine arthritis encephalitis (CAEV), equine infectious anaemia virus (EIAV), simian immunodeficiency virus (SIV) and visna maedi virus (VMV)) in the Gag-CasX variation (VI 04, VI 05, VI 15, VI 16 and VI 17, FIG. 63B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 63B and listed in Table 33 . The pXDP129, pXDP130, pXDP140, pXDP141 and pXDP142 plasmid contains the Gag polyprotein sequence followed by the CasX 491 protein fused at the C-terminus. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudo-typing the XDP were also incorporated into the constructs. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33.

[001236] XDPs derived Spumaretrovirinae family (bovine foamy virus (BFV), equine foamy virus (EFV), feline foamy virus (FFV), Brown greater galago prosimian foamy virus (BGPFV), Rhesus macaque simian foamy virus (RHSFV) and Simian foamy virus (SFV)) in the Gag-CasX variation (V80a, V81a, V82a, V83a, V84a, V85a and V86a; see FIG. 62A) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 62A and listed in Table 33 . The pXDP78, pXDP79, pXDP80, pXDP81, pXDP82, pXDP83 and pXDP84 plasmid contains the Gag polyprotein sequence followed by the CasX protein fused at the C-terminus. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide cassette having scaffold 174 and spacer components (targeted to tdTomato) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also incorporated into the constructs. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33.

Table 33: XDP Plasmid and Encoding Sequences

Transfection

[001237] The steps for creation of the XDP are depicted graphically in FIG. 24. HEK293T Lenti-X cells were maintained in 10% FBS supplemented DMEM with HEPES, penicillin/streptomycin (Pen/Step), sodium pyruvate, and 2-mercaptoethanol. Cells were seeded in TWO 15 cm dishes at 8e6 cells per dish in 10 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection cells were 70-90% confluent. For transfection, the following plasmid amounts were used for the structural plasmid individually: pXDP127 (146 pg), pXDP129 (141 pg), pXDP130 (143 pg), pXDP131 (145 pg), pXDP132 (143 pg), pXDP135 (145 pg), pXDP136 (152 pg), pXDP138 (149 pg), pXDP139 (146 pg), pXDP140 (143 pg), pXDP141 (143 pg), pXDP142 (141 pg), pXDP143 (146 pg), pXDP78 (145 pg), pXDP81 (141 pg), pXDP82 (139 pg), pXDP83 (145 pg), pXDP0017 (122 pg). Along with the structural plasmid, each transfection also received 26.3 pg of pStx42.174.12.7, and the 5 pg of pGP2 in 3800 pi of Opti-MEM media. 1 mg/ml linear polyethylenimine (PEI, MW=25,000 Da) was then added to the plasmid mixture at 1:3 DNA:PEI concentration, mixed, and allowed to incubate at room temperature before being added to the cell culture.

Collection and concentration

[001238] Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 pM filter using a 60 mL syringe. The filtered supernatant was concentrated by centrifugation at 17,000 x g at 4°C for 4h using a 10% sucrose buffer in NTE. The concentrated XDPs were held at -20°C until use.

Editing of tdTomato neural progenitor cells using XDP

[001239] tdTomato neural progenitor cells (tdT NPCs) were grown in DMEM F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using a Takara Biosciences Neuron Dissociation Kit and seeded on PLF coated 96 well plates. Cells were allowed to grow at 37°C for 48 hours before being treated with targeting XDPs (having spacer 12.7 for tdTomato) as a lOx concentrate from the sucrose buffer concentrates using half-log dilutions. NPCs were grown for 96 hours before analysis of fluorescence as a marker of editing of tdTomato. Version 18 with pXDP32 serves as the control for these experiments.

[001240] Results: The results of the editing assay are shown in FIGS. 75 A and B, FIG. 76 and in Table 34 and Table 35 below. FIGS. 75 A and B shows the percentage editing efficacy for specific amounts of the various XDP versions in tdTomato NPCs. Tables 34 and 35 represent the results showing % editing of the tdTomato target sequence when 50 ul and 16.6 ul of the concentrated XDP prep were used to treat NPCs. The results indicate that, under the conditions of the assay, XDPs constructed using members of the Retroviridae in Gag-CasX configuration of the XDP, were able, for the majority of the genera, to result in significant editing of the target nucleic acid in the NPC cells, with several editing above 4%.

Table 34: Results of Editing Assay for the first dilution (50ul)

Table 35: Results of Editing Assay for the second dilution (16.6ul)

Example 26: Transfection and recovery of XDP constructs derived from Spumaretrovirinae.

[001241] Editing efficiency and specificity can be altered and enhanced with the method of CasX delivery that is employed. A wide variety of viral vector families, including those of retroviral origin, can be engineered for the transient delivery of CasX RNPs. In addition to potentially enhancing editing with altered cell and tissue tropism, use of RNPs packaged within these viral vectors also offers the unique advantage of negating the potential risks of insertional mutagenesis and long-term transgene expression. The purpose of the following experiment was to build upon the previous example and to create and identify unique CasX delivery particles derived from different genera of the Retroviridae family using different architectures. The genera investigated in the following experiments include Spumaretroviruses in a Gag-CasX + Gag-(-l)-Protease-CasX configuration. Here we hypothesized that by adding in different amounts of the protease with the Gag-Protease-CasX polyprotein along with the Gag-CasX polyproteins, we could potentially improve XDP particle formation and maturation, mediated by proteolytic cleavage.

Methods

Method for the generation of XDPs

[001242] XDPs derived from Spumaretrovirinae family (BFV, EFV, FFV, BGPFV, RHSFV and SFV) in the 90% Gag-CasX + 10% Gag-(-l)-Protease-CasX variation (V80b, V81b, V82b,

V83b, V84b, V85b and V86b; see FIG. 62A) were produced by transient transfection of LentiX HEK293T cells (Takara Biosciences) using the plasmids portrayed in FIG. 62A and listed in Table 36. The plasmids pXDP54, pXDP55, pXDP56, pXDP57, pXDP58, pXDP59 and pXDP60 have been described in previous examples. The pStx42.174.12.7 plasmid was created with a human U6 promoter upstream of a CasX guide RNA cassette having scaffold 174 and spacer components (targeted to tdTomato: CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 825) in a single-guide format. Plasmids containing VSV-G (pGP2) for pseudotyping the XDP were also used. All plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 36 and A .

Table 36: Plasmid Sequences

Transfection

[001243] The steps for creation of the XDP are depicted graphically in FIG. 24. HEK293T

Lenti-X cells were maintained in 10% FBS supplemented DMEM with HEPES, penicillin/streptomycin (Pen/Step), sodium pyruvate, and 2-mercaptoethanol. Cells were seeded in two 15 cm dishes at 8e6 cells per dish in 10 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection cells were 70-90% confluent.

For transfection, the following plasmid amounts were used for the structural plasmid individually: pXDP78 + pXDP54 (146 pg + 15 pg), pXDP81 + pXDP57 (150 pg + 15 pg), pXDP82 + pXDP58 (146 pg + 15 pg), pXDP83 + pXDP59 (154 pg + 15.4 pg). Along with the structural plasmid, each transfection also received 26.3 pg of pStx42.174.12.7, and the 5 pg of pGP2 in 3800 mΐ of Opti-MEM media. 1 mg/ml linear polyethylenimine (PEI, MW=25,000 Da) was then added to the plasmid mixture at 1 :3 DNA:PEI concentration, mixed, and allowed to incubate at room temperature before being added to the cell culture.

Collection and concentration

[001244] Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 mM filter using a 60 mL syringe. The filtered supernatant was concentrated by centrifugation at 17,000 x g at 4°C for 4h using a 10% sucrose buffer in NTE. The concentrated XDPs were held at -20°C until use.

Editing of tdTomato neural progenitor cells using XDP

[001245] tdTomato neural progenitor cells (tdT NPCs) were grown in DMEM F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using a Takara Biosciences Neuron Dissociation Kit and seeded on PLF coated 96 well plates. Cells were allowed to grow at 37°C for 48 hours before being treated with targeting XDPs (having a spacer for tdTomato) as a lOx concentrate from the sucrose buffer concentrates using half-log dilutions. NPCs were grown for 96 hours before analysis of fluorescence as a marker of editing of tdTomato. Version 18 with pXDP32 serves as the control for these experiments.

[001246] Results: The results of the editing assay are shown in FIGS. 73 A and B, FIG. 74 and in Table 37 and Table 38 below. FIGS. 73 A and B shows the percentage editing efficacy for specific amounts of the various XDP versions in tdTomato NPCs. Fig 74 shows specifically the editing efficacy of the various XDP versions when 16.6 mΐ of the concentrated XDP prep is used to treat tdTomato NPCs. Tables 37 and 38 represent the results showing % editing of the dtTomato target sequence when 50 mΐ and 16.6 mΐ of the concentrated XDP prep were used to treat NPCs. The results indicate that, under the conditions of the assay, XDPs constructed using members of the Retroviridae in 90% Gag-CasX + 10% Gag-protease-CasX configuration of the XDP, were able, for the majority of the genera, to result in significant editing of the target nucleic acid in the NPC cells, with several editing above 10%.

Table 37: Results of Editing Assay for the first dilution (50ul)

Table 38: Results of Editing Assay for the second dilution (16.6ul)

Claims

CLAIMS What is claimed is:

1. A delivery particle (XDP) system comprising one or more nucleic acids encoding:

(a) one or more retroviral components;

(b) a therapeutic payload; and

(c) a tropism factor

2. The XDP system of claim 1, 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.

3. The XDP system of claim 2, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483,

485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,

523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559,

561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595 as set forth in Table 4, 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.

4. The XDP system of claim 2, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559,

561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595.

5. The XDP system of any one of the preceding claims, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

6. The XDP system of claim 5, wherein the protein payload 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, and an anti-cancer modality.

7. The XDP system of claim 6, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

8. The XDP system of claim 7, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of a Type II, a Type V, or a Type VI protein.

9. The XDP system of claim 8, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

10. The XDP system of claim 9, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

11. The XDP system of claim 5, wherein the therapeutic payload comprises a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

12. The XDP system of claim 11, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 14 and 30 nucleotides and is complementary to a target nucleic acid sequence.

13. The XDP system of claim 12, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781 as set forth in Table 3, 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.

14. The XDP system of claim 13, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781.

15. The XDP system of any one of the preceding claims, wherein the nucleic acids further encode one or more components selected from:

(a) all or a portion of a retroviral gag polyprotein;

(b) one or more protease cleavage sites; (c) a gag-transframe region-pol protease polyprotein (gag-TFR-PR);

(d) a retroviral gag-pol polyprotein; and

(e) a non-retroviral protease capable of cleaving the protease cleavage sites.

16. The XDP system of any one of the preceding claims, wherein one or more of the retroviral components are derived from an Orthoretrovirinae virus or a Spumaretrovirinae virus.

17. The XDP system of claim 16, wherein the Orthoretrovirinae virus is selected from the group consisting of an Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus.

18. The XDP system of claim 16, wherein the Spumaretrovirinae virus is selected from the group consisting of Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, or Spumavirus.

19. The XDP system of any one of the preceding claims, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoded on two nucleic acids;

(c) the components are encoded on three nucleic acids;

(d) the components are encoded on four nucleic acids; or

(e) the components are encoded on five nucleic acids.

20. The XDP system of claim 19, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

21. The XDP system of claim 19 or claim 20, wherein the one or more of the retroviral components are encoded by a nucleic acid selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, and 234-339 as set forth in Table 5.

22. The XDP system of any one of the preceding claims, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

23. The XDP of claim 22, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

24. The XDP system of claim 23, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

25. The XDP of claim 22, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

26. The XDP system of claim 25, wherein the tropism factor confers preferential interaction of the XDP with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

27. An XDP system comprising one or more nucleic acids encoding components:

(a) all or a portion of an Alpharetrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

28. The XDP system of claim 27, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a P2A peptide, a P2B peptide, a P10 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

29. The XDP system of claim 28, wherein the gag polyprotein comprises, from N-terminus to C-terminus, a matrix polypeptide (MA), a P2A peptide, a P2B peptide, a P10 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

30. The XDP system of any one of claims 27-29, wherein the one or more nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

31. The XDP system of any one of claims 27-30, 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.

32. The XDP system of claim 31, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595, 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.

33. The XDP system of claim 31, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group of sequences consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477,

479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,

517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553,

555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591,

593 and 595 as set forth in Table 4.

34. The XDP system of claim 33, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G), optionally wherein the VSV-G glycoprotein comprises a sequence of SEQ ID NO: 438.

35. The XDP system of any one of claims 27-34, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

36. The XDP system of claim 35, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti cancer modality.

37. The XDP system of claim 36, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

38. The XDP system of claim 37, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

39. The XDP system of claim 38, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

40. The XDP system of claim 39, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

41. The XDP system of claim 39, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

42. The XDP system of any one of claims 39-41, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of PKKKRKV (SEQ ID NO: 130), KRPAATKKAGQAKKKK (SEQ ID NO: 131), PAAKRVKLD (SEQ ID NO: 132), RQRRNELKRSP (SEQ ID NO: 133),

NQ S SNF GPMKGGNF GGRS S GP Y GGGGQ YF AKPRNQGGY (SEQ ID NO: 134), RMRIZFKNKGKDTAELRRRRVEV S VELRKAKKDEQILKRRNV (SEQ ID NO: 135), VSRKRPRP (SEQ ID NO: 136), PPKKARED (SEQ ID NO: 137), PQPKKKPL (SEQ ID NO: 138), SALIKKKKKMAP (SEQ ID NO: 139), DRLRR (SEQ ID NO: 140), PKQKKRK (SEQ ID NO: 141), RKLKKKIKKL (SEQ ID NO: 142), REKKKFLKRR (SEQ ID NO: 143), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 144), RKCLQAGMNLEARKTKK (SEQ ID NO: 145), PRPRKIPR (SEQ ID NO: 146), PPRKKRTVV (SEQ ID NO: 147),

NL SKKKKRKREK (SEQ ID NO: 148), RRPSRPFRKP (SEQ ID NO: 149), KRPRSPSS (SEQ ID NO: 150), KRGINDRNFWRGENERKTR (SEQ ID NO: 151), PRPPKMARYDN (SEQ ID NO: 152), KRSFSKAF (SEQ ID NO: 153), KLKIKRPVK (SEQ ID NO: 154), PKTRRRPRRSQRKRPPT (SEQ ID NO: 156), RRKKRRPRRKKRR (SEQ ID NO: 159), PKKK SRKPKKK SRK (SEQ ID NO: 160), HKKKHPD AS VNF SEF SK (SEQ ID NO: 161), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 162), LSPSLSPLLSPSLSPL (SEQ ID NO: 163), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 164), PKRGRGRPKRGRGR (SEQ ID NO: 165), M SRRRK ANPTKL SENAKKL AKEVEN (SEQ ID NO: 157), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 155), and PKKKRKVPPPPKKKRKV (SEQ ID NO: 166), wherein the NLS are located at or near the N-terminus and/or the C-terminus.

43. The XDP system of claim 35, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double- stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

44. The XDP system of claim 43, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

45. The XDP system of claim 44, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

46. The XDP system of claim 45, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

47. The XDP system of any one of claims 44-46, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

48. The XDP system of any one of claims 27-47, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

49. The XDP system of claim 48, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

50. The XDP system of claim 48 or claim 49, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27,

30, and 33, or sequences 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.

51. The XDP system of any one of claims 27-50, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

52. The XDP of claim 51, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

53. The XDP system of claim 52, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

54. The XDP of claim 51, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

55. The XDP system of claim 54, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

56. An XDP system comprising one or more nucleic acids encoding components:

(a) all or a portion of an Betaretrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

57. The XDP system of claim 56, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a PP21/24 peptide, a P12/P3/P8 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

58. The XDP system of claim 56, wherein the gag polyprotein comprises, from N-terminus to C-terminus, a matrix polypeptide (MA), a PP21/24 peptide, a P12/P3/P8 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

59. The XDP system of any one of claims 56-58, wherein the nucleic acids further encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

60. The XDP system of any one of claims 56-59, 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.

61. The XDP system of claim 60, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595, 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.

62. The XDP system of claim 61, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595.

63. The XDP system of claim 62, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

64. The XDP system of any one of claims 56-63, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

65. The XDP system of claim 64, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti cancer modality.

66. The XDP system of claim 65, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

67. The XDP system of claim 66, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

68. The XDP system of claim 67, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

69. The XDP system of claim 68, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, or 11, 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.

70. The XDP system of claim 68, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

71. The XDP system of any one of claims 68-70, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

72. The XDP system of claim 64, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double- stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

73. The XDP system of claim 72, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

74. The XDP system of claim 73, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

75. The XDP system of claim 73, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

76. The XDP system of any one of claims 73-75, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

77. The XDP system of any one of claims 56-76, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

78. The XDP system of claim 77, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

79. The XDP system of claim 77 or claim 78, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27,

30, and 33, or sequences 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.

80. The XDP system of any one of claims 56-79, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

81. The XDP of claim 80, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

82. The XDP system of claim 81, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

83. The XDP of claim 80, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

84. The XDP system of claim 83, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

85. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Deltaretrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

86. The XDP system of claim 85, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

87. The XDP system of claim 86, wherein the gag polyprotein comprises, from N-terminus to C-terminus, matrix polypeptide (MA), a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

88. The XDP system of any one of claims 85-87, wherein the nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

89. The XDP system of any one of claims 85-88, 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.

90. The XDP system of claim 89, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559,

561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595, 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.

91. The XDP system of claim 89, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,

565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595.

92. The XDP system of claim 91, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

93. The XDP system of any one of claims 85-92, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

94. The XDP system of claim 93, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti cancer modality.

95. The XDP system of claim 94, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

96. The XDP system of claim 95, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

97. The XDP system of claim 96, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

98. The XDP system of claim 97, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

99. The XDP system of claim 97, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

100. The XDP system of any one of claims 97-99, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

101. The XDP system of claim 93, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double- stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

102. The XDP system of claim 101, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

103. The XDP system of claim 102, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

104. The XDP system of claim 102, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

105. The XDP system of any one of claims 102-104, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

106. The XDP system of any one of claims 85-105, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

107. The XDP system of claim 106, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

108. The XDP system of claim 106 or claim 107, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

109. The XDP system of any one of claims 85-108, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

110. The XDP of claim 109, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

111. The XDP system of claim 110, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

112. The XDP of claim 109, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

113. The XDP system of claim 112, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

114. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Epsilonretrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

115. The XDP system of claim 114, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a p20 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

116. The XDP system of claim 114, wherein the gag polyprotein comprises, from N-terminus to C-terminus, matrix polypeptide (MA), a p20 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

117. The XDP system of any one of claims 114-116, wherein the nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and (f) a gag-transframe region-pol protease polyprotein.

118. The XDP system of any one of claims 114-117, 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.

119. The XDP system of claim 118, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559,

561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595, 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.

120. The XDP system of claim 118, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,

565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595.

121. The XDP system of claim 120, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

122. The XDP system of any one of claims 114-121, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

123. The XDP system of claim 122, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti cancer modality.

124. The XDP system of claim 123, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

125. The XDP system of claim 124, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

126. The XDP system of claim 125, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

127. The XDP system of claim 126, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

128. The XDP system of claim 126, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

129. The XDP system of any one of claims 126-128, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

130. The XDP system of claim 122, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double- stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

131. The XDP system of claim 130, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

132. The XDP system of claim 131, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781or 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.

133. The XDP system of claim 131, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

134. The XDP system of any one of claims 131-133, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

135. The XDP system of any one of claims 114-134, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids; (c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

136. The XDP system of claim 135, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

137. The XDP system of claim 135 or claim 136, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

138. The XDP system of any one of claims 114-137, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

139. The XDP of claim 138, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

140. The XDP system of claim 139, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

141. The XDP of claim 139, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

142. The XDP system of claim 141, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

143. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Gammaretrovirus gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

144. The XDP system of claim 143, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a pl2 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

145. The XDP system of claim 144, wherein the gag polyprotein comprises, from N-terminus to C-terminus, matrix polypeptide (MA), a pl2 peptide, a capsid polypeptide (CA), and a nucleocapsid polypeptide (NC).

146. The XDP system of any one of claims 143-145, wherein the nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

147. The XDP system of any one of claims 143-146, 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.

148. The XDP system of claim 147, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595, 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.

149. The XDP system of claim 147, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487,

489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525,

527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,

565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595.

150. The XDP system of claim 149, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

151. The XDP system of any one of claims 143-150, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

152. The XDP system of claim 151, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti cancer modality.

153. The XDP system of claim 152, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

154. The XDP system of claim 153, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

155. The XDP system of claim 154, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

156. The XDP system of claim 155, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

157. The XDP system of claim 155, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

158. The XDP system of any one of claims 155-157, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

159. The XDP system of claim 151, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double- stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

160. The XDP system of claim 159, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

161. The XDP system of claim 160, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

162. The XDP system of claim 160, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

163. The XDP system of any one of claims 160-162, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

164. The XDP system of any one of claims 143-163, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

165. The XDP system of claim 164, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

166. The XDP system of claim 164 or claim 165, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

167. The XDP system of any one of claims 164-166, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

168. The XDP of claim 167, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

169. The XDP system of claim 168, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

170. The XDP of claim 167, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

171. The XDP system of claim 170, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

172. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Lentivirus gag polyprotein; (b) a therapeutic payload; and

(c) a tropism factor.

173. The XDP system of claim 172, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a capsid polypeptide (CA), a p2 peptide, a nucleocapsid polypeptide (NC), a pi peptide, and a p6 peptide.

174. The XDP system of claim 173, wherein the gag polyprotein comprises, from N-terminus to C-terminus, matrix polypeptide (MA), a capsid polypeptide (CA), a p2 peptide, a nucleocapsid polypeptide (NC), a pi peptide, and a p6 peptide.

175. The XDP system of any one of claims 172-173, wherein the nucleic acids encode one or more components selected from

(a) a Gag-Pol polyprotein;

(b) one or more protease cleavage sites;

(c) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(d) a gag-transframe region-pol protease polyprotein.

176. The XDP system of any one of claims 172-175, wherein the lentivirus is selected from the group consisting of human immunodeficiency- 1 (HIV-1), human immunodeficiency-2 (HIV- 2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

177. The XDP system of claim 176, wherein the lentivirus is HIV-1

178. The XDP system of any one of claims 172-177, 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.

179. The XDP system of claim 178, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595, 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.

180. The XDP system of claim 178, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487,

489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525,

527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,

565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595.

181. The XDP system of claim 180, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

182. The XDP system of any one of claims 172-181, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

183. The XDP system of claim 182, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti cancer modality.

184. The XDP system of claim 183, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

185. The XDP system of claim 184, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

186. The XDP system of claim 185, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

187. The XDP system of claim 186, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

188. The XDP system of claim 186, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

189. The XDP system of any one of claims 186-188, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

190. The XDP system of claim 182, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double- stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

191. The XDP system of claim 190, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

192. The XDP system of claim 191, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781or 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.

193. The XDP system of claim 191, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

194. The XDP system of any one of claims 191-193, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

195. The XDP system of any one of claims 172-194, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

196. The XDP system of claim 195, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

197. The XDP system of claim 195 or claim 196, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

198. The XDP system of any one of claims 195-197, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

199. The XDP of claim 198, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

200. The XDP system of claim 198, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

201. The XDP of claim 198, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

202. The XDP system of claim 201, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

203. An XDP system comprising one or more nucleic acid encoding components:

(a) all or a portion of an Spumaretrovirinae gag polyprotein;

(b) a therapeutic payload; and

(c) a tropism factor.

204. The XDP system of claim 203, wherein the gag polyprotein comprises one or more components selected from the group consisting of a p68 Gag polypeptide and a p3 Gag polypeptide.

205. The XDP system of claim 204, wherein the gag polyprotein comprises, from N-terminus to C-terminus, p68 Gag polypeptide and a p3 Gag polypeptide.

206. The XDP system of any one of claims 203-205, wherein the nucleic acids encode one or more components selected from

(a) an HIV pi peptide;

(b) an HIV p6 peptide;

(c) a Gag-Pol polyprotein;

(d) one or more protease cleavage sites;

(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and

(f) a gag-transframe region-pol protease polyprotein.

207. The XDP system of any one of claims 203-206, 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.

208. The XDP system of claim 207, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559,

561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595, 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.

209. The XDP system of claim 207, wherein the tropism factor is a glycoprotein having a sequence selected from the group consisting of SEQ ID NOS: 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501„ 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,

565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 and 595.

210. The XDP system of claim 209, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).

211. The XDP system of any one of claims 203-210, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

212. The XDP system of claim 211, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti cancer modality.

213. The XDP system of claim 212, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

214. The XDP system of claim 213, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

215. The XDP system of claim 214, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

216. The XDP system of claim 215, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

217. The XDP system of claim 216, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397.

218. The XDP system of any one of claims 203-217, wherein the CasX further comprises one or more NLS selected from the group of sequences consisting of SEQ ID NOS: 130-166, wherein the NLS are located at or near the N-terminus and/or the C-terminus.

219. The XDP system of claim 211, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double- stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

220. The XDP system of claim 219, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence is complementary to a target nucleic acid sequence.

221. The XDP system of claim 220, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

222. The XDP system of claim 221, wherein the scaffold sequence of the guide RNA comprises a sequence of SEQ ID NOS: 597-781.

223. The XDP system of any one of claims 220-222, wherein the targeting sequence of the guide RNA consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides.

224. The XDP system of any one of claims 203-223, wherein

(a) the components are encoded on a single nucleic acid;

(b) the components are encoding on two nucleic acids;

(c) the components are encoding on three nucleic acids;

(d) the components are encoding on four nucleic acids; or

(e) the components are encoding on five nucleic acids.

225. The XDP system of claim 224, wherein the one or more of the components encoded by the nucleic acids are configured according to any one of FIGS. 36-68.

226. The XDP system of claim 224 or claim 225, wherein the one or more of the components are encoded by nucleic acids selected from the group of sequences consisting of SEQ ID NOS: 192, 193, 195, 196, 198-201, 782, 234-339, 880-933, and 947-1000 as set forth in Tables 5, 24, 27, 30, and 33, or sequences 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.

227. The XDP system of any one of claims 224-226, wherein the components are capable of self-assembling into an XDP when the one or more nucleic acids are introduced into a eukaryotic host cell and are expressed.

228. The XDP of claim 227, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.

229. The XDP system of claim 228, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

230. The XDP of claim 227, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.

231. The XDP system of claim 230, wherein the tropism factor confers preferential interaction with the cell surface of a target cell and facilitates entry of the XDP into the target cell.

232. The XDP system of any one of the preceding claims, wherein the gag polyprotein and the therapeutic payload is expressed as a fusion protein.

233. The XDP system of claim 232, wherein the fusion protein does not comprise a protease cleavage site between the gag polyprotein and the therapeutic payload.

234. The XDP system of claim 232, wherein the fusion protein comprises a protease cleavage site between the gag polyprotein and the therapeutic payload.

235. The XDP system of any one of claims 232-234, wherein the fusion protein comprises protease cleavage sites between the components of the gag polyprotein.

236. The XDP system of claim 234 and/or claim 235, wherein the cleavage sites are capable of being cleaved by the protease of the Gag-Pol polyprotein, the protease of the gag-transframe region-pol protease polyprotein, or the non-retroviral, heterologous protease.

237. The XDP system of claim 236, wherein the cleavage sites are capable of being cleaved by the protease of the gag-transframe region-pol protease polyprotein.

238. The XDP system of claim 236, wherein the cleavage sites are capable of being cleaved by the protease of the Gag-Pol polyprotein

239. The XDP system of claim 236, wherein the non-retroviral, heterologous protease is selected from the group consisting of tobacco etch virus protease (TEV), potyvirus HC protease, potyvirus PI protease, PreScission (HRV3C protease), b virus NIa protease, B virus RNA-2- encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C4ike protease, parsnip yellow fleck virus protease, 3C-like protease, heparin, cathepsin, thrombin, factor Xa, metalloproteinase, and enterokinase.

240. The XDP system of claim 239, wherein the non-retroviral, heterologous protease is PreScission (HRV3C protease).

241. The XDP system of claim 239, wherein the non-retroviral, heterologous protease is tobacco etch virus protease (TEV).

242. The XDP system of any one of claims 12-13, 44-47, 73-76, 96-99, 103-106, 132-135, 161-164, 192-195 or 221-224, wherein the guide RNA further comprises one or more ribozymes.

243. The XDP system of claim 242, wherein the one or more ribozymes are independently fused to a terminus of the guide RNA.

244. The XDP system of claim 242 or claim 243, wherein at least one of the one or more ribozymes is a hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

245. The XDP system of any one of claims 12-13, 44-47, 73-76, 96-99, 103-106, 132-135, 161-164, 192-195 or 221-224, wherein the guide RNA is chemically modified.

246. The XDP system of any one of claims 12-13, 44-47, 73-76, 96-99, 103-106, 132-135, 161-164, 192-195 or 221-224, wherein the guide RNA comprises an element selected from the group consisting of a Psi packaging element, kissing loop a, kissing loop bl, kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, sarcin-ricin loop, or pseudoknot, wherein the element has affinity to a protein incorporated into the CasX selected from the group consisting of MS2, PP7, Qbeta, U1A, and phage R-loop.

247. A eukaryotic cell comprising the XDP system of any one of the preceding claims.

248. The eukaryotic cell of claim 247, wherein the cell is a packaging cell.

249. The eukaryotic cell of claim 247 or claim 248, wherein the eukaryotic cell is selected from the group consisting of HEK293 cells, Lenti-X 293T cells, BHK cells, HepG2, Saos-2, HuH7, NSO cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO, NIH3T3 cells, COS, WI38, MRC5, A549, HeLa cells, CHO cells, and HT1080 cells.

250. The eukaryotic cell of claim 248 or claim 249, wherein the packaging cell comprises one or more mutations to reduce expression of a cell surface marker.

251. The eukaryotic cell of any one of claims 247-250, wherein all or a portion of the nucleic acids encoding the XDP system are integrated into the genome of the eukaryotic cell.

252. A method of making an XDP comprising a therapeutic payload, the method comprising:

(a) propagating the packaging cell of any one of claims 248-251 under conditions such that XDPs are produced; and

(b) harvesting the XDPs produced by the packaging cell.

253. An XDP produced by the method of claim 252.

254. The XDP of claim 253, comprising a therapeutic payload of an RNP of a CasX and guide RNA and, optionally, a donor template.

255. A method of method of modifying a target nucleic acid sequence in a cell, the method comprising contacting the cell with the XDP of claim 254, wherein said contacting comprises introducing into the cell the RNP and, optionally, the donor template nucleic acid sequence, wherein the target nucleic acid targeted by the guide RNA is modified by the CasX.

256. The method of claim 255, wherein the modification comprises introducing one or more single- stranded breaks in the target nucleic acid sequence.

257. The method of claim 255, wherein the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence.

258. The method of any one of claims 255-257, wherein the modification comprises insertion of the donor template into the target nucleic acid sequence.

259. The method of any one of claims 255-258, wherein the cell is modified in vitro or ex vivo.

260. The method of any one of claims 255-258, wherein the cell is modified in vivo.

261. The method of claim 260, wherein the XDP is administered to a subject.

262. The method of claim 261, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

263. The method of claim 261 or 262, wherein the XDP is administered by a route of administration selected from the group consisting of subcutaneous, intradermal, intraneural, intranodal, intramedullary, intramuscular, intravenous, intracerebroventricular, intracistemal, intrathecal, intracranial, intralumbar, intratracheal, intraosseous, inhalatory, intracontralateral striatum, intraocular, intravitreal, intralymphatical, intraperitoneal routes and sub-retinal routes.

264. The method of any one of claims 261-263, wherein the XDP is administered to the subject using a therapeutically effective dose.

265. The method of claim 264, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles/kg, or at least about 1 x 10⁶ particles/kg, or at least about 1 x 10⁷ particles/kg, or at least about 1 x 10⁸ particles/kg, or at least about 1 x 10⁹ particles/kg, or at least about 1 x 10¹⁰ particles/kg, or at least about 1 x 10¹¹ particles/kg, or at least about 1 x 10¹² particles/kg, or at least about 1 x 10¹³ particles/kg, or at least about 1 x 10¹⁴ particles/kg, or at least about 1 x 10¹⁵ particles/kg, or at least about 1 x 10¹⁶ particles/kg.

266. The method of any one of claims 261-265, wherein the XDP is administered to the subject according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose of the XDP.

267. The method of claim 266, 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.

268. A method for introducing a CasX and gNA RNP into a cell having a target nucleic acid, comprising contacting the cell with the XDP of claim 253 or claim 254, such that the RNP enters the cell.

269. The method of claim 268, wherein the RNP binds to the target nucleic acid.

270. The method of claim 269, wherein the target nucleic acid is cleaved by the CasX.

271. The method of any one of claims 268-270, wherein the cell is modified in vitro.

272. The method of any one of claims 268-270, wherein the cell is modified in vivo.

273. The method of claim 272, wherein the XDP is administered to a subject.

274. The method of claim 273, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

275. The method of any one of claims 272-274, wherein the XDP is administered to the subject using a therapeutically effective dose.

276. The method of claim 275, wherein the XDP is administered at a dose of at least about 1 x 10⁵ particles/kg, or at least about 1 x 10⁶ particles/kg, or at least about 1 x 10⁷ particles/kg, or at least about 1 x 10⁸ particles/kg, or at least about 1 x 10⁹ particles/kg, or at least about 1 x 10¹⁰ particles/kg, or at least about 1 x 10¹¹ particles/kg, or at least about 1 x 10¹² particles/kg, or at least about 1 x 10¹³ particles/kg, or at least about 1 x 10¹⁴ particles/kg, or at least about 1 x 10¹⁵ particles/kg, or at least about 1 x 10¹⁶ particles/kg.

277. A XDP particle comprising:

(a) a retroviral matrix (MA) polypeptide;

(b) a therapeutic payload encapsidated within the XDP; and

(c) a tropism factor incorporated on the XDP surface.

278. The XDP particle of claim 277, further comprising one or more retroviral components selected from:

(a) a capsid polypeptide (CA);

(b) a nucleocapsid polypeptide (NC);

(c) a P2A peptide, a P2B peptide;

(d) a P10 peptide;

(e) a p 12 peptide

(f) a PP21/24 peptide;

(g) a P12/P3/P8 peptide;

(h) a P20 peptide;

(i) a pi peptide; and

(j) a p6 peptide

279. The XDP particle of claim 277 or claim 278, 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.

280. The XDP particle of claim 279, wherein the tropism factor is a glycoprotein having an sequence selected from the group consisting of SEQ ID NOS: 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488,

490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,

528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564,

566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594 and 596, 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.

281. The XDP particle of claim 279, wherein the tropism factor is a glycoprotein having an encoding sequence selected from the group consisting of SEQ ID NOS: 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484,

486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522,

524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560,

562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594 and 596.

282. The XDP particle of any one of claims 277-281, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.

283. The XDP particle of claim 282, wherein the protein payload is selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, RNAse, DNAse, a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, and an anti cancer modality.

284. The XDP particle of claim 283, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.

285. The XDP particle of claim 284, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of Type II, Type V, or Type VI protein.

286. The XDP particle of claim 285, wherein the CRISPR protein is a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX.

287. The XDP particle of claim 286, wherein the CRISPR protein is a CasX comprising a sequence of SEQ ID NOS: 21-233, 343-345, 350-353, 355-367 or 388-397, 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.

288. The XDP particle of claim 282, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASOs), a double- stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

289. The XDP particle of claim 288, wherein the CRISPR guide nucleic acid is a single molecule guide RNA comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 14 and 30 nucleotides and is complementary to a target nucleic acid sequence.

290. The XDP particle of claim 289, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781, 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.

291. The XDP particle of claim 290, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781.

292. The XDP particle of any one of claims 286-291, wherein the therapeutic payload comprises a CasX and a guide RNA complexed as a ribonucleoprotein complex (RNP) and, optionally, a donor template.

293. The XDP particle of any one of claims 277-292, wherein the retroviral components are derived from a Orthoretrovirinae virus or a Spumaretrovirinae virus.

294. The XDP particle of claim 293, wherein the Orthoretrovirinae virus is selected from the group consisting of Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus.

295. The XDP particle of claim 293, wherein the Spumaretrovirinae virus is selected from the group consisting of Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, and Spumavirus.

296. The XDP particles, or the XDP systems of any one of the preceding claims, for use as a medicament for the treatment of a subject having a disease.