CA3159320A1

CA3159320A1 - Particle delivery systems

Info

Publication number: CA3159320A1
Application number: CA3159320A
Authority: CA
Inventors: Benjamin OAKES; Sean Higgins; Hannah SPINNER; Sarah DENNY; Brett T. STAAHL; Kian TAYLOR; Katherine BANEY; Isabel COLIN; Maroof ADIL; Matthew Gardner; Trent GOMBERG; Gayathri VIJAYAKUMAR
Original assignee: Scribe Therapeutics Inc
Current assignee: Scribe Therapeutics Inc
Priority date: 2019-12-06
Filing date: 2020-12-04
Publication date: 2021-06-10
Also published as: JP2023504536A; EP4069716A1; AU2020398658A1; IL293591A; CN115175921A; WO2021113772A1; KR20220113442A

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

2 PARTICLE DELIVERY SYSTEMS
CROSS REFERENCE TO RELATED APPLICATIONS
100011 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
100021 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 05W0 SeqList ST25.txt and is 114 MB in size.
BACKGROUND
100031 The delivery of protein or nucleic acid therapeutics to particular cells or organs of the body generally requires complex systems in which a targeting modality or vehicle is linked to or contains the therapeutic. 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.
100041 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 Spurnaretrovirinae . The best known lentivirus is the Human Immunodeficiency Virus (HIV), which causes acquired immune deficiency syndrome (AIDS). As with all retroviruses, lentiviruses have gag, pot 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 at. 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
100051 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.
100061 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.
100071 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 anti sense oligonucleotide (AS0s), 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-poi 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 Orthoretrovirthae virus or a Spumaretrovirinae virus wherein the Orthoretrovirinae virus is selected from the group consisting of Alpharetrovirus, Befaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus, and the Spumaretrovirinae virus is selected from the group consisting of Bovispurnavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, and Spumavirus.
100091 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 p1 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 Betcweirovirus 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 pl peptide, an I-11V 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 Deharetrovirus gag polyprotein, wherein the gag

4 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 p1 peptide, an HW 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.
100141 In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of a Epsdonretrovirus 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 p1 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.
100151 In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of a Ganunattreirovirus gag polyprotein, wherein the gag polyprotein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a p12 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 p1 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-transfranie region-pol protease polyprotein.
100161 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 pl 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.
100171 In some embodiments of the XDP system, the nucleic acids encoding the retroviral components are all or a portion of a Sputnaretrovirinae 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 pl peptide, an 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-pot protease polyprotein.
100181 In some embodiments of the CasX system, the CasX further comprises one or more NLS selected from the group of sequences consisting of PICKICRKV (SEQ ID NO:
130), KRPAATKKAGQAICICKK (SEQ ID NO: 131), PAAKRVKLD (SEQ ID NO: 132), RQRRNELICRSP (SEQ ID NO: 133), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 134), RMRIZFKNKGKDTAELRRRRVEVSVELRKAICKDEQILICRRNV (SEQ ID NO: 135), VSRKRPRP (SEQ ID NO: 136), PPKKARED (SEQ ID NO: 137), PQPICKKPL (SEQ ID NO:
138), SALIKKICKKMAP (SEQ ID NO: 139), DRLRR. (SEQ ID NO: 140), PKQKKRK (SEQ
ID NO: 141), RICL,KICKIKKL (SEQ ID NO: 142), REICICKFLICRR (SEQ ID NO: 143), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 144), RKCLQAGMNLEARKTICK (SEQ ID
NO: 145), PRPRKIPR (SEQ ID NO: 146), PPRKKRTVV (SEQ ID NO: 147), NLSKICKKRKREK (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), KLICIKRPVK (SEQ ID NO: 154), PKTRRRPRRSQRKRPPT (SEQ ID NO: 156), RRICKRRPRRKKRR (SEQ ID NO: 159), PICICKSRKPKKKSRK (SEQ ID NO: 160), HICKKITPDASVNFSEFSK (SEQ ID NO: 161), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 162), LSPSLSPLLSPSLSPL (SEQ ID NO: 163), RGKGGKGLGKGGAKR.HRIC (SEQ ID NO: 164), PKRGRGRPKR.GRGR (SEQ ID NO: 165), MSRRRKANPTKLSENAKKLAICEVEN (SEQ ID NO: 157), PICKKRICVPPPPAAKRVKLD
(SEQ ID NO: 155), and PICKICRKVPPPPICKICRKV (SEQ ID NO: 166), wherein the NLS
are located at or near the N-terminus and/or the C-terminus.
100191 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 P1 protease, PreScission (HRV3C protease), b virus Ma protease, B
virus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picoma 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.
[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 1-IEK293 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, MRCS, 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 viva 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, intracistemal, 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 103 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 10" particles/kg, or at least about 1 x 1012 particles/kg,, or at least about 1 x 10" particles/kg, or at least about 1 x 10" 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/U52020/036505, 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
100251 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 p5TX34 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-FreeTm 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 StainFreeTM 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 StainFreeTM 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 Na2, [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 sgR_NAs 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.

228 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 lx 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 ref alive 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. 40A 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. 41A 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 43A 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. 47A 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. 48A 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. 49A 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.

100921 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. 51A 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. 51B 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. 53A 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 )(DP_ [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, 6%, 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.

1001081 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 736 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 751, 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. 61A 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.
1001141 FIG. 61B 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. 63A 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 698 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. 71A 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. 73A 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-c,ovalently 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-Fintron A, SV40, RSV, HIV-Ltr, elongation factor 1 alpha (EF la), MMLV-ltr, internal ribosome entry site (lRES) 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 Sled 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 Ku=[L] [P]/[LP], where [P], [L] and [LP]
represent molar concentrations of the protein, ligand and complex, respectively.

1001521 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).
1001571 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.
1001631 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.
1001641 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(abs)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 1001731 The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996);
Nonviral Vectors for Gene Therapy (Wagner et al_ eds., Academic Press 1999); Viral Vectors (Kaplift &
Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle &
Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.
[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.

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 frilly 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 11 CRISPR/Cas effector polypeptides such as Cas9. In other cases, the nucleases include Class 2, Type V CRISPR/Cas effector polypeptides such as a Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12J, 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 subdomthns), 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 p10 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 Betaretrovints, 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 aBovaspumcrvirus, an Equispumavirus, aFel/spun/a-virus, a Prosimiispumavirus or a Sitniispumavirus.
b. Retroviral Components [00184] The Retroviridae family of viruses have different subfamilies, including Orthoretrovirinae, Sputnaretrovirinae, 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 ahareirovirus, 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 Deltareirovirus 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 Gcrmmaretrovints include murine leukemia virus, Maloney murine leukemia virus, and feline leukemia virus, as well as viruses that infect other animal species. Lent/virus is a genus of retroviruses that cause chronic and deadly diseases, including 1-1IV-1 and HIV-2, the cause of the disease AIDS, and also includes Simian immunodeficiency virus. The subfamily Spurnaretrovirinae include the genera Bovispuntavirus, Equispuntavirus, Felispumavirus, Prositniispurnavirus, Sitniispumavirus, 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 p10 spacer peptide; a capsid polypeptide (CA);
a nueleocapsid polypeptide (NC), a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), p2A, p214, p10, a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-Pol polyprotein; a Gag-transframe region-Pal 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, p10, 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 more 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 Alphcrretrovirus 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.
1001861 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/p12 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/p12, a nucleocapsid polypeptide (NC); a therapeutic payload; a tropism factor; a Gag-Po1 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/p12 spacer, and NC), 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.
1001871 In some embodiments, the XDP viral components are derived from Deltaretrovirus, including but not limited to bovine leukemia virus (BLV) and the human T-Iymphotropie 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 Deftaretrovirus, 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 more Deftaretrovirus 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 Deftaretrovirus 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 giycoprotein embodiment described herein.
1001881 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-Pot 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 pp12 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a pp12 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, pp12, 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 Gamtnaretrovirus 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.
1001901 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 pi/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-Pot 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 Lent/virus, including but not limited to HIV-1, HIV-2, and Simian immunodeficiency virus (SW). 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 Lent/virus 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 Lent/virus 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 g,NA
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 Bovisputnavirus, Equisputnavirtts, Felispuntavirus, 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 Sputnaretrovirinae including but not limited to Bovispumavirus, Equispumavirus, Felispuma-virus, Prosimiisputnavirus, 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 Spurnaretrovirinae 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 Sputnareirovirinae 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 welt 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.
1001921 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 p10 spacer peptide, a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a pp21/24 spacer peptide, a p3-P8 spacer peptide, a pp12 spacer peptide, a p20 spacer peptide, a pi/p6 spacer peptide, a p68 Gag, a p3 Gag, a cleavage site(s), a Gag-Pol polyprotein; a Gag-transframe region-Pot protease polyprotein; and a non-retroviral, heterologous protease capable of cleaving the protease cleavage sites wherein the components are derived from Alpharetrovirus, Betaretrovirus, Deharetrovirus, Epsdonretrovirus, Gattunaretrovirus, Lent/virus, Bovisputnavirus, Equispumavirus, Felisputnavirus, Prositniisputnavirus, Simlispumavirus, or Spumavirus..
1001931 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-Pot 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, aphthovims L protease, enterovirus 2A
protease, rhinovirus 2A 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 enterokinasa 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 p6po1, separated by a protease cleavage site.
Cleavage at the p6po1-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, .11µ4. Et at. 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 TER 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.
1001951 In a corresponding fashion, wherein pro-tease 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 protease, the encoded mV-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 Gln 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., 11IV-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., 1FNs a, 13, and 7, TNF-ct, G-CSF, GM-CSF)), interleukins (e.g., IL-1 to IL-40), growth factors (e.g., VEGF, PDGF, IGF-1, EGF, and TGF-I3), 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 biologics 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 (AS0s), 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 1D 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 1-INH 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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.
1001991 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, , Planctornycetes, or Candidatus (as described in US20180346927A1 and W02018064371A1, incorporated herein by reference). Exemplary embodiments of CasX variants envisaged as being within the scope of the disclosure are described herein, below.
1002001 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:

EVMPQVISNN

LKPEMDEKGN

LKPEKDS DEA

ACMGT IAS FL

DAYNEVIARV

KKL I DAKRDM

LLYLEKKYAG

FVLERLKEMD

LLAWKYLENG

DEQL I I L PLA

T FE RREVVDP

Y KE KORA I QA

LSRGFGRQGK

TTADYDGMLV

GNND I SKWTK

SNSTEFKSYK
961 SGKQPFVGAW QAFYKRRLKE VWKPNA (SEQ ID NO: 1).
1002011 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:

KPENIPQPIS

RKL I PVKDGN

SPHKPEANDE

DACMGAVAS F
241 LTKYQDI ILE HQKVIKKNEK RLANLKDIAS .ANGLAFPKIT LPPQPHTKEG
I EAYNNVVAQ

VKKL I NEKKE

GE DWGKVYDE

ADKDE FORCE

L I I NY FKGGK

RQGRE F I WND

KPMNL I G I DR

VEQRRAGGYS

MAE RQYTRME

KTATGWMTT I

GEALSLLKKR
901 FSHRPVQEKF VCLNCGFETH ADEQ.AALNIA RSWLFLRSQE YKKYQTNKTT
GNTDKRAFVE
961 TWQSFYRKKL KEVWKPAV ( SEQ ID NO: 2 ) .
1002021 In some cases, a Type V reference CasX protein is isolated or derived from Candidaius 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 FAAVEAARER

ALRHKAEGAM

LNTCLAPEYD

RLRFFNGRIN

KPGSAVPLPQ

ARYMDIISFR

MALAKDANAP

SFDEYPASGV

LFFHMVISGP

KEYIDQLIET

ERLDDQFHGR

CTQCGTVWLA

RLTPRYSRVM

AATNLARRAI
841 SLIRRLPDTD TPPTP (SEQ ID NO: 3).
1002031 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, glutarnic 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, 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 01 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.
1002081 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 11 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, ODD, 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, 5890, or 5932 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 1D NO:2. In some embodiments, the CasX variant comprises at least one modification in the ODD domain. In some embodiments, the at least one modification in the ODD 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, 6695, 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 A7081C; (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 Siting 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 protein:gNA (RNP) complex stability, improved protein solubility, improved protein:gNA
(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:1, 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 1D 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, E3-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:!, 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 700/s, 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.
1002161 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 glutarnic 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.
I 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 H, OBD
and RuvC domains. In some embodiments, the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical H, 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
II) 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 1, 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 11. 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 Descriptio Amino Acid Sequence n*
TSL, MQEIKRINKI
RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
Helical I, PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVA
Helical II, QPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGK
OBD and AYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIH
RuvC
VTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILE
domains HQKVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVI
from SEQ VVVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERIDANEVDVVVVDMVCNV
ID NO:2 KKLINEKKEDGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQFG
and an DLLLHLEKKHGEDWGIWYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSK

Descriptio Amino Acid Sequence n*
NTSB
AALTDVVLRAKASFVIEGLKEADKDEFCRCELKLQIONYGDLRGKPFAIEAE
domain NSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
from SEQ NRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLE
ID NO: 1 TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN
IKPMNLI
GI DRGEN IPAVIALTDPEGC PLS RFKDSLGNPTHILRIGESYKEKQ RTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQ'YTRMEDVVLTAKLAYEGLSKTYLSKTLAQ'YTS

RQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAF
VETVVQSFYRKKLKEVWKPAV (SEQ ID NO: 21) NTSB, MQE
IKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
Helical I, PEN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYWE EFQ KDPVGLMS RVA
Helical II, Q PAP KN IDQ RKLIPVKDGNERLTSSGFACSO CCQ PLYVYKLEQVN DKGKP
OBD and HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RuvC
RESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQ
domains KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
from SEQ NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVVVDMVCNVKKL
ID NO:2 INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
and a TSL LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
domain TDVVLRAKAS FVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAI
EAENSIL
from SEQ DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
ID NO: 1 . YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGS
LKLANGRVIE KTLYNRRTRQDEPALFVALTFERREVLDSSNIKPM NLIGIDR
GEN IPAVIALTDP EGC P LSRFKDSLGNPTH ILRIGESYKEKQ RTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGROGKRTFMAERQ'YTRMED1NLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITTADYDGMLVRLKKTSDGWATTLNNKELKAEGQITYYNRYKRQT
VEKELSAELDRLSEESGNNDISKVVI-KGRRDEALFLLKKRFSHRPVQEQFV

TVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 22) TSL, MEKRINKIRKKLSADNATKPVSRSGPMKTUVRVMTDDLKKRLEKRRKKP
Helical I, EVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFA
Helical II, Q PAP KN IDQ RKLIPVKDGNERLTSSGFAC SQ CCQ PLYVYKLEQVN DKGKP
OBD and HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLOKFGQRALDFYSIHVT
RuvC KESTHPVKPLAQ
IAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKV
domains VKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
from SEQ WVNLNLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWVVNTIN EVK
lD NO:1 KLIDAKRDMGRVFVVSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPICK
and an PAKRQFGDLLLYLEKKYAGDWGKVFDEAVVERIDKKIAGLTSHIEREEARN
NTSB
AEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKVVYGDLRG
domain from SEQ GKKGR IRFTDGTDIKKSGKWQGLLYGGGKAKVIDLTFDP DDE QV ILP LAF
ID NO:2 GTRQGREFIVVNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFE
RREVVDPSN IKPVNLIGVDRGEN IPAVIALTDPEGCP LP EFKDSSGGPTDI L

Descriptio Amino Acid Sequence n*
RIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARD

GLTSKTYLSKTLAQYTSKTCSNCGFTITTADYDGMLVRLKKTSDGWATTL
NNKELKAEGQ ITYYNRYKRQTVEKELSAELDRLSEESGNNDIS KVVTKGRR
DEALF LLKKRFS H RPVQ EQ FVC LDCG H EVHADEQAALN IARSWLFL NS NS
TEFKSYKSGKQPFVGAWQAFYKRRLKEV1NKPNA (SEQ ID NO: 23) NTSB, MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKP
Helical I, EVMPQVISNNAAN NLRMLLDDYTKMKEAILQVYWQ EFKDDHVGLMCKFA
Helical II, QPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGK
ODD and AYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIH
RuvC VTKESTHPVKPLAQ IAG N RYASGPVG KALSDACMGTIASFLS
KYQ D I IIEHQ
domains KWKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVR

from SEQ MWVN L N LWQ KLKLSRD DAKP L LRLKG F PS FPVVE RR EN EVDWVVNTIN EV
ID NO:! KKLIDAKR DMGRVFWSGVTAE KR NTILEGYNYLP N E N
DHKKREGS LE N PK
and an TSL KPAKRQFGDLLLYLEKKYAGDWGKVFDEAVVERIDKKIAGLTSH IE RE EAR
domain NAEDAQS KAVLTDWLRAKASFVLE RLKEM DE KEFYACE IQ LQ
KWYG D LR
from SEQ GN PFAVEAENRVVD ISG FS I GS DGHS IQYRNLLAWKYLENGKREFYLLMN
ID NO:2. YOKKG RIRFTDGTDIKKSGKVVQ GLLYGGGKAKVIDLTFDP DD EQ LI I LPLAF
GTRQGREFIVVNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFE
RREVVDPS N IKPVN L IGVD RGE N IPAVIALTDP EGC PLP EF KDS SGG PTD IL
RIGEGYKEKQRAIQAAKEVEQ RRAGGYSR KFAS KS RN LADDMVRNSARD
LFYHAVTHDAVLVFENLSRGFGRQGKRTFMTERQYTKMEDVVLTAKLAYE
GLTSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTIN
GKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSG
EALSLLKKRFS HRPVQEKFVCLNCGFETHADEQAALN IARSWLFLN S N ST
EFKSYKSGKQPFVGAVVQAFYKRRLKEVWKPNA (SEQ ID NO: 24) NTSB, MQE IKRI N KI RRRLVKDS NTKKAGKTG PM
KTLLVRVMTPDLRE RLE NLRKK
TSL, PEN I PQ P ISNTS RAN LN KLLTDYTEM KKAILHVYVVE EFQ
KD PVG LMS RVA
Helical I, QPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCQPLYVYKLEQVNDKGKP
Helical II HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
and OBD RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVAS FLTKYQD I I LEHQ
domains KVIKKNEKRLANLKDIASANGLAFPKITLPPQ PHTKEG I EAYN
NVVAQ IVIVVV
SEQ ID NLNLWQ KLKI GRDEAKP LQ RLKGFPS F PLVE ROAN
EVDVVWDMVCNVKKL
NO:2 and INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
an LH LE KKHG E DWG KVYDEAWERIDKKVEG LS KH I KLEE
ERRS EDAQ S KAAL
exogenous TDWLRAKASFVI EGLKEADKDEFCRCELKLQ KVVYG D LRGKPFA I EAENSI L
RuvC DISGFSKOYNCAFIWQKDGVKKLNLYLI I NYFKGG KLRFKKI KP
EAFEAN RF
domain or a YTV I NKKSG E IVPMEVN FN FDD P N LIILPLAFGKRQG RE FIVVN D LLS
LETGS
portion LKLANGRVIEKTLYNRRTRODEPALEVALTFERREVLDSSN I KPVN
LIGVDR
thereof GEN IPAVIALTD PEGG P LP E FKDSS GG PTDILR IG
EGYKEKQ RAI QAAKEVE
from a OR RAGGYSRKFAS KS RN LADDMVRNSAR DLFYHAVTH
DAVLVFE N LSRG
second FGRQGKRTFMTERQYTKMEDVVLTAKLAYEGLTSKTYLSKTLAQYTSKTC
CasX SNCGFTITSADYD RVLEKLKKTATGVVMTTI NC KELKVEGQ
ITYYNRYKRQ
protein.

Descriptio Amino Acid Sequence n*
NVVKDLSVELD RLS EESVN N D IS SVVTKGRSGEALS L LKKRFSH R PVQ E KF
VCLNCGFETHA (SEQ ID NO: 25) MQEIKRINKI RRR LVKDSNTKKAG KTGPM KTLLVRVMTPD LRE R LEN LRKK
PEN I PQ P ISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVA
Q PAPKN I DQRKL IPVKDGN ERLTSSGFACS QCCQ P LYVYKL EQVN DKGKP
HTNYFG RC NVS EH ERL I LLSP H KP EAN D ELVTYSLGKFGQ RALDFYSI HVT
RES N HP VK P LEQIGG N SCASG PVG KALS DAC MGAVAS FLTKYQ D I ILEHQ
KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIINV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVVVDMVCNVKKL
I NE KKEDG KVFWQN LAGYKRQ EALRPYLSSEE DRKKGKKFARYQ FGDLL
LH LE KKH GEDWGIWYD EAWE RI D KKVE GLSKH IKL EE ERRS E DAQS KAAL
TDWLRAKASFVIEGLKEADKDEFC RCELKLQ KVVYGDLRG KP FAI EAE N S IL
DISGFSKQYNCAFIWQKDGVKKLN LYL II NYFKGGKLRFKKIKP EAFEAN R F
YTVI N KKSGEIVP MEVN FN FDDP N LI I LP LAFG KRQ GRE F IWN DLLSLETGS
LKLANGRVI E KTLYNRRTRQ D EPALFVALTFE RREVLDS SN I KP M N L IG IDR
G EN I PAVIALTD PEGCP LS RFKDSLGN PTH I LR IG ESYKE KQ RTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDINLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGINMTTINGKELKVEGQ ITYYNRYKRQ N
VVKDLSVE L DR LSE ESVN N D I SSVVTKGRSGEALSLLKKRF SH RPVQ EKFV
CLNCGFETHA (SEQ ID NO: 26) NTSB, MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
TSL, PEN I PQ P ISN NAANN LRM LLD DYTKM KEAILQVYINQ
EFKDDHVGLMCKFA
Helical II, Q PAPKN I DQRKL IPVKDGN ERLTSSGFACS QCC Q P LYVYKL EQVN DKGKP
OBD and HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RuvC KESTHPVKPLAQ IAGN RYASGPVG KALS DACM GTIAS F LS
KYQ D II I EH Q KV
domains VKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
from SEQ INVNLNLWQ KLKLS RDDAKP LL RLKGFPS FP LVER QAN EVDWINDMVCNV
ID NO:2 KKL I N EKKE DGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQFG
and a D LLLH LEKKHGEDWG KVYD EAVVE RID KKVEG LSKH IKL
E E ERRSEDAQS K
Helical I
AALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVY'GDLRGKPFAIEAE
domain N S ILD ISG FSKQYN CAF IWQ KDGVKKLN LYL I INYF
KGG KLRF KKI KPEAFEA
from SEQ N RFYTVI N KKSGE IVP M EVN FN FDD PN L I I LPLAFGKRQG REFIVVN D L LS
L E
ID NO: 1 TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN I
KPM N L I
G IDRGEN IPAV IALTD PEGCP LS RFKDSLGN PTH I LR I GESYKEKQRTIQAK
KEVEQ RRAGGYSRKYASKAKN LAD DMVR NTARD L LYYAVTQ DAM LIFE N
LSRGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ ITYYNRYK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSWLFLRSQ EYKKYQTNKTTGNTDKRAF
VETWQSFYRKKLKEVWKPAV (SEQ ID NO: 27) NTSB, MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
TSL, PEN I PQ P
ISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVA
Helical I, QPAPKNIDORKLIPVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP

Descriptio Amino Acid Sequence n*
OBD and HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RuvC RESNH PVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQ
DIILEHQ
domains KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
from SEQ NLNLWQKLKIGRDEAKPLQRLKGFPSFPVVERRENEVDWVVNTINEVKKLI
ID NO:2 DAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPA
and a KRQFGDLLLYLEKKYAGDWGKVFDEAVVERIDKKIAGLTSHIEREEARNAE
Helical II DAQSKAVLTDVVLRAKASFVLE RLKEM D EKE FYAC E I Q LQ KVVYGDLRG N P
domain FAVEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIK
from SEQ P EAFEANRFYTVINKKSGE IVP MEVNFNFDDPNLI I LPLAFGKRQGREFIWN
ID NO:
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIK
PMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQR
TIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAM

QYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYY
NRYKRQNWKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHR
PVQEKFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNT
DKRAFVETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 28) NTSB, MISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQPAPKN
TSL, IDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYFG
Helical I, RC NVSEH ERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTRESNH P
Helical II VKPLEQ IGG N SCASG PVGKALS DACMGAVASFLTKYQ D I ILEHQ KVI KKNE
and RuvC KRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVI1NVNLNLWQ
domains KLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVWVDMVCNVKKLINEKKE
from a first DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLLLHLEKK
CasX
HGEDWGIWYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLR
protein and AKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENRVVDISGF
an SIGSDGHSIQYRNLLAVVICYLENGKREFYLLMNYGICKGRIRFTDGTDIKKS
exogenous GKINQGLLYGGGKAKVIDLTFDPDDEQUILPLAFGTROGREFIWNDLLSLE
OBD or a TGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPMNLIGI
part thereof DRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKE
from a VEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLS
second RGFGRQGKRTFMAERQ'YTRMEDWLTAKLAYEGLSKTYLSKTLAQ'YTSKT
CasX
CSNCGFTITSADYDRVLEKLKICATGWMTTINGKELKVEGQITYYNRYKR
protein QNWKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFV
ETINQSFYRKKLKEVVVKPAV (SEQ ID NO: 29) MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKP
EVMPQVISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ
PAPKNIDORKLIPVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKPH
TNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTR
ESNHPVKP LEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQ DI ILEHQK
VI KKN EKRLAN LKD IASANGLAF PKITLP PQ P HTKE G I EAYN NVVAQ IVIWV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWVVDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LH LE KKHGE DWG KVYDEAVVE RI D KKVEG LS KH I KLE E E RRS EDAQ S KAAL

Descriptio Amino Acid Sequence n*
TDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GEN IPAVIALTDPEGCP LSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYAS KAKN LADDMVRNTARDLLYYAVTQ DAM LI FE N LSRG
FGRQGKRTFMAERQ'YTRMEDVVLTAKLAYEGLSICYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGINMTTINGKELKVEGQITYYNRYKRQN
VVKDLSVE LDR LSE ESVNN D IS SWTKG RSG EALS LLKKRFSH RPVQ EKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WOSFYRKKLKEVVVKPAV (SEQ ID NO: 30) MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
PEN IPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA
QPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVAS FLTKYQDI I LEK/
KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVVVDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDWLRAKAS FVI EG LKEADKD E FCRC E LKLQ KWYG D LRGKPFA I EAEN RV
VDISGFSIGSDGHSIQYRNLLAVVKYLENGKREFYLLMNYGKKGRIRFTDGT
DIKKSGKWQGLLYGGGKAKVIDLTFDP DDEQ LI ILP LAFGTRQGREFIWN D
LLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPM
NLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQ
AKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIF
ENLSRGFGROGKRTFMAERQ'YTRMEDWLTAKLAYEGLSKTYLSKTLAQY
TS KTCS NCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ ITYYNR
YKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPV
QEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDK
RAFVETWQSFYRKKLKEV1A/KPAV (SEQ ID NO: 31) substitution MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of L379R, PEN IPQP ISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA
a QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGORALDFYSIHVT
of C477K, RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVAS FLTKYQDI I LEHQ
a KVIKKNEKRLANLKDIASANGLAFPKITLPPQ PHTKEGIEAYNNVVAQ
IVIVVV
substitution NLNLWQ KLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVVVDMVCNVKKL
of A708K, INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
a deletion LH LEKKHGEDWGIWYDEAWERIDKKVEGLS KH IKLEEERRSEDAQSKAAL
of P at TDWLRAKASFVIEGLKEADKDEFKRCELKLQKVVYGDLRGKPFAIEAENSIL
position DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
793 and a YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
substitution LKLANGRVIEKPLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
of T62013 GEN IPAVIALTDPEGCP LSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE

Descriptio Amino Acid Sequence n*
of SEQ ID QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
NO:2 FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NC GFT ITSADYD RVLEKLKKTATGINMTTI NG KELKVE GQ ITYYNRYKRQ N
VVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALN IARSWLFLRSQ EYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 32) substitution MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of M771A PE N I PQ P ISNTS RAN L N KLLTDYTEM KKAI LHVYVVE EF Q KDPVGLMSRVA
of SEQ ID QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
NO:2.
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVAS FLTICYQDI I LEHQ
KVI KKN EKRLAN LKDIASANGLAF PKITLP PQ PHTKEG I EAYN NVVAQ IVIWV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDINVVDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDWGIWYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GEN IPAVIALTDPEGCP LSRFKDSLGNPTH ILRIGESYKEKQRTIQAAKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFAAERQ'YTRMEDWLTAKLAYEGLPSKTYLSKTLAQ'YTSKTC
SNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQ ITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVVVKPA (SEQ ID NO: 33) substitution MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of L379R, PEN I PQ P ISNTS RAN LN KLLTDYTEM KKAI LHVYVVE EFQ KD PVG LMS RVA
a QPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCQPLYVYKLEQVNDKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of A708K, RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVAS FLTICYQD11 LEHQ
a deletion KVI KKN EKRLAN LKDIASANGLAF PKITLP PQ PHTKEG I EAYN NVVAQ IVIVVV
of P at NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDININDMVCNVKKL
position INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
793 and a LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
substitution TDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL
of D732N DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
of SEQ ID YTV I NKKSGE IVPMEVNFN FDD PN LIILPLAFGKRQG RE FIWN D LLS LETGS
NO:2. LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN
IKPMNLIGIDR
GEN IPAVIALTDPEGCP LSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLANDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQ'YTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQN
VVKDLSVELDRLSEESVNNDISSVITTKGRSGEALSLLKKRFSHRPVQEKFV

Descriptio Amino Acid Sequence n*
CLNCGFETHADEQAALNIARSWLFLRSOEYKKYOTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 34) substitution MQ E I KRI N KI RRRLVKDSNTKKAG KTGPM laLLVRVMTPD LRE RLENLRKK
of W782Q PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYINEEFOKDPVGLMSRVA
of SEQ ID OPAPKNIDORKLIPVKDGNERLTSSGFACSOCCePLYVYKLEQVNDKGKP
NO:2.
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGORALDFYSIHVT

KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDININDMVCNVKKL
INEKKEDGKVFWONLAGYKRQEALLPYLSSEEDRKKGKKFARYOFGDLLL
HLEKKHGEDWGIWYDEAVVERIDKKVEGLSKHIKLEEERRSEDAGSKAAL
TDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIINNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKORTIOAAKEVE
ORRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTODAMLIFENLSRG
FGRQGKRTFMAEROYTRMEDQLTAKLAYEGLPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGINMTTINGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHR PVQ EKF
VCLNCGFETHADEQAALNIARSINLFLRSOEYKKYOTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 35) substitution MOEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of M77 1 Q PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYINEEFQKDPVGLMSRVA
of SEQ ID QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
NO:2 HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYODIILEHO
KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQ IVIWV
NLNLWOKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVIANDMVCNVKKL
INEKKEDGKVFVVQNLAGYKROEALLPYLSSEEDRKKGKKFARYOFGDLLL
HLEKKHGEDWGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDINLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL
DISGFSKOYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIINNDLLSLETGS
LKLANGRVIEKTLYNRRTRODEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIOAAKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTODAMLIFENLSRG
FGRQGKRTFQAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGINMTTINGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHR PVe EKF
VCLNCGFETHADEGAALNIARSINLFLRSQEYKKYOTNKTTGNTDKRAFVE
TWOSFYRKKLKEVVVKPAV (SEQ ID NO: 36) substitution MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of R4581 PENIPOP ISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA

Descriptio Amino Acid Sequence n*
and a Q PAP KN IDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVN
DKGKP
substitution HTNYFG RCNVS EH E RLI LLSP H KP EAN D ELVTYS LG KFGQ RALDFYSI HVT

of A739V RESNH PVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQ DIILEHQ
of SEQ ID KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQ IVIWV
NO:2. NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWVVDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDWGIWYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDINLIAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL
D ISGFSKQYNCAFIWQ KDGVKKLN LYLI I NYFKGGKLR FKKIKPEAFEANR F
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIE KTLYNRRTRQDEPALFVALTFERREVLDSSNIKPM NLIGIDR
GEN IPAVIALTDP EGC P LSRFKDSLGNPTH I LR IGESYKEKQ RTIQAAKEVE
QRRAGGYSRKYASKAKNLADDMVRNIVRDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVILTAKLAYEGLPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQ EKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 37) L379R, a MQE IKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
substitution PEN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYVVE EFQKDPVGLMS RVA
of A708K, Q PAP KN IDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVN DKGKP
a deletion HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of P at position KVI KKN EKRLAN LKD IASAN GLAFP KITLPP Q PHTKEG I
EAYN NVVAQ IVIWV
793 and a NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVVVVDMVCNVKKL
substitution INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
of M771N LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
of SEQ ID TDWLRAKAS FVIEGLKEADKDE FCRCE LKLQKVVYGDLRGKPFAI EAE NS I L
NO:2 DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIE KTLYNRRTRQDEPALFVALTFERREVLDSSNIKPM NLIGIDR
G EN I PAVIALTD P EGC P LSRFKDS LG N PTH I LRIG ESYKE KQ RTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFNAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQN
VVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
VVQSFYRKKLKEVVVKPAV (SEQ ID NO: 38) substitution MOE IKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of L379R, PEN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYVVE EFQKDPVGLMS RVA
a Q PAP KN IDQ R KLIPVKDGNERLTSSGFAC S QCCQ
PLYVYKLEQVN DKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of A708K, RESNH PVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQ DIILEHQ
a deletion KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQ IVIWV
of P at NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWVVDMVCNVKKL

Descriptio Amino Acid Sequence n*
position INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
793 and a LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
substitution TDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL
of A739T DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
of SEQ ID YWINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
NO:2 LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GEN I PAVIALTD PEGCP LS RFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTTRDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 39) substitution MQE I KR I N KI RRR LVKDSNTKKAGKTGPMKTLLVRVMTPD LR ER LE N LRKK
of L379R, PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFOKDPVGLMSRVA
a 0 PAPKNIDQRKLIPVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of C477K, RESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYODIILEHO
a KVIKKN EKRLAN LKD IASANG LAFP KITLPPQ PHTKEG I
EAYN NVVAQ IVIWV
substitution NLNLWQKLKIGRDEAKPLQRLKGF PSFPLVEROANEVDWVVDMVCNVKKL
of A708K, INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
a deletion LH LE KKHGE DWGIWYD EAWE R I DKKVEGLS KH I KLE EE RRS EDAQS KAAL
of P at TDV'VLRAKASFVIEGLKEADKDEFKRCELKLQKVVYGSLRGKPFAIEAENSIL
position DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
793 and a YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
substitution LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
of D4895 GEN I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
of SEQ ID Q RRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
NO: 2.
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 40) substitution MQE I KR I N KI RRRLVKDSNTKKAGKTGPMKTLLVRVMTPD LRERLE N LRKK
of L379R, PENIPQPISNTSRANLNKLLTD'YTEMKKAILHVYWEEFQKDPVGLMSRVA
a CIPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCQPLYVYKLEQVNDKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of C477K, RESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQ
a KVIKKN EKRLAN LKD IASANG LAFP KITLPPQ PHTKEG I
EAYN NVVAQ IVIWV
substitution NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVEROANEVDWWDMVCNVKKL
of A708K, INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
a deletion LHLE KKHGEDWGIONDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
of P at TDVVLRAKASFVIEGLKEADKDEFKRCELKLQKVVYGDLRGKPFAIEAENSIL
position DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
793 and a 'YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS

Descriptio Amino Acid Sequence n*
substitution LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
of D732N GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
of SEQ ID QRRAGGYSRKYASKAKNLANDMVRNTARDLLYYAVTQDAMLIFENLSRG
NO:2. FGRQ
GKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTI NG KE L KVE GQ ITYYNRYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLN CG FE THAD EQAALN IARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 41) substitution MQ E I KRI N KIRRRLVKDS NTKKAGKTG P M KTLLVRVMTP DLRERL EN LRKK
of V71 1K PEN IP Q P I SNTS RANL N KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMSRVA
of SEQ ID QPAPKN I DQ RKL I PVKDG N ER LTSSGFACSQC CQ P LYVYKLE QVN DKGKP
NO:2.
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQ
KVI KKN E KR LANL KD IASANG LAF P KITLP PQ PHTKEG I EAYNNVVAQ IVIWV
N LN LWQ KLKI GRD EAKP LQ R L KGF PS FP LVE RQAN EVDVVVVDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
H LE KKHG EDWG KVY DEAVVERI DKKVEG LS KH I KLE EERRS EDAQS KAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDD P N LI I L PLAFGKRQ GRE FIWNDL LSL ETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEIWRTIQAAKEKE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLPSKTYLSKTLAQYTS KTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTI NGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNN DISSWTKG RSG EALS LL KKR FS H RPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNICTGNTDKRAFVE
TVVQSFYRKKLKEV'VVKPAV (SEQ ID NO: 42) substitution MQ E I KRI N KIRRRLVKDS NTKKAGKTG P M KTLLVRVMTP DLRERL EN LRKK
of L379R, PEN IP Q P I SNTS RANL N KLLTDYTEM KKAI LHVYWEE FQ KDPVG LMS RVA
a QPAPKN I DQ RKL I PVKDG N ER LTSSGFACSQC C Q P
LYVYKLE QVN DKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of C477K, RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQD1ILEHQ
a KVIKKNEKRLANLKD IASANG LAF P KITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
substitution NLN LWQ KLKI GRD EAKP LQ R L KGF PS FP LVE ROAN EVDVVVVDMVCNVKKL
of A708K, INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
a deletion LH L E KKHGE DWGINY DEAWER I DKKVEGLSKH I KLEE ERRS EDAQS KAAL
of P at TDVVLRAKASFVIEGLKEADKDEFKRCELKLQKVVYGDLRGKPFAIEAE NS IL
position DISGFSKQYNCAFIWQKDGVKKLNLYLI
INYFKGGKLRFKKIKPEAFEANRF
793 and a YIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
substitution LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
of Y797L GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
of SEQ ID QRRAGGYSRICY'ASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
NO: 2. FGRQ
GKRTFMAERQYTRMEDVVLTAKLAYEGLSKTLLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTI NC KE L KVE GO ITYYNRYKRQ N

Descriptio Amino Acid Sequence n*
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 43) 119:
MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
substitution PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA
of L379R, Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
a HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
substitution RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
of A708K KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
and a N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN
EVDVVVVDMVC NVKKL
deletion of INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
P at LHLEKKHGE DWGKVYDEAVVER I DKKVE GLSKH IKLEE
ERRSEDAQSKAAL
position TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ
KVVYGDLRGKPFAIEAE NS I L
793 of SEQ DISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIKPEAFEANR F
ID NO:2. YTV INKKSGEIVPMEVNFN FDD P NLI I LPLAFGKRQ GRE FIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQD EPALFVALTFE RR EVLDSSN IKPMNLIGID R
GENIPAVIALTDPEGC P LSRFKDSLGNPTH ILR IGESYKEKQRTIQAKKEVE
ORRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQ'YTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTI NC KE LKVE GO ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WOSFYRKKLKEVVVKPAV (SEQ ID NO: 44) substitution MQ E I KRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLE N LRKK
of L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
a Q PAPKN IDQRKL I PVKDGNER LTSSGFACSQCCQP
LYVYKLEQVNDKGKP
substitution HTNYFGRCNVSEH ERL ILLSP HKP EAN D E LVTYSLGKFGQRALD FYS I HVT
of C477K, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
a KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIVVV
substitution NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVVVVDMVCNVKKL
of A708K, INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
a deletion LHLEKKHGE DWGIWYDEAWER I DKKVE GLSKH IKLEE ERRSEDAOSKAAL
of P at TDWLRAKASFVIEGLKEADKDEFKRCELKLQKVVYGDLRGKPFAIEAE
NS IL
position DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
793 and a YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIWNDLLSLETGS
substitution LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
of M77 1N GENIPAVIALTDPEGCP LSRFKDSLGNPTH ILR IGESYKEKQRTIQAKKEVE
of SEQ ID QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
NO: 2.
FGROGKRTFNAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CO FTITSADYD RVLE KLKKTATGVVMTTI NO KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
C LN CG FETHAD EQAALN IARSWLFLRSQ EYKKYQTN KTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 45) Descriptio Amino Acid Sequence n*
substitution MQ E I KR I N KIRRRLVKDS NTKKAGKTG P M KTLLVRVMTP DLRERL EN LRKK
of A708K, PEN IPQ P I SNTS RANL N KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
a deletion Q PAPKN I DQRKL I PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
of P at HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
position RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
793 and a KVIKKNEKRLANLKD IASANGLAFPKITLPPQPHTKEGIEAYNNVVAQ IVIWV
substitution N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN EVDWVVDMVC NVKKL
of E386S INEKKEDGKVFWQNLAGYKRIDEALLPYLSSESDRKKGKKFARYQFGDLLL
of SEQ ID H LE KKHG EDVVG KVYDEAVVER I DKKVEG LS KH I KLE EER RS EDAQS KAAL
NO:2. TDVVLRAKAS FVI EG LKEADKDEFC RC ELKLQ
KVVYGDLRGKPFAIEAENS I L
DISGFSKOYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVI N KKSG E IVP MEVNFN FDD P N LI I L PLAFGKR Q GR E FIWNDL LSL ETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RREVLDSS N IKPM NLIG I D R
GEN I PAVIALTDPEGCP LS RFKDSLGN PTH ILR I GESYKE KQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CC FTITSADYD RVLE KLKKTATGVVMTTI NC KE LKVE GQ ITYYNRYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLN CGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 46) substitution MQ E I KR I N KIRRRLVKDS NTKKAGKTG P M KTLLVRVMTP DLRERL EN LRKK
of L379R, PE N IPQ P I SNTS RANL N KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
a Q PAPKN I DQRKL I PVKDG N ER LTSSGFACSQCCQ P
LYVYKLEQVN DKGKP
substitution HTNYFGRCNVSEHERL ILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of C477K, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
a KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIWV
substitution N LN LWQ KLKI GRD EAKP LORLKGFPS FP LVE ROAN EVDWNDMVC NVKKL
of A708K INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
and a LH L E KKHGE DWGKVYDEAWER I DKKVE GLS KH IKLEE
ERRS EDAQS KAAL
deletion of TDVVLRAKASFVIEGLKEADKDEFKRCELKLQKVVYGDLRGKPFAIEAE NS IL
P at DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
position YTVI N KKSG E IVP MEVNFN FDD P N LI I L PLAFGKRQ
GRE FIWNDL LSL ETGS
793 of SEQ LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RREVLDSS N IKPM NLIG I D R
ID NO:2, GEN I PAVIALTDPEGCP LS RFKDSLGN PTH ILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGROGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTI NC KE LKVE GO ITYYNRYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLN CG FETHAD EQAALN IARSVVLFL RSQ EYKKYQTN KTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 47) substitution MQ E I KR I N KIRRRLVKDS NTKKAGKTG P M KTLLVRVMTP DLRERL EN LRKK
of L792D PE N IPQ P I SNTS RANL N KLLTDYTEM KKAI LHVYWEE FQ KDPVG LMSRVA
of SEQ Q PAPKN I DQRKL I PVKDG N ER LTSSGFACSQCCQ P
LYVYKLEQVN DKGKP
NO:2.
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNH PVKP LEG IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H

Descriptio Amino Acid Sequence n*
KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
NLNLVVQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVVVDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDWGIWYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQICNYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGDPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 48) substitution MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of G791F PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVA
of SEQ ID OPAPKNIDORKLIPVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKOKP
NO:2.
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQ
KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
NLNLVVQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWNDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDVVGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEIWRTIQAAKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEFLPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF

TVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 49) substitution MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of A708K, PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA
a deletion QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
of P at HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKEGORALDFYSIHVT
position RESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQ
793 and a KVIKKNEKRLANLKD IASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
substitution NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVEROANEVDWNDMVCNVKKL
of A739V INEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDVVGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL

Descriptio Amino Acid Sequence n*
of SEQ ID DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
NO:2. YTVINKKSGEIVPMEVNFN FDDPNLI I
LPLAFGKRQGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTVRDLLYYAVTQDAMLIFENLSRG
FGROGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGVVMTTI NC KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 50) substitution MQ E IKR I N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
of L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
a QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of A708K, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
a deletion KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIVVV
of P at NLNLWQKLKIGRDEAKP LQRLKGFPS FP LVE ROAN
EVDWVVDMVC NVKKL
position INEKKEDGKVFVVONLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
793 and a LHLEKKHGEDWGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
substitution TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAENSIL
of A739V DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
of SEQ ID 'YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIWNDLLSLETGS
NO:2. LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN
IKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
ORRAGGYSRKYASKAKNLADDMVRNIVRDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGVVMTTI NC KE LKVE GO ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 51) substitution MQ E I KRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
of C477K, PEN IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
a Q PAPKN IDQRKL I
PVKDGNERLTSSGFACSOCCCIPLYV'YKLEQVNDKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of A708K RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQDI1LEHQ
and a KVI KKN E KR LANLKD !MANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
deletion of NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVVVVDMVCNVKKL
P at INEKKEDGKVFVVQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
position HLEKKHG
EDWGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
793 of SEQ TDVVLRAKASFVIEGLKEADKDEFKRCELKLQKVVYGDLRGKPFAIEAE NS IL
ID NO:2. DISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG

Descriptio Amino Acid Sequence n*
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQN
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 52) substitution MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of L249I PEN !POP I SNTS RANL N KLLTDYTEM KKAI LHVYWEE FQ
KDPVG LMS RVA
and a QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of M77 IN RESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIIIEHQK
of SEQ ID VIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIVVV
NO:2.
NLNLVVQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDVVGIONDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFNAERQYTRMEDVVLTAKLAYEGLPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWOS FYRKKLKEVVVKPAV (SEQ ID NO: 53) substitution MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
of V747K PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA
of SEQ ID QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
NO:2.
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT

KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDWWDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDVVGIONDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDWLRAKASFVIEGLKEADKDEFCRCELKLQIONYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKORTIQAAKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAKTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF

Descriptio Amino Acid Sequence n*
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWQSFYRKKLKEVVVKPAV (SEQ ID NO: 54) substitution MQ E I KR I N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
of L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
a OPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCIDPLYVYKLEQVNDKGKP
substitution HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
of C477K, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
a KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIVVV
substitution NLNLVVQKLKIGRDEAKPLQRLKGFPSFPLVEROANEVDWNDMVCNVKKL
of A708K, INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
a deletion LH LE KKHGE DWGKVYDEAWER I DKKVE GLSKH IKLEE ER RS EDAQSKAAL
of P at TDVVLRAKASFVIEGLKEADKDEFKRCELKLQKVVYGDLRGKPFAIEAE NS IL
position DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
793 and a YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIWNDLLSLETGS
substitution LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
of M779N GEN I PAVIALTDPEGCP LS RFKDSLGN PTH ILR I GESYKE KORTIQAKKEVE
of SEQ ID QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
NO: 2.
FGROGKRTFMAERQYTRNEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTI NC KE LKVE GQ ITYYNRYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 55) MQ EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA

PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIVVV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVEROANEVDWVVDMVCNVKKL
INEKKEDGIWFVVONLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDVVGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KWY'GD LRG KP FAI EAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAAKEVE
ORRAGGYSRICY'ASKAKNLADDMVRNTARDLLYYAVTODAMLIMENLSRG
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGOITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWOSFYRKKLKEVVVICPAV (SEQ ID NO: 56) 429: MQ
EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA

Descriptio Amino Acid Sequence n*
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT

D I I LE H Q
KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN EVDVIANDMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL

DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
ORRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGINMTTI NG KE LKVE GQ ITYYN RRKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVINKPAV (SEQ ID NO: 57) 430: MQ E IKRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V KVI KKN E KR LANLKD !MANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVVVVDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAOSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIVVNDLLSLETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RR EVLDSS N IKPM NLIGVD
RGEN IPAVIALTDPEGCPLSRFKDSLGN PTH ILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDINLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVIVITTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSVITIKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWQSFYRKKLKEVWKPAV (SEQ ID NO: 58) 431: MQ E IKRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
KVI KKN E KRLANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERGANEVDVVVVDMVCNVKKL

Descriptio Amino Acid Sequence n*
I658V, INEKKEDGKVFWQNLAGYKRQEALRPYLSSENDRKKGKKFARYQFGDLL

RSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTV INKKSGE IVPMEVNFN FDD P NLI I LPLAFGKRQ GRE FIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQD EPALFVALTFE RREVLDSSN IKPMNLIGVD
RGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEV
EQRRAGGYS RKYASKAKNLADDMVRNTARDLLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDVILTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTI N GKELKVEGQITYYN RRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWOSFYRKKLKEVWKPAV (SEQ ID NO: 59) 432: MQ EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I PVKDGNER LTSSGFACSQCCQP
LYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ

I658V, KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNWAQ IVIWV

EVDWNDMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
KHLEKKH GE DWGKVYD EAVVE R IDKKVEGLSKH IKLEEERRSEDAQSKAA
LTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSI
LD I SG FS KQYNCAFIWQ KDGVKKLNLYL I I NYF KGGKLRF KKI KPEAFEAN R
FYIVINKKSGEIVPMEVNFNFDDPNLI ILP LAFGKRQ GRE Fl WN DLLSLETG
SLKLANGRVIEKTLYNRRTRQ DE PALFVALTFER REVLDSSN I KPMNLIGV
DRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKE
VEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLS
RGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKT
C SNC GFTITSADYD RVLEKLKKTATGWMTTI NG KELKVEGQ ITYYNRRKR
QNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 60) 433: MQ E I KRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQVRALDFYSIHV
Y857R, TRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQD I
ILE H
I658V, Q KVIKKN E KRLAN LKD IASANGLAFPKITLPPQ P HTKEG I
EAYN NVVAQ IVI

KKL I N EKKEDGKVFWQ N LAGYKRQ EALRPYLSS E E DRKKG KKFARYQFG
DLLLHLEKKH GE DWGKVYD EAWER IDKKVEGLSKH IKLEEERRSEDAQSK
AALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAE
NSILDISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSGE IVPMEVNFNFD DP NLI ILPLAFGKRQGRE FIWND LLSLE

Descriptio Amino Acid Sequence n*
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTC S N CGFTITSADYD RVLEKLKKTATGVVMTTI NG KE LKVEGQ ITYYN RRK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAF
VETWQSFYRKKLKEVVVKPAV (SEQ ID NO: 61) 434: MQ E I KRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERL EN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIWV
L404K, NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVEROANEVDVVVVDMVCNVKKL

INEKKEDGKVFVVQNLAGYKRQEALRPYLSSENDRKKGKKFARYQFGDLL
KHLEKKHGEDWGIWYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAA
LTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSI
LD I SG FS KQYNCAFIWQ KDGVKKLNLYL I I NYF KGGKLRF KKIKPEAFEAN R
FYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETG
SLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN I KPMNLIGV
DRGEN IPAVIALTDPEGGPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKE
VEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLS
RGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKT
CSNCGFTITSADYD RVLEKLKKTATGVVMTTI NG KELKVEGQ ITYYN RRKR
QNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFV
ETWQSFYRKKLKEVWKPAV (SEQ ID NO: 62) 435: MQEIKRINKIRRRLVKDSNTKKAGICTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ
IGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQ
I658V, KVI KKN E KRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV

NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVWDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLL
LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFGRCELKLQ KVVYGDLRG KP FAIEAE NS IL
DISGFSKOYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVD
RGEN IPAVIALTDPEGCPLSRFKDSLGN PTH ILRIGESYKEKORTIOAKKEV
EQ R RAGGYS RKYASKAKN LADDMVR NTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ

Descriptio Amino Acid Sequence n*
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVWKPAV (SEQ ID NO: 63) 436: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PEN IP Q P I SNTS RANL N KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
F399L, N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN
EVDVVWDMVC NVKKL

INEKKEDGKVFWQNLAGYKRQEALRPYLSSENDRKKGKKFARYQLGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KWYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIVVNDLLSLETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RR EVLDSS N IKPM NLIGVD
RGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVWKPAV (SEQ ID NO: 64) 437: MQ E IKRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLE N LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVI KKN E KRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIVVV
F399L, NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVVVVDMVCNVKKL

INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLL
LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAOSKAAL
TDWLRAKASFVIEGLKEADKDEFSRCELKLQKVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKROGREFIVVNDLLSLETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RR EVLDSS N IKPM NLIGVD
RGEN IPAVIALTDPEGCPLSRFKDSLGN PTH ILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAEROYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWQSFYRKKLKEVWKPAV (SEQ ID NO: 65) Descriptio Amino Acid Sequence n*
438: MQ EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
F399L, N LN LVVQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN
EVDWWDMVC NVKKL

LGDLL
KHLEKKHGEDWGKVYDEAVVERIDKKVEGLSKH IKLEEERRSEDAQSKAA
LTDWLRAKAS FVI E GLKEADKDE FC RC E LKLQ KVVYGDLRG KP FAI EAE N S I
LD I SG FS KOYNCAFIWQ KDGVKKLNLYL I I NYF KGGKLRF KKI KPEAFEAN R
FYTVINKKSGEIVPMEVNFNFDDPNLI ILP LAFGKRQ GREFI WN DLLSLETG
SLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSN I KPMNLIGV
DRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKE
VEQ RRAGGYSRKYASKAKN LAD DMVRNTARDLLYYAVTQ DAMLI FE NLS
RGFGRQGKRTFMAE RQYTRM E DVVLTAKLAYE GLSKTYLSKTLAQYTS KT
C SNC GFTITSADYD RVLEKLKKTATGWMTTI NG KELKVEGQ ITYYN RRKR
QNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVINKPAV (SEQ ID NO: 66) 439: MQ EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIWV
F399L, N LN LWQ KLKI GRD EAKP LORLKGFPS FP LVE ROAN
EVDWWDMVC NVKKL
E386N, INEKKEDGKVFWQNLAGYKRQEALRPYLSSENDRKKGKKFARYQLGDLL
C477S, KHLEKKHGEDWGKVYDEAVVERIDKKVEGLSKH
IKLEEERRSEDAQSKAA

LTDWLRAKASFVIEGLKEADKDEFSRCELKLQKVVYGDLRGKPFAIEAENSI
LD I SG FS KQYNCAFIWQ KDGVKKLNLYL I I NYF KGGKLRF KKI KPEAFEAN R
FYTVINKKSGEIVPMEVNFNFDDPNLI ILPLAFGKROGREFIWN DLLSLETG
SLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSN I KPMNLIGV
DRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKE
VEQ RRAGGYSRKYASKAKN LAD DMVRNTARDLLYYAVTQ DAMLI FEN LS
RGFGRQGKRTFMAE RQYTRM EDWLTAKLAYEGLSKTYLSKTLAQYTS KT
C SNC GFTITSADYD RVLEKLKKTATGWMTTI NG KELKVEGO ITYYN RRKR
QNVVKDLSVELDRLSEESVNNDISSWIKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 67) 440: MQ EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG WS RVA
A708K, Q PAPKN IDQRKL I
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q

Descriptio Amino Acid Sequence n*
I658V, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIWV
F399L, NLNLWOKLKIGRDEAKPLORLKGFPSFPLVERDANEVDWWDMVCNVKKL

INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLL
LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKAS FVI EG LKEADKDEFC RC ELKLQ KlNYGD LRG KP FAI EAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVD
RGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTLLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 68) 441: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A7081C, OPAPKNIDORKLIPVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKOKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIVVV
F399L, N LN LVVQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN
EVDWWDMVC NVKKL
Y797L, INEKKEDGKVFVVQNLAGYKRQEALRPYLSSENDRKKGKKFARYQLGDLL

LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KWYGD LRG KP FAI EAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVD
RGEN IPAVIALTDPEGCPLSRFKDSLGN PTH ILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDVILTAKLAYEGLSKTLLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVINKPAV (SEQ ID NO: 69) 442: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGORALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIVVV
F399L, N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN
EVDWNDMVC NVKKL
Y797L, INEKKEDGKVFWQNLAGYKRQEALRPYLSSENDRKKGKKFARYQLGDLL
E386N, KHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAA
LTDVVLRAKASFVIEGLKEADKDEFSRCELKLQKVVYGDLRGKPFAIEAENSI

Descriptio Amino Acid Sequence n*
C477S, LD I SG FS KQYNCAFIWQ KDGVKKLNLYL I I NYF
KGGKLRF KKI KPEAFEAN R
L4041( FYTVINKKSGEIVPMEVNFNFDDPNLI ILPLAFGKRQGREFIWN
DLLS LETG
SLKLANGRVIEKTLYNRRTRQ DE PALFVALTFERREVLDSSN I KPMNLIGV
DRG EN IPAVIALTDPEGCPLS RF KDS LG NPTH ILR IG ESYKEKQ RTI QAKKE
VEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLS
RGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTLLSKTLAQYTSKT
C SNC GFTITSADYD RVLEKLKKTATGWMTTI NG KELKVEGQ ITYYNRRKR
QNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVVVISPAV (SEQ ID NO: 70) 443: MQ EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIVVV

EVDWNDMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LH LE KKHGE DWGKVYDEAWER I DKKVE GLSKH IKLEE ERRS EDAQSKAAL
TDWLRAKAS FVI EG LKEAD KD E FC RC ELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSG E IVPMEVNFN FDD P NLI I LPLAFGKRQ GRE FIVVNDLLSLETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RR EVLDSS N IKPM NLIGVD
RGEN IPAVIALTD P EGCP LS R FKDS LG N PTH ILRIGESYKEKQRTIQAKKEV
EQRRAGGYS RKYASKAKNLADDMVRNTARDLLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTLLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTI N GKELKVEGQITYYN RRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWOSFYRKKLKENNVKPAV (SEQ ID NO: 71) 444: MQ E I KRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMSRVA
A708K, Q PAPKN IDQRKL I
PVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H O
I658V, KVI KKN E KR LANLKD !MANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
Y797L, NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVIDVVVVDMVCNVKKL

INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
KH LE KKH GE DWGKVYD EAVVE R IDKKVEGLSKH IKLEEERRSEDAQSKAA
LTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSI
LD I SG FS KQYNCAFIWQ KDGVKKLNLYL I I NYF KGGKLRF KKI KPEAFEAN R
FYIVINKKSGEIVPMEVNENFDDPNLI ILPLAFGKRQGREFIVVN DLLS LETG
SLKLANGRVIEKTLYNRRTRQ DE PALFVALTFER REVLDSSN I KPMNLIGV
DRG EN IPAVIALTDPEGCPLS RF KDS LG NPTH ILRIG ESYKEKQ RTI QAKKE
VEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLS

Descriptio Amino Acid Sequence n*
RGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTLLSKTLAQYTSKT
CSNCGFTITSADYD RVLEKLKKTATGWMTTI NG KELKVEGQ ITYYN R R KR
QNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 72) 445: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IN) P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQKDPVG LMS RVA
A708K, QPAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQP
LYVYKLEQVNDKGKP
P793, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIWV
Y797L, N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN
EVDWWDMVC NVKKL

INEKKEDGKVFVVQNLAGYKRQEALRPYLSSENDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVD
RGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTLLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWOSFYRKKLKEVWKPAV (SEQ ID NO: 73) 446: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQP
LYVYKLEQVNDKGKP
P793, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
I658V, KVI KKN E KRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIVVV
Y797L, N LN LWQ KLKI GRD EAKP LORLKGFPS FP LVE ROAN
EVDWWDMVC NVKKL
E386N, INEKKEDGKVFWQNLAGYKRQEALRPYLSSENDRKKGKKFARYQFGDLL
C477S, KHLEKKHGEDWGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAA

LTDVVLRAKASFVIEGLKEADKDEFSRCELKLQKWYGDLRGKPFAIEAENSI
LD I SG FS KeYNCAFIWQ KDGVKKLNLYL I I NYF KGGKLRF KKI KPEAFEAN R
FYWINKKSGEIVPMEVNFNFDDPNLI ILPLAFGKROGREFIWN DLLSLETG
SLKLANGRVIEKTLYNRRTRODEPALFVALTFERREVLDSSN I KPMNLIGV
DRGENIPAVIALTDPEGCPLSRAWSLGNPTHILRIGESYKEKORTIOAKKE
VEQ RRAGGYSRKYASKAKN LAD DMVRNTARDLLYYAVTO DAMLI FE NLS
RGFGROGKRTFMAERQYTRMEDWLTAKLAYEGLSKTLLSKTLAWTSKT
C SNC GFTITSADYD RVLEKLKKTATGWMTTI NG KELKVEGO ITYYN RRKR
Q NVVKDLSVELDRLSEESVN NDISSWTKGRSGEALSLLKKR FSHR PVC)EK

Descriptio Amino Acid Sequence n*
FVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVWKPAV (SEQ ID NO: 74) 447: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, OPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q

EAYNNVVAQ IVIWV
NLNLVVQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSENDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGROGKRTFMAERQYTRMEDWLTAKLAYEGLSICTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGVVMTTI NG KE LKVE GQ ITYYN RRKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 75) 448: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Y857R, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
E386N, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIWV

NVKKL
INEKKEDGKVFWONLAGYKRQEALRPYLSSENDRKKGKKFARYQFGDLL
KHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAA
LTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSI
LDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANR
FYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVN DLLSLETG
SLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSN I KP MNLIG ID
RGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEV
EQRRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAM LI FENLSR
GFGRQGKRTFIVIAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGOITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWOS FYRKKLKEVVVKPAV (SEQ ID NO: 76) 449: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMSRVA

Descriptio Amino Acid Sequence n*
A708K, QPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
D732N, RESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQ
E385P, KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVIWV

EVDVIANDMVC NVKKL
INEKKEDGKVFWQ NLAGYKRQEALRPYLSSP EDRKKGKKFARYQ FGDLL
LHLEKKHGEDWGKVYDEAWER I DKKVE GLSKH IKLEE ERRSEDAQSKAAL

DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCP LSRFKDSLGNPTH ILR IGESYKEKQRTIQAKKEVE

FGRQ GKRTFMAE RQYTRME DVVLTAKLAYEG LS KTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGINMTTI NG KE LKVE GQ ITYYNRRKRQN
VVKD LSVE LDRLS E ESVN ND IS SVVTKGRSGEALS LLKKRFSH RPVQE KFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVINKPAV (SEQ ID NO: 77) 450: MQ E I KRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I PVKDGNER LTSSGFACSQCCQP
LYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
D732N, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
E385P, KVI KKN E KR LANLKD !MANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
Y857R, N LN LWQ KLKI GRD EAKP LORLKGFPS FP LVE ROAN
EVDVINVDMVC NVKKL

INEKKEDGKVFVVQNLAGYKRQEALRPYLSSPEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWER I DKKVE GLSKH IKLEE ERRSEDAOSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIVVNDLLSLETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RR EVLDSS N I KPM NLIGVD
RGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEV
EQRRAGGYS RKYASKAKN LAN DMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDINLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVIVITTINGKELKVEGQITYYNRRKRQ
NVVKDLSVE LDRLS E ESVNN DISSWTKG RSG EALS LLKKRFS H R PVQ E KF
VC LNC GFETHAD EQAALNIARSVVLFLRSQ EYKKYQTN KTTG NTD KRAFVE
TWOS FYRKKLKEVWKPAV (SEQ ID NO: 78) 451: MQ E I KRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I PVKDGNER LTSSGFACSQC C Q P
LYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERL ILLSP HKP
EANDELVTYSLGKFGQRALDFYSIHVT
D732N, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
E385P, KVI KKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
Y857R, N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE RQAN
EVDVVVVDMVC NVKKL

Descriptio Amino Acid Sequence n*
I658V, INEKKEDGKVFWQNLAGYKRQEALRPYLSSPEDRKKGKKFARYQLGDLL

LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGVD
RGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LAN DMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWOSFYRKKLKEVWKPAV (SEQ ID NO: 79) 452: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
D732N, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
E385P, KVI KKN E KRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNWAQ IVIWV
Y857R, N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE RQAN
EVDWA/DMVC NVKKL
I658V, INEKKEDGKVFWQNLAGYKRQEALRPYLSSPNDRKKGKKFARYQFGDLL

LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KWYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKROGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGVD
RGEN IPAVIALTDPEGCPLSRFKDSLGN PTH ILRIGESYKEKORTIOAKKEV
EQ RRAGGYS RKYASKAKN LAN DMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWOS FYRKKLKEVWKPAV (SEQ ID NO: 80) 453: MQ E IKRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMSRVA
A708K, Q PAPKN IDQRKL I PVKDGNER LTSSGFACSQCCQ P
LYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
D73N, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
E385P, KVI KKN E KRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIVVV
Y857R, NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVWDMVCNVKKL
I658V, INEKKEDGKVFWQNLAGYKRQEALRPYLSSPEDRKKGKKFARYQFGDLL

IKLEEERRSEDAQSKAA
LTDWLRAKAS FVI E GLKEADKDE FC RCE LKLQ KWYGDLRG KP FAI EAE N S I
LO I SG FS KQYNCAFIWQ KDGVKKLNLYL I I NYF KGGKLRF KKI KPEAFEAN R
FYTVINKKSGEIVPMEVNFNFDDPNLI ILPLAFGKRQGREFIWN DLLSLETG

Descriptio Amino Acid Sequence n*
SLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSN I KP MNLIGV
DRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKE
VEQRRAGGYSRKYASKAKNLANDMVRNTARDLLYYAVTQDAMLIFENLS
RGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKT
C SNC GFTITSADYD RVLEKLKKTATGWMTTI NG KELKVEGQ ITYYN RRKR
ONVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 81) 454: MCIEIKRINKIRRRLVKDSNTKKAGKTGPMKTUNRVMTPDLRERLENLRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQ P
LYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
T620P, RESNHPVKPLEQ
IGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHK
E385P, KVI KKN E KR LANLKD
IASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
Y857R, NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVVVDMVCNVKKL

INEKKEDGKVFVVQNLAGYKRQEALRPYLSSPEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KWYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIVVNDLLSLETGS
LKLANGRVIEKPLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEIWRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGVVMTTI NG KE LKVE GQ ITYYN RRKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 82) 455: MQ E I KRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA
A708K, Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQ P
LYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGORALDFYSIHVT
T620P, RESNHPVKPLEQ
IGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHK
E385P, KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
Y857R, NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVWDMVCNVKKL
I658V, INEKKEDGKVFWQNLAGYKRQEALRPYLSSPEDRKKGKKFARYQFGDLL

LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKOYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIVVNDLLSLETGS
LKLANG RVIE KPLYN RRTRQ D E PALFVALTFE RR EVLDSS N IKP MN LIGVD
RGENIPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKORTIOAKKEV
EQRRAGGYS RKYASKAKN LADDMVR NTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ

Descriptio Amino Acid Sequence n*
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVWKPAV (SEQ ID NO: 83) 456: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PEN IP Q P I SNTS RANL N KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
T620P, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H K
E385P, KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIWV
Y857R, N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN
EVDVVWDMVC NVKKL
I658V, INEKKEDGKVFWQNLAGYKRQEALRPYLSSPNDRKKGKKFARYQFGDLL
E386N, LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL

KWYGDLRGKPFAIEAE NS I L
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIVVNDLLSLETGS
LKLANG RVIE KPLYN RRTRQ D E PALFVALTFE RR EVLDSS N IKP MN LIGVD
RGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVWKPAV (SEQ ID NO: 84) 457: MQ E IKRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP DLRERLEN LRKK
L379R, PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE
FQ KDPVG LMS RVA
A708K, QPAPKN IDQRKLI
PVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
T620P, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H K
E385P, KVI KKN E KRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVIVVV
Y857R, NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVVVVDMVCNVKKL
I658V, INEKKEDGKVFWQNLAGYKRQEALRPYLSSPEDRKKGKKFARYQLGDLL
F399L, LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAOSKAAL

NS I L
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKROGREFIVVNDLLSLETGS
LKLANG RVIE KPLYN RRTRQ D E PALFVALTFE RR EVLDSS N IKP MN LIGVD
RGEN IPAVIALTDPEGCPLSRFKDSLGN PTH ILRIGESYKEKQRTIQAKKEV
EQ RRAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSR
GFGRQGKRTFMAEROYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TWQSFYRKKLKEVWKPAV (SEQ ID NO: 85) Descriptio Amino Acid Sequence n*
458: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA
A708K, OPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP

HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
T620P, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H K
E385P, KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I
EAYNNVVAQ IVI WV
Y857R, N LN LVVQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN
EVDWVVDMVC NVKKL
I658V, INEKKEDGKVFWQNLAGYKRQEALRPYLSSPEDRKKGKKFARYQFGDLL
L404K, KH L E KKH GE DWGKVYD EAVVE R IDKKVEGLSKH
IKLEEERRSEDAQSKAA

LTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSI
LDISGFSKOYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANR
FYN! N KKSGEIVPMEVN F N F DDP N LI I LP LAFG KRQ GRE Fl WN DLLS LETG
SLKLANGRVIEKPLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGV
DRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKE
VEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLS
RGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKT
CSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRRKR
QNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVINKPAV (SEQ ID NO: 86) 459: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVA
A708K, QPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
T620P, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ

Y857R, KVI KKN E KR LANL KD IASANG LAF PKITLP PQ PHTKEG
I EAYNNVVAQ IVI WV
I658V, N LN LWQ KLKI GRD EAKP LORLKGFPS FP LVE ROAN
EVDWWDMVC NVKKL

INEKKEDGKVFWQNLAGYKRQEALRPYLSSENDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKAS FVI EG LKEADKD E FC RC ELKLQ KWYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIVVNDLLSLETGS
LKLANGRVIEKPLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVD
RGEN IPAVIALTD P EGCP LS R FKDS LG N PTH ILR I GESYKE KORTIQAKKEV
EQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSR
GFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRKRQ
NVVKDLSVELDRLSEESVNNDISSVITTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 87) 460: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R, PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA
A708K, QPAPKNIDQRKLIPVKDGNERLTSSGFACSOCCQPLYVYKLEQVNDKGKP
P793_, HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
T620P, RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H K

Descriptio Amino Acid Sequence n*
E385P, KVIKKNEKRLANLKD
IASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV

EVDWWDMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSPEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWER I DKKVE GLSKH IKLEE ER RSEDAQSKAAL
TDVVLRAKAS FVI EG LKEADKDEFC RC ELKLQ KlNYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIWNDLLSLETGS
LKLANGRVIEKPLYN RRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKORTIQAKKEVE
Q RRAGGYSRKYAS KAKN LAD DMVRNTARDLLYYAVTQ DAM LI FEN LS RG
FGRQ GKRTFMAE RQYTRME DVVLTAKLAYEG LS KTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGWMTTI NC KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 88) LENLRKKP
EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYWE E FQ KDPVGLM SRVAQ
PAPKNIDORKLIPVKDONERLTSSGFACSOCCOPLYVYKLEQVNDKGKPH

ESN HPVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQD I ILEHQK
VI KKN EKR LAN LKD IASAN GLAFP KITLPPQ PHTKEG I EAYN NVVAQ IVIVIN

INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWER I DKKVE GLSKH IKLEE ER RSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KWYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIKPEAFEANR F
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
Q RRAGGYSRKYAS KAKN LAD DMVRNTARDLLYYAVTQ DAM LI FEN LS RG
FGRQ GKRTFMAE RQYTRME DVVLTAKLAYEG LS KTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTI NC KE LKVE GQ ITYYN RYKRQ N
VVKD LSVE LDRLS E ESVN ND IS SVVTKGRSGEALS LLKKRFSH RPVQE KFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 89) EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE FQ KDPVG LMS RVA
Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGORALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN EVDWNDMVC NVKKL
IN EKKEDGKVFWQ N LAGYKRQEALR PYLS SE E D R KKGKKFARYQ FG DLL
LHLEKKHGEDWGKVYDEAWER I DKKVE GLSKH IKLEE ERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL

Descriptio Amino Acid Sequence n*
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVI N KKSG E IVP MEVNFN FDD P N LI I L PLAFGKR Q GR E FIWNDL LSL ETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RREVLDSS N IKPM NLIG I D R
GEN I PAVIALTDPEGCP LS RFKDSLGN PTH ILR I GESYKE KQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGROGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTI NC KE L KVE GQ ITYYNRYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 90) DLRERL EN LRKK
PE N IP Q P I SNTS RANL N KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
Q PAPKN I DQRKL I PVKDG N ER LTSSGFACSQCCQ P LYVYKLEQVN DKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANL KD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIVVV
N LN LWQ KLKI GRD EAKP LQRL KGF PS FP LVE ROAN EVDWNDMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LH L E KKHGE DINGKVYDEAWER I DKKVE GLS KH IKLEE ERRS EDAQS KAAL
TDINLRAKAS FVI EG LKEADKD E FC RC ELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVI N KKSG E IVP MEVNFN FDD P N LI I L PLAFGKRQ GRE FIVVNDL LSL ETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RR EVLDSS N IKPM NLIG I D R
GEN I PAVIALTDPEGCP LS RFKDSLGN PTH ILR I GESYKE KQRTIQAKKEVE
ORRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTODAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTI NC KE L KVE GO ITYYNRYKRQN
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 91) DLRERL EN LRKK
PE N IP Q P I SNTS RANL N KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
Q PAPKN I DQRKL I PVKDG N ER LTSSGFACSQCCQ P LYVYKLEQVN DKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANL KD !MANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVIDVINVDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LH L E KKHGE DWGKVYDEAWER I DKKVE GLS KH IKLEE ERRS EDAQS KAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVI N KKSG E IVP MEVNFN FDD P N LI I L PLAFGKRQ GRE FIWNDL LSL ETGS
LKLANG RVIE KTLYNRRTRQD EPALFVALTFE RR EVLDSS N IKPM NLIG I D R
GEN I PAVIALTDPEGC P LS RFKDSLGN PTH ILR I GESYKE KQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG

Descriptio Amino Acid Sequence n*
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGWMTTI NC KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 92) MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQ P LYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
NLNLVVQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CC FTITSADYD RVLE KLKKTATGVVMTTI NC KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 93) MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE FQ KDPVG LMS RVA
Q PAPKN IDQRKL I PVKDGNER LTSSGFACSQCCQ P LYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDWWDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSIL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKROGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKORTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGVVMTTI NC KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV

Descriptio Amino Acid Sequence n*
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVWKPAV (SEQ ID NO: 94) MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVA
OPAPKNIDQRKLIPVKDGNERLTMSSGFACSOCCOPLYVYKLEQVNDKG
KPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIH
VTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILE
HQKVIKKNEKRLANLKDIASANGLAFPKITLPPQ P HTKEG I EAYN NVVAQ IVI
VVVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVWDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFG
DLL LH L E KKH GE DWGKVYD EAWER IDKKVEGLSKH IKLEE ERRS EDAQS K
AALTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAE
N S I LD ISGF SKQYN CAF IVVQ KDGVKKL N LYLI I NYFKGG KLRFKKIKP EAF EA
NRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSROFGROGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYK
RQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALN IARSWLFLRSQEYKKYQTNKTTGNTDKRAF
VETWQSFYRKKLKEVVVKPAV (SEQ ID NO: 95) MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVA
QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANL KD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIVVV
N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN EVDWVVDMVC NVKKL
INEKKEDGKVFWONLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKAS FVI EG LKEAD KD E FC RC ELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
ORRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTODAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
NCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQN
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 96) MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFOKDPVGLMSRVA

Descriptio Amino Acid Sequence n*
Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQ P LYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN EVDVIANDMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL

DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCP LSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
ORRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGINMTTI NC KE LKVE GO ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 97) DLRERLEN LRKK
PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQP LYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANLKD !MANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVINVDMVCNVKKL
INEKKEDGKVFVVQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAOSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCP LSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CC FTITSADYD RVLE KLKKTATGWMTTI NC KE LKVE GO ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
C LN CG FETHAD EQAALN IARSWLFLRSQ EYKKYQTN KTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 98) DLRERLEN LRKK
PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQ P LYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERGANEVDVVVVDMVCNVKKL

Descriptio Amino Acid Sequence n*
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KWYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CO FTITSADYD RVLE KLKKTATGVVMTTI NO KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
CLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 99) MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQP LYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNWAQ IVIWV
N LN LWQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN EVDWA/DMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KWYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKROGREFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCP LSRFKDSLGNPTH ILRIGESYKEKORTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGVVMTTI NC KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
C LN CG FETHAD EQAALN IARSWLFLRSQ EYKKYQTN KTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 100) DLRERLEN LRKK
PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE FQ KDPVG LMS RVA
Q PAPKN IDQRKL I PVKDGNERLTSSGFACSQCCQ P LYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVIKKNEKRLANLKD IASANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIVVV
NLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWVVDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDWLRAKASFVIEGLKEADKDEFCRCELKLQ KWYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKROGREFIVVNDLLSLETGS

Descriptio Amino Acid Sequence n*
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCP LSRFKDSLGNPTH ILRIGESYKEKQRTIQAKKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG
FGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSKTCS
N CG FTITSADYD RVLE KLKKTATGVVMTTI NG KE LKVE GQ ITYYN RYKRQ N
VVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKFV
C LN CG FETHAD EQAALN IARSVVLFLRSQ EYKKYQTN KTTGNTDKRAFVET
WQSFYRKKLKEVVVKPAV (SEQ ID NO: 101) 387:
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
NTSB EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E
FQ KDPVGLM SRVAQ
swa from PASKKIDQ NKLKPEM DE KGNLTTAGFACSQCGQP LFVYKLEQVSEKGKA
p SEQ ID YTNYFGRCNVAE
HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
NO:1 TRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQD I
ILEH
Q KVIKKN E KR LAN LKD IASANGLAFPKITLPPQ P HTKEG I EAYN NVVAQ IVI
WVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDINVVDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
NSI LDISGFSKQYNCAF IWQKDGVKKLN LYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSGE IVPMEVNFNFDDP NLI ILPLAFGKRQGREFIINNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSSN IKPMNL I
GIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTCS N CGFTITSADYD RVLEKLKKTATGVVMTTI NG KE LKVEGQ ITYYN RYK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYOTNKTTGNTDKRAF
VETWQSFYRKKLKEVWKPAV (SEQ ID NO: 102) 395:
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
Helical 1B EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E FQ KDPVGLM SRVAQ
swa from PAPKN IDQRKL IPVKDGN ERLTSSGFACSQCCQP LYVYKLEQVNDKGKPH
p SEQ ID TNYFGRC NVS EH E
RLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTK
NO:1 ESTH PVKP LAQIAGN RYASGPVG KALSDACM GTIASFLS KYQ
DI I IEHQKVV
KGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMW
VN LN LWQ KLKLSRDDAKPLLRLKG FPSF PLVE ROAN EVDWVVDMVC NVK
KLI N EKKEDGKVFWQ N LAGYKRQ EALR PYLS SE E D RKKGKKFARYQFGD
LLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKH IKLEEERRSEDAQSKA
ALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAEN
S I LD I SG FSKQYNCAF IWQ KDGVKKLN LYL I I NYFKGG KLRFKKIKP EAFEAN
RFYTVINKKSGE IVPMEVNFNFDDPNLI ILPLAFGKRQGREFIWNDLLSLET
GSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN I KPMNLIGI
DRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKE
VEQ RRAGGYSR KYASKAKN LAD DMVRNTAR DLLYYAVTQ DAMLI FE NLS
RGFGRQGKRTFMAE RQYTRM EDWLTAKLAYEGLSKTYLSKTLAQYTS KT
CSNCGFTITSADYD RVLEKLKKTATGWMTTI NG KELKVEGQ ITYYN RYKR

Descriptio Amino Acid Sequence n*
QNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEV1NKPAV (SEQ ID NO: 103) 485: QEIKRINKIRRRLVKOSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
Helical 1B EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E FQ KDPVGLM SRVAQ
swa from PAPKN IDQRKL IPVKDGN ERLTSS GFACSQCCQ P LYVYKLEQVNDKGKPH
p SEQ ID TNYFGRC NVS EH E RL ILLSP H KP EAN D E LVTYS
LGKFGQ RALD FYSIHVTK
NO:1 ESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVV
KGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMW
VNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVWVDMVCNVK
KLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGD
LLLHLEKKHG EDWG KVYDEAVVE RID KKVEG LS KH IKLE EERRSE DAQSKA
ALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAEN
SILDISGFSKQYNCAF IWQ KDGVKKLNLYL I INYFKGGKLRFKKIKP EAFEAN
RFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLET
GSLKLAN GRVIEKTLYNR RTRQ D EPALFVALTFERREVLDSSN I KPMNLIG
VDRGENIPAVIALTDPE GCPLSRFKDSLGN PTH ILR IGESYKEKQRTIQAKK
EVEQR RAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENL
SRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSK
TCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQ ITYYNRRK
RQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALN IARSVVLFLRSQEYKKYQTNKTTGNTDKRAF
VETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 104) 486: QEIKRINKI RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
Helical 1B EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E FQ KDPVGLM SRVAQ
swa from PAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKPH
p SEQ ID TNYFGRCNVS EH E RL ILLSP H KP EAN D E LVTYS
LGKFGQ RALD FYSIHVTK
NO:1 ESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVV
KGNQKRLESLRELAGKENLEYPSVTLPPCIPHTKEGVDAYNEVIARVRMW
VNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVEROANEVDVINVDMVCNVK
KLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGD
LLKHLEKKHGEDWGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKA
ALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAEN
S I LD I SG FSKQYNCAF IWQ KDGVKKLN LYL I I NYFKGG KLRFKKIKP EAFEAN
RFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLET
GSLKLAN GRVIEKTLYNR RTRQ D EPALFVALTFERREVLDSSN I KPMNLIG
VDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKORTIQAKK
EVEQR RAGGYS RKYASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENL
SRG FGRQ GKRTFMAERQYTRM E DVVLTAKLAYEGLS KTYLS KTLAQYTS K
TCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQ ITYYNRRK
RQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALN IARSVVLFLRSQEYKKYQTNKTTGNTDKRAF
VETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 105) Descriptio Amino Acid Sequence n*
487: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
Helical 1B ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQ
swa from PAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKPH
p SEQ ID
TNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVIK
Nal ESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVV
KGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMW
VNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDINVVDMVCNVK
KLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGD
LLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKA
ALTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAEN
SILDISGFSKOYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEAN
RFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLET
GSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIG
VDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKElaRTIQAKK
EVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENL
SRGFGRQGKRTFMAERQYTRMEDINLTAKLAYEGLSKTYLSKTLAQYTSK
TCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKR
QNVVKDLSVELDRLSEESVNNDISSINTKGRSGEALSLLKKRFSHRPVQEK
FVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV
ETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 106) 488: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
NTSB and ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ
Helical 1B PASKKIDQ NKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKA
swa from YTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
p SEQ ID
TKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQK
NO:1 VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVVVDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFG
DLLLHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
NSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
NRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEIWRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAF
VETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 107) 489: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
NTSB and ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ
Helical 1B PASKKIDQ NKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKA
swa from YTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
p TKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQK

Descriptio Amino Acid Sequence n*
SEQ ID
VVKGNOKRLESLRELAGKENLEYPSVTLPPOPHTKEGVDAYNEVIARVRM
NO:1 WVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVVVDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
NSILDISGFSKQYNCAFIVVQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
NRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRK
RQNVVKDLSVELDRLSEESVNNDISSINTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAF
VETWQSFYRKKLKEVVVKPAV (SEQ ID NO: 108) 490: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
NTSB and ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ
Helical 1B PASKKIDQNKLKPEMDEKGNLTTAGFACSQCGOPLFVYKLEQVSEKGKA
swa from YTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
p SEQ ID
TKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQK
NO:1 VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWVVDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLKHLEKKHGEDWGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQS
KAALTDINLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEA
ENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFE
ANRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSL
ETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRRK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAF
VETWQSFYRKKLKEVVVKPAV (SEQ ID NO: 109) 491: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
NTSB and ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ
Helical 1B PASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKA
swa from YTNYFGRCNVAEHEKLILLAOLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
p SEQ ID
TKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQK
NO:1 VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM

KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE

Descriptio Amino Acid Sequence n*
NSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
NRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTS
KTC S N CGFTITSADYD RVLEKLKKTATGVVMTTI NG KE LKVEGQ ITYYN RYK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAF
VETWQSFYRKKLKEVVVKPAV (SEQ ID NO: 110) 494:
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
NTSB EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E
FQ KDPVGLM SRVAQ
swa from PASKKIDQ NKLKPEM DE KGNLTTAGFACSQCGQPLFVYKLEQVSEKGKA
p SEQ ID YTNYFGRCNVAE
HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
NO:1 TRESN HPVKPLEQIGGNSCASGPVGKALS DACMGAVASFLTKYQD
I ILEH
Q KVIKKN E KR LAN LKD IASANGLAFPKITLPPQ P HTKEG I EAYN NVVAQ IVI

KKLINEKKEDGKVFWONLAGYKRQEALRPYLSSEEDRKKOKKFARYQLG
DLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKH IKLEEERRSEDAQSK
AALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
NSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
NRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTC S N CGFTITSADYD RVLEKLKKTATGVVMTTI NG KE LKVEGQ ITYYN RYK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAF
VETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 111) 328: MQ E IKRI N KIRRRLVKDS NTKKAGKTG PM KTLLVRVMTP
DLRERLEN LRKK

FQ KDPVG LMS RVA
QPAPKN IDQRKLI PVKDGNERLTSSGFACSOCCOPLYVYKLEQVNDKGKP
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
KVI KKN E KR LANLKD !MANG LAF PKITLP PQ PHTKEG I EAYNNVVAQ IVIWV
NLNLWQKLKIGRDEAKPLORLKGFPSFPLVEROANEVDVVVVDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLL
HLEKKHGEDWGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKASFVIEGLKEADKDEFCRCELKLQ KVVYGD LRG KP FAI EAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDPNLI I LPLAFGKRQGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAAKEVE
QRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG

Descriptio Amino Acid Sequence n*
FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGINMTTINGKELKVEGQITYYNRYKRQ
NVVKDLGVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVINKPAV (SEQ ID NO: 112) 388: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R+A7 PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVA
08K+
QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
[P793] + HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
Xi RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ
D I I LE H Q
Helical2 KVIKKNEKRLANLKD
IASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
swap NLNLVVQKLKIGRDEAKPLQRLKGFPSFPVVERRENEVDWINNTINEVKKLI
DAKRDMGRVFVVSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPA
KRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAE
DAQSKAVLTDVVLRAKASFVLERLKEMDEKEFYACEIQLQKVVYGDLRGNP
FAVEAENSILDISGFSKQYNCAFIVVQKDGVKKLNLYLIINYFKGGKLRFKKIK
PEAFEANRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWN
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIK
PMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQR
TIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAM
LIFENLSRGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLA
QYTSKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ ITYY
NRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHR
PVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNT
DKRAFVETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 113) 389: MQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R+A7 PENIPQPISNTSRANLNKLLTDYTEMKKAILHVYINEEFQKDPVGLMSRVA
08K+
QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCOPLYVYKLEQVNDKGKP
[P793] +
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
X1 RuvC1 RESNHPVKPLEQ IGG N SCASGPVGKALSDACMGAVAS FLTKYQ D I I LE H Q
swap KVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIVVV
NLNLVVQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDIMNDMVCNVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAAL
TDVVLRAKAS FVI EG LKEADKDEFC RC ELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPVNLIGVDR
GENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVE
ORRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFENLSRG
FGRQGKRTFMTERQYTKIVIEDINLTAKLAYEGLTSKTYLSKTLAQYTSKTC
SNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYKRQ
NVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKF
VCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRAFVE
TVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 114) Descriptio Amino Acid Sequence n*
390: MQ
EIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKK
L379R+A7 PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYVVEE FQ KDPVG LMS RVA
08K+ Q PAPKN IDQRKL I
PVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKP
[P793] +
HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVT
X1 RuvC2 RESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQ DI ILEH Q
swap KVIKKNEKRLANLKD
IASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWV
N LN LVVQ KLKI GRD EAKP LQRLKGFPS FP LVE ROAN EVDWWDMVC NVKKL
INEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQFGDLL
LHLEKKHGEDWGKVYDEAWER I DKKVE GLSKH IKLEE ER RSEDAQSKAAL
TDVVLRAKAS FVI EG LKEADKDEFC RC ELKLQ KVVYGDLRGKPFAIEAE NS IL
DISGFSKOYNCAFIWQKDGVKKLNLYLIINYFKGOKLRFKKIKPEAFEANRF
YTVINKKSGEIVPMEVNFN FDDP NLI I LPLAFGKRQ GR EFIVVNDLLSLETGS
LKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN IKPMNLIGIDR
GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVE
Q RRAGGYSRKYAS KAKN LAD DMVRNTARDLLYYAVTQ DAM LI FEN LS RG
FGRQ GKRTFMAE RQ YTRME DWLTAKLAYEG LS KTYLSKTLAQYTSKTCS
N CC FTITSADYD RVLE KLKKTATGVVMTTI NC KE LKVE GQ ITYYN RYKRQ N
VVKD LSVE LDRLS E ESVN ND IS SVVTKGRSGEALS LLKKRFSH RPVQE KFV
CLNCGFETHADEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAF
YKRRLKEVVVKPNA (SEQ ID NO: 115) 514: QEIKRINKI RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
AH817 in EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E FQ KDPVGLM SRVAQ

LFVYKLEQVSEKGKA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
WVNLN LWQKLKLS RD DAKPLLRLKG FPSFP LVE RQANEVDVVWDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKH IKLEEERRSEDAQSK
AALTDVVLRAKASFVI EGLKEADKDE FC RC E LKLQ KVVYGDLRG KPFAI EAE
NSI LDISGFSKQYNCAF I VVQ KDGVKKLN LYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSSN IKPMNL I
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKCIRTIQAK
KEVEQRRAGGYSR KYASKAKN LAD DMVRNTAR DLLYYAVTQDAM LI FEN
LS RG FGRQGKRTFMAERQYTRM EDVVLTAKLAYEGLSKTYLS KTLAQYTS
KTCSNCGFTIHTSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRY
KRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVID
EKFVC LN CC FETHADEQAALN IARSWLF LRSQ EYKKYQTNKTTGNTDKRA
FVETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 217) 515: QEIKRINKI RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
AP793 in EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E
FQ KDPVGLM SRVAQ

LFVYKLEQVSEKGKA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK

Desc riptio Amino Acid Sequence n*
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVWDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
NSILDISGFSKQYNCAFIVVQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
NRFYTVIN KKSGE IVPMEVNFNFDDPNLI ILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRY
KRONVVKDLSVELDRLSEESVNNDISSWTKORSGEALSLLKKRFSHRPVQ
EKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRA
FVETVVQSFYRKKLKEVVVKPAV (SEQ lin NO: 218) 516: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
L307H in ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ

PASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKA
YTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNHNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVVVDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIONDEAWERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
NSILDISGFSKQYNCAFIVVQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
NRFYTVIN KKSGE IVPMEVNFNFDDPNLI ILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYK
RQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAF
VETVVQSFYRKKLKEVVVKPAV (SEQ 1:13 NO: 219) 517: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
AA224 in ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ

PASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKA
YTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTHPVKPLAQIAGNRYASGAPVGKALSDACMGTIASFLSKYQDIIIEHQ
KVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVR
MVINNLNLVVQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVWVDMVGN
VKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQL
GDLLLHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQ
SKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIE

Descriptio Amino Acid Sequence n*
AE N S I LD ISGFSKQYNCAF IWQ KDGVKKLN LYL I INYF KGG KLRF KKI KP EAF
EAN RFYTVINKKSGE IVPMEVNFNFDDPNL I ILPLAFGKRQGREF IWNDLLS
LETGSLKLANGRVIE KTLYN RRTRQ DE PALFVALTFE RREVLDSSN IKPM N
LIGVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQ
AKKEVEQ RRAGGYS RKYASKAKN LAD DMVRNTARDLLYYAVTQ DAM LIF
EN LS RGFGRQGKRTFMAE RQYTRM EDWLTAKLAYE GLS KTYLSKTLAQY
TS KTC S N CC FTITSADYDRVLE KLKKTATGWMTTI NC KE LKVEGQ ITYYNR
YKRQ NVVKDLSVELDRLS E ESVN ND I SSVVTKGRSGEALSLLKKRFSH RPV
QEKFVCLNCGFETHADEQAALN IARSVVLFLRSQEYKKYQTNKTTGNTDK
RAFVETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 220) 518: RQ E I KRIN KI RRRLVKDSNTKKAG KTG PMKTLLVRVMTPDLRE RLEN LRKK
AR! in 491 PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE FQ KDPVG LMS RVA
Q PASKKI DON KLKP EMDE KG N LTTAG FAC SQC GQ P LFVYKLEQVSE KGK
AYTNYFG RC NVAE H E KLI LLAQ LKPE KDS DEAVTYSLGKFGQ RALD FYS I H
VTKESTHPVKPLAQ IAGN RYASGAPVGKALSDACMGTIASFLSKYQ D I II E H
Q KVVKGNQ KRLES LRE LAGKE N LEYPSVTLPPQ P HTKEGVDAYN EVIARV
RMWVN LNLWQ KLKLSRD DAKP LLRLKGFPS FP LVERQAN EVDVVVVDMVC
NVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
LGDLLLHLEKKHGEDWGKVYDEAVVERIDKKVEGLSKHIKLEEERRSEDAQ
SKAALTDWLRAKASFVI EGLKEAD KDE FC RC E LKLQ KVVYGDLRGKP FAIE
AE N S I LD ISGFSKQYNCAF IWQ KDGVKKLN LYL I INYF KGG KLRF KKI KP EAF
EAN RFYTVINKKSGE IVPMEVNFNFDDPNL I ILPLAFGKRQGREF IVVNDLLS
LETGSLKLANGRVIE KTLYN RRTRQ DE PALFVALTFE RREVLDSSN IKPM N
LIGVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQ
AKKEVEQ RRAGGYS RKYASKAKN LAD DMVRNTAR DLLYYAVTQ DAM LIF
EN LS RGFGRQGKRTFMAE RQYTRM EDWLTAKLAYE GLS KTYLSKTLAQY
TS KTCS N CG FTITSADYDRVLE KLKKTATGWMTTI NC KE LKVEGQ ITYYNR
YKRQ NVVKDLSVELDRLS E ESVN ND I SSVVTKGRSGEALSLLKKRFSH RPV
QEKFVCLNCGFETHADEQAALN IARSWLFLRSQEYKKYQTNKTTGNTDK
RAFVETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 221) 519: QEIKRINKI RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
AQ692 i EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E FQ KDPVGLM
SRVAQ
n LEVYKLEOVSEKGKA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLN LWQKLKLS RD DAKPLLRLKG FPSFP LVE RQANEVDVVWDMVCNV
KKL IN EKKEDGKVFWQ N LAGYKRQ EALRPYLSS E E DRKKG KKFARYQLG
DLLLHLEKKHGEDWGKVYDEAINERIDKKVEGLSKH IKLEEERRSEDAQSK
AALTDWLRAKASFVI EGLKEADKDE FC RC E LKLQ KVVYGDLRG KPFAI EAE
NSI LDISGFSKQYNCAF I VVQ KDGVKKLN LYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSGE IVPMEVNFNFDDP NLI ILPLAFGKRQGREFIWNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSSN IKPMNL I
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH IQ LRIGESYKEKQRTIQA
KKEVEQRRAGGYSRKYAS KAKN LADDMVRNTARDLLYYAVTQ DAM L IFE

Descriptio Amino Acid Sequence n*
NLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRY
KRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQ
EKFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRA
FVETVVQSFYRKKLKEVWKPAV (SEQ ID NO: 222) 520:
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
1705T in EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E
FQ KDPVGLM SRVAQ

LFVYKLEQVSEKGKA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLN LWQKLKLS RD DAKPLLRLKG FPSFP LVE RQANEVDVVVVDMVC NV
KKL IN EKKEDGKVFWQ N LAGYKRQ EALRPYLSS E E DRKKG KKFARYOLG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKH IKLEEERRSEDAQSK
AALTDWLRAKASFVI EGLKEADKDE FC RC E LKLQ KVVYGDLRG KPFAI EAE
NSI LDISGFSKQYNCAF I VVQ KDGVKKLN LYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSGE IVPMEVNFNFDDP NLI ILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSSN IKPMNL I
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTTQA
KKEVEQRRAGGYSRKYAS KAKN LADDMVRNTARDLLYYAVTQ DAM L IFE
N LS RGFGRQGKRTFMAERQYTRME DWLTAKLAYEG LS KTYLS KTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ ITYYNRY
KRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQ
EKFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDKRA
FVETWQSFYRKKLKEVWKPAV (SEQ ID NO: 223) 522: QEIKRINKI
RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
D683R in EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E FQ KDPVGLM SRVAQ

LFVYKLEQVSEKGKA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLN LWQKLKLS RD DAKPLLRLKG FPSFP LVE RQANEVDVVWDMVCNV
KKL IN EKKEDGKVFWQ N LAGYKRQ EALRPYLSS E E DRKKG KKFARYOLG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKH IKLEEERRSEDAQSK
AALTDVVLRAKASFVI EGLKEADKDE FC RC E LKLQ KVVYGDLRG KPFAI EAE
NSILDISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSSN IKPMNL I
GVDRGEN IPAVIALTDPEGCPLSRFKRSLGNPTH ILRIGESYKEKQRTIQAK
KEVEQRRAGGYSR KYASKAKN LAD DMVRNTAR DLLYYAVTQDAM LI FEN
LS RG FGRQGKRTFMAERQYTRM EDWLTAKLAYEGLSKTYLS KTLAQYTS
KTCS N CGFTITSADYD RVLEKLKKTATGVVMTTI NG KE LKVEGQ ITYYNRYK
RQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALN IARSVVLFLRSQEYKKYQTNKTTGNTDKRAF
VETWQSFYRKKLKEVVVKPAV (SEQ ID NO: 224) Descriptio Amino Acid Sequence n*
523: Q E I KRI NKI RR R LVKDSNTKKAGKTYP MKTLLVRVMTPDLR E R LE NLR KKP
G26Y in EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E
FQ KDPVGLM SRVAQ

LFVYKLEQVSEKGKA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK
VVKGNQ KRLESLRELAGKEN LEYPSVTLP PO PHTKEGVDAYN EVIARVRM
VVVNLN LWQKLKLS RD DAKPLLRLKG FPSFP LVE RQANEVDVVVVDMVC NV
KKL IN EKKEDGKVFWQ N LAGYKRQ EALRPYLSS E E DRKKG KKFARYQLG
DLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKH IKLEEERRSEDAQSK
AALTDWLRAKASFVI EGLKEADKDE FC RC E LKLQ KVVYGDLRG KPFAI EAE
NSILDISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSGE IVPMEVNFNFDDP NLI ILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSSN IKPIVINL I
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILR IGESYKEKQ RTIQAK
KEVEQRRAGGYSRKYASKAKN LAD DMVRNTARDLLYYAVTQDAM LI FEN
LS RG FGRQGKRTFMAERQYTRM EDVVLTAKLAYEGLSKTYLS KTLAQYTS
KTC S N CGFTITSADYD RVLEKLKKTATGVVMTTI NG KE LKVEGQ ITYYNRYK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALN IARSWLFLRSQEYKKYQTNKTTGNTDKRAF
VETVVQSFYRKKLKEVVVKPAV (SEQ 1:13 NO: 225) 524: QEIKRINKI RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
T817H in EN I PQ PI S NTS RAN LN KLLTDYTEM KKAILHVYVVE E FQ KDPVGLM SRVAQ

LFVYKLEQVSEKGKA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
WVNLN LWQKLKLS RD DAKPLLRLKG FPSFP LVE RQANEVDVVWDMVCNV
KKL IN EKKEDGKVFWQ N LAGYKRQ EALRPYLSS E E DRKKG KKFARYQLG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKH IKLEEERRSEDAQSK
AALTDVVLRAKASFVI EGLKEADKDE FC RC E LKLQ KVVYGDLRG KPFAI EAE
NSI LDISGFSKQYNCAF I VVQ KDGVKKLN LYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSGE IVPMEVNFNFDDP NLI ILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSSN IKPMNL I
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILRIGESYKEKQRTIQAK
KEVEQRRAGGYSR KYASKAKN LAD DMVRNTAR DLLYYAVTQDAM LI FEN
LS RG FGRQGKRTFMAERQYTRM EDVVLTAKLAYEGLSKTYLS KTLAQYTS
KTCSNCGFTIHSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYN RY
KRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVICI
EKFVC LN CC FETHADEQAALN IARSWLF LRSQ EYKKYQTNKTTGNTDKRA
FVETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 226) 525; QEIKRINKI
RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
V746A in EN I PQ PIS NTS RAN LN KLLTDYTEMKKAILHVYVVEEFQ KDPVGLMSRVAQ

LFVYKLEQVSEKGKA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK

Desc riptio Amino Acid Sequence n*
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VWNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVWDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDVVLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
NSILDISGFSKQYNCAFIVVQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
NRFYTVIN KKSGE IVPMEVNFNFDDPNLI ILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLI
GVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAATQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRYK
RQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAF
VETWQSFYRKKLKEVWKPAV (SEQ ID NO: 227) 526: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
K708A in ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ

PASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKA
YTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVVVDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIONDEAWERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
NSILDISGFSKQYNCAFIVVQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEA
NRFYTVIN KKSGE IVPMEVNFNFDDPNLI ILPLAFGKRQGREFIVVNDLLSLE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSS NIKPM N L I
GVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAA
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYK
RQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQE
KFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAF
VETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 228) 527: QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKK
AR26 in PE N IPQ P I SNTS RANLN KLLTDYTEM KKAI LHVYWEE
FQ KDPVG LMS RVA

QPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGK
AYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIH
VTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQ
KVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVR
MVINNLNLVVQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVWVDMVGN
VKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQL
GDLLLHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDAQ
SKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIE

Descriptio Amino Acid Sequence n*
AE N S I LD ISGFSKQYNCAF IWQ KDGVKKLN LYL I INYF KGG KLRF KKI KP EAF
EAN RFYTVINKKSGE IVPMEVNFNFD DPNL I ILPLAFGKRQG REF IWND LLS
LETGSLKLANGRVIE KTLYN RRTRQ DE PALFVALTFE RREVLDSSN IKPM N
LIGVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQ
AKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIF
ENLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQY
TSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNR
YKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPV
QEKFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTDK
RAFVETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 229) 528: QEIKRINKI RRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP

ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ
in LEVYKLEOVS EKGKA
YTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTHPVKPLAQIAGNRYASYPVGKALSDACMGTIASFLSKYQDIIIEHQK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
WVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVWDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDVVLRAKASFVI EGLKEADKDE FC RC E LKLQ KVVYGDLRG KPFAI EAE
N S I LD ISGFSKQYN CAF I VVQ KDGVKKLN LYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSG E IVPMEVNFNFD DP NLI ILPLAFGKRQGRE FIVVND LLS LE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSS N I KPM N L I
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILR I GE SYKE KQ RTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDVVLTAKLAYEGLPSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQITYYNRY
KRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQ
EKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRA
FVETVVQSFYRKKLKEVVVKPAV (SEQ ID NO: 230) 529: QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKP
G223N in ENIPQPISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQKDPVGLMSRVAQ

LEVYKLEOVS EKGKA
YTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYAS N PVGKALS DACMGTIAS F LS KYQ D III E H QK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVWDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG
DLLLHLEKKHGEDWGIWYDEAINERIDKKVEGLSKHIKLEEERRSEDAQSK
AALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAE
N S I LD ISGFSKQYN CAF I VVQ KDGVKKLN LYLI INYFKGGKLRFKKIKPEAF EA
NRFYTVIN KKSG E IVPMEVNFNFD DP NLI ILPLAFGKRQGRE FIWND LLS LE
TGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTF ERREVLDSSN IKPMNL I
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILRI GE SYKE KQ RTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN

Descriptio Amino Acid Sequence n*
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ ITYYNRY
KRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKRFSHRPVQ
EKFVCLNCGFETHADEQAALN IARSVVLFLRSQEYKKYQTNKTTGNTDKRA
FVETVVQSFYRKKLKEVWKPAV (SEQ ID NO: 231) 530: Q E I KRI NKI RRRLVKDSNTKKAGKTGPM KTLLVRVMTPDLRERLENLRKKP
Aw539 in EN I PQ P I S NTS RAN LN KLLTDYTEM KKAILHVYWE E FQ KDPVGLM SRVAQ

LFVYKLEQVS EKG KA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VsA/NLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVWDMVCNV
KKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYOLG

AALTDWLRAKASFVIEGLKEADKDEFC RC E LKLQ KVVYGDLRG KP FA I EAE
N S I LD ISGF SKQYN CAF I VVQ KDGVKKL N LYLI I NYFKGWG KLRF KKI KPEAF
EAN RFYTVINKKSGE IVPMEVN FN FD DPN L I I LP LAFG KRQG REF IVVN D LLS
LETGSLKLANGRVIE KTLYN RRTRQ DE PAL FVALTFE RREVLDSSN IKPM N
LIGVD R GEN I PAVIALTDP EGC PLS RF KDS LG N PTH I L RIG ESYKE KQ RTIQ
AKKEVEQ RRAGGYS RKYASKAKN LAD DMVRNTARDLLYYAVTQ DAM LIF
ENLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQ
YTS KTCSNC G FTITSADY DRVL EKLKKTATGVVMTTI N GKELKVEGQ ITYYN
RYKRQNVVKDLSVE LDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRP
VQEKFVC LNCGFETHADEQAALNIARSVVLFLRSQEYKKYQTNKTTGNTD
KRAFVETWQSFYRKKLKEVWKPAV (SEQ ID NO: 232) 531: Q E I KRI NKI RRRLVKDSNTKKAGKTGPM KTLLVRVMTPDLRERLENLRKKP
Ay539 in EN I PQ P I S NTS RAN LN KLLTDYTEM KKAILHVYVVE E FQ KDPVGLM SRVAQ

LFVYKLEQVS EKG KA
YTNYFGRCNVAE HEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHV
TKESTH PVKPLAQ IAG N RYASGPVGKALS DACMGTIAS FLS KYQ D I IIE H QK
VVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM
VVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDVVWDMVCNV
KKLINEKKEDGKVFMNLAGYKRQEALRPYLSSEEDRKKGKKFARYOLG

AALTDVVLRAKASFVIEGLKEADKDEFC RC E LKLQ KVVYGDLRG KP FA I EAE
N S I LD ISGF SKQYN CAF IWQ KDGVKKL N LYLI I NYFKGYGKLRFKKIKP EAF E
ANRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSL
ETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSN I KPMNLI
GVDRGEN IPAVIALTDPEGCPLSRFKDSLGNPTH ILR I GE SYKE KQ RTIQAK
KEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN
LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ ITYYNRY
KRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQ
EKFVC LN CC F ETHADEQAAL N IARSWLFLRSQEYKKYQTNKTTGNTDKRA
FVETVVQSFYRKKLKEVWKPAV (SEQ ID NO: 233) * Where a number is indicated in the left column, it designates the CasX
variant numerically;
changes, where indicated, are relative to SEQ ID NO2 g. CasX Fusion Proteins 1002201 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).
1002211 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.
1002221 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, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).
[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, SUIVIOylating 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 VP16, 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, MILLI to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JIIDM2a/b, UTX, IMID3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TETICD), TETI, DME, DML I, DML2, ROS I, 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 51(D); 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- 20111, RIZ1, and the like; histone lysine demethylases such as JNIJD2A/JHDM3A, JN1JD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JAME) 1C/SMCX, JARID1D/SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, FIDAC5, HDAC7, HDAC9, SIRTI, SIRT2, HDAC 11, and the like; DNA

methylases such as HhaI DNA m5c-methyltransferase (M.HhaI), DNA
methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), HETI, 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., Fold nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA
methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DR1VI3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET 1 CD), TETI, DME, DML1, DML2, ROS1, and the like), DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme, e.g., an APOBEC protein such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and/or 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 g,lycosylase 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 VP16 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 Fold nuclease), and a base editor (e.g., cytidine deaminase such as APOBEC1).
[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 (FINIT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB
1, and the like, SET1A, SET1B, MILLI to 5, ASH1, SYMD2, NSD1, DOT IL, Pr-SET7/8, 20H1, EZH2, R1Z1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase lA (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JIVLID2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JAR1D1D/SMCY, lUTX, 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, FIB01/MYST2, HMOF/MYST1, SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., FIDAC1, HDAC2, HDAC3, MACS, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, S1RT2, HDAC11, 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.
1002301 Suitable chloroplast transit peptides include, but are not limited to:

MASMISSSAVTTVSRASRGQSAANIAPFGGLKSMTGFPVRICVNTDITSITSNGGR
VKCMQVWPPIGKICKFETLSYLPPLTRDSRA (SEQ ID NO: 116);

(SEQ ID NO: 117);
MASSMILSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQV
WPPIEKKKFETLSYLPDLTDSGGRVNC (SEQ ID NO: 118;

SELRPLKVMSSVSTAC (SEQ ID NO: 119);
MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQIIPRAYPISSSWGLICKSGMTLIG
SELRPLKVMSSVSTAC (SEQ ID NO: 120);
MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKICLKNSANSMiLVLICKDSIFMQLF
CSFR1SASVATAC (SEQ ID NO: 121);

RFDRRCLSMVV (SEQ ID NO: 122);
MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLICPRSPAGGDATSLSVTTSARATPICQ
QRSVQRGSRRFPSVVVC (SEQ ID NO: 123);
MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQC
(SEQ ID NO: 124);
MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVKCSAAVTPQASPVIS
RSAAAA (SEQ ID NO: 125); and MGAAATSMQSLICFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCASSWNSTING
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 GLFHALLHLLEISLWHILLLHA (SEQ ID
NO:
128), or FILIHRHIII-IFIH (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., elF4G); 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 IINT3); proteins and protein domains responsible for nonsense mediated RNA
decay (for example UPF1, UPF2, UPF3, UPF3b, RNP SI, Y14, DEK, REF2, and SRm160); 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., e1F4G); proteins and protein domains responsible for polyadenylation of RNA (for example PAP1, GLD-2, and Star-PAP); proteins and protein domains responsible for polyuridinylation of RNA
(for example CI
DI and terminal uridylate transferase); proteins and protein domains responsible for RNA
localization (for example from 111/1P1, 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 My); 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., elF4G); 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 W02012068627, which is hereby incorporated by reference in its entirety.
1002341 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 Al 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
Al 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 Bc1-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.
1002351 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.).
1002361 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).
1002371 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).
1002381 Non-limiting examples of NLSs include sequences derived from: the NLS
of the SV40 virus large T-antigen, having the amino acid sequence PKICICRICV (SEQ ID NO:
130); the NLS
from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAICKKK (SEQ ID NO: 131); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 132) or RQRRNELICRSP (SEQ ID NO, 133); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ
ID NO: 134); the sequence RMRIZFICNKGKDTAELRRRRVEVSVELRKAKKDEQ1LICRRNV (SEQ ID NO: 135) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 136) and PP1CKARED (SEQ ID NO: 137) of the myoma T protein; the sequence PQPKICICPL
(SEQ ID
NO: 138) of human p53; the sequence SALIKKICKKMAP (SEQ ID NO: 139) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 140) and PKQICKRK (SEQ ID NO: 141) of the influenza virus NS1; the sequence RICLICICKIKKL (SEQ ID NO: 142) of the Hepatitis virus delta antigen; the sequence REKKKFLICRR (SEQ ID NO: 143) of the mouse Mxl protein; the sequence ICRKGDEVDGVDEVAKICKSKK (SEQ ID NO: 144) of the human poly(ADP-ribose) polymerase; the sequence RKCLQAGMNLEARKT1CK (SEQ ID NO: 145) of the steroid hormone receptors (human) glucocorticoid; the sequence PRPRICIPR (SEQ ID NO:
146) of Boma disease virus P protein (BDV-P1); the sequence PPRKICRTVV (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 (RI-IA); the sequence KRSFSKAF (SEQ ID NO: 153) of nucleolar RNA helicase II; the sequence KLKIKRPVIC (SEQ
ID NO: 154) of TUS-protein; the sequence PKKICRKVPPPPAAICRVICLD (SEQ ID NO:
155) associated with importin-alpha; the sequence PKTRRRPRRSQRICRPPT (SEQ ID NO:
156) from the Rex protein in HTLV-I; the sequence MSRRRKANPTKLSENAKKLAKEVEN (SEQ
ID NO: 157) from the EGL-13 protein of Caenorhabditis elegans; and the sequences KTRRRPRRSQRICRPPT (SEQ ID NO: 158), RRICKRRPRRKKRR (SEQ ID NO: 159), PKKKSRKPKKKSRK (SEQ ID NO: 160), TIKKKHPDASVNFSEFSK (SEQ ID NO: 161), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 162), LSPSLSPLLSPSLSPL (SEQ ID NO: 163), RGKGGKGLGKGGAKRIIRIC (SEQ ID NO: 164), PKRGRGRPKRGRGR (SEQ ID NO: 165), and PKKKRKVPPPPICKICRKV (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.

002391 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 YGRKKRRQRRR (SEQ ID NO: 167), RICKRRQRR (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 at. (2004) Pharm. Research 21 :1248-1256); polylysine (Wender et al. (2000) Proc.
Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSICLM1CR (SEQ ID NO: 173);
Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 174);
KALAWEAICLAKALAICALAKHLAKALAKALKCEA (SEQ ID NO: 175); and RQIICIWFQNRRMKWIC_K (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 Na 184), GSSSG (SEQ ID NO: 185),GPGP (SEQ ID NO: 186), GOP, 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, Matteson:ewe/es, or Candidatus (as described in US20180346927A1 and W02018064371A1, 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". hi 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 thymidina 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 ("reframing" strategy).
When the reframing 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).
1002441 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 8NA 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.
1002451 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.
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.
f. 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, Q13, Ul 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.
It. Extended Stem Loop 1002501 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 GCGCTTATTTATCGGAGAGAAATCCGATAAATAAGAAGC (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, Q13, Ul 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.

L Targeting Sequence (a.k.a. Spacer) 1002511 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 hobo 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.
1002521 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.
gNA scaffolds 1002551 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.
1002561 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:

AUGGACGAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 6) and ACAUCUGGCGCGUUUAUUCCAUUACUIJUGGAGCCAGUCCCAGCGACUAUGUCGU
AUGGACGAAGCGCIUUAUUUAUCGG (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:
UACUGGCGCULTUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA
UGGGUAAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 8) and UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA

UGGGUAAAGCGCUUAUUUAUCGG (SEQ ID NO: 9). Exemplary crRNA sequences isolated or derived from Planctotnycetes 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.
1002571 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: GUULTACACACUCCCUCUCAUAGGGU (SEQ ID
NO: 10), GUUUACACACUCCCUCUCAUGAGGU (SEQ ID NO: 11), UUUUACAUACCCCCUCUCAUGGGAU (SEQ ID NO: 12) and GUUUACACACUCCCUCUCAUGGGGG (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 SEQ ID NO. Nucleotide Sequence ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCG
ACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCG
AUAAGUAAAACGCAUCAAAG
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGA
CUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCG
AUAAAUAAGAAGCAUCAAAG

6 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCG
ACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGA

7 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCG
ACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGG

8 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGA
CUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGA

9 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGA
CUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGG
GUUUACACACUCCCUCUCAUAGGGU

GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC

GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAU
GUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
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 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 664U. In the foregoing embodiment, the gNA scaffold comprises the sequence ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG
UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG (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 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, 13-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.
1002631 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.

1002661 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 start: stop 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 SEQ
NUCLEOTIDE SEQUENCE
NAME or ID, NO: Modification 597 phage UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
replication AGCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGACGA
stable CCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
598 Kissing UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
loop_bl AGCGACUAUGUCGUAUGGGUAAAGCGCUGCUCGACGCG
UCCUCGAGCAGAAGCAUCAAAG
599 Kissing UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
loop_a AGCGACUAUGUCGUAUGGGUAAAGCGCUGCUCGCUCCG
UUCGAGCAGAAGCAUCAAAG
600 32: uvsX
GUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCAC
hairpin CAGCGACUAUGUCGUAUGGGUAAAGCOCCCUCUUCGGA
GGGAAGCAUCAAAG

UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCAGGAGUUUCUAU
GGAAACCCUGAAGGAUCAAAG
602 64: trip mut, GUACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCAC
extended stem CAGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUC
truncation GGUCCGUAAGAAGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification 603 hyperstable UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
tetraloop AGCGACUAUGUCGUAUGGGUAAAGCGCUGCGCUUGGGC
AGAAGCAUCAAAG

UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

UACUGGCGCUUUUAUCGCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

loop AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG

UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCACAUGAGGAUUA
CCCAUGUGAAGCAUCAAAG

-1, A2G, -78, GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCA

GCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGUGA
GAAAUCCGAUAAAUAAGAAGCAUCAAAG

UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUGCAUGUCUAAG
ACAGCAGAAGCAUCAAAG

45,44 hairpin UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCAGGGCUUCGGC
CGAAGCAUCAAAG

UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCAAUCCAUUGCAC
UCCGGAUUGAAGCAUCAAAG

A14C, U17G
UACUGGCGCUUUUCUCGCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

loop modified AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
CUUCGGUCCGAUAAAUAAGAAGCAUCAAAG
614 Kissing UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
loop ¨b2 AGCGACUAUGUCGUAUGGGUAAAGCGCUGCUCGUUUGC
GGCUACGAGCAGAAGCAUCAAAG

-76:78, -83:87 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGAG
AGAUAAAUAAGAAGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
in NAME or NO: Modification UACGGCGCU
UUUAUCUCAUUACUUUGAGAGCCAUCACCA
GCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

extended stem UACUGGCGCCUUUUAUCUCAUUACUUUGAGAGCCAUCAC
truncation CAGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACU UC
GGUCCGUAAGAAGCAUCAAAG

UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUCGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
619 trip mut UAC
UGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
CUUCGGUCCGAUAAAUAAGAAGCAUCAAAG
620 -76:78 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGAG
AAAU CC GAUAAAUAAGAAGCAUCAAAG
621 -1:5 GCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGA
CUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAA
AUCCGAUAAAUAAGAAGCAUCAAAG
622 -83:87 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAGAUAAAUAAGAAGCAUCAAAG
623 =+G28, UACUGGCGCUUUUAUCUCAUUACUUUGGAGAGCCAUCAC
A82U, -84, CAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGG
AGAGUAUCCGAUAAAUAAGAAGCAUCAAAG
624 =+51U

UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUUCGUAUGGGUAAAGCGCUUAUUUAUCGG
AGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

-1:4, +GSA, AGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCG
+G86, ACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGA
AAUGCCGAUAAAUAAGAAGCAUCAAAG
626 =+A94 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAAUAAGAAGCAUCAAAG
627 =+G72 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUGUAUCG
GAGAGAAAU CC GAUAAAUAAGAAG CAU CAAAG

shorten front, GCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGA
CLTUCGG
CUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGACUUCG
loop modified. GUCCGAUAAAUAAGCGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
in NAME or NO: Modification extend extended UACUGGCGCUUUUCUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
630 -1:3, +63 GUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAG
CGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAU CGGAGA
GAAAUCCGAUAAAUAAGAAGCAUCAAAG
631 =+C45, +U46 UAC UGGCGC UUUUAUCUCAUUAC U U UGAGAGC CAU
CAC C
AGC GAC C UUAUG U CGUAU GGG UAAAGC GC U UAUU UAUCG
GAGAGAAAU CC GAUAAAUAAGAAG CAU CAAAG

loop modified, GCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAC
fun start UUCGGUCCGAUAAAUAAGAAGCAUCAAAG
633 -93:94 UAC UGGCGC U U U UAUCU CAU UAC U
U UGAGAGCCAU CAC C
AGC GAC UAU GUCGUAUGGGUAAAGC GC U UAUU UAUCGGA
GAGAAAUCC GAUAAAAGAAGCAUCAAAG
634 =+U45 UAC UGGCGC UUUUAUCUCAUUAC U U
UGAGAGC CAU CAC C
AGCGAUCUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGG
AGA GAAA U CCGAUAAAUAAGAAG CAU CAAAG
635 -69, -94 UAC UGGCGC U U U UAUCU CAU UAC U
U UGAGAGCCAU CAC C
AGCGACUAUGUCGUAUGGGUAAAGGCUUAUUUAUCGGAG
AGAAAU C C GAUAAAAAGAAGCAU CAAAG

U UGAGAGCCAU CAC C
AGC GAC UAU GUCGUAUGGGUAAAGC GC U UAUU UAUCGGA
GAGAAAUCC GAUAAAAAGAAGCAU CAAAG
637 modified UACUGGCGCUUUAUCUCAUUACUUUGAGAGCCAUCACCA
CUUCGG, GCOACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUGGGAC
minus U in 1st UUCGGUCCGAUAAAUAAGAAGCAUCAAAG
triplex 638 -1:4, +C4, CGGCGCU UUUC
UCGCAUUACUUUGAGAGCCAUCACCAGC
A14C, U17G, GACUAUGUCGUAUGGGUAAAGCGCUUAUUGUAUCGAGAG
+G72, -76:78, AUAAAUAAGAAGCAUCAAAG
-83:87 639 U1C, -73 CAC UGGCGC U U U UAUCU CAU UAC U
U UGAGAGCCAU CAC C
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
640 Scaffold UACUGGCGCUUUAUCUCAUUACUUUGAGAGCCAUCACCA
uuCG, stem GCGACUUCGGUCGUAUGGGUAAAGCGCUUAUGUAUCGG
uuCG. Stem CUUCGGCCGAUACAUAAGAAGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification swap, t shorten 641 Scaffold UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
uuCG, stem AGCGACUUCGGUCGUAUGGGUAAAGCGCUUAUGUAUCG
uuCG. Stem GCUUCGGCCGAUACAUAAGAAGCAUCAAAG
swap 642 =+G60 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUGAAAGCGCUUAUUUAUCG
GAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
643 no stem UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
Scaffold AGCGACUUCGGUCGUAUGGGUAAAG
uuCG
644 no stem GAUGGGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAG
Scaffold CGACUUCGGUCGUAUGGGUAAAG
uuCG, fun start 645 Scaffold GAUGGGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAG
uuCG, stem CGACUUCGGUCGUAUGGGUAAAGCGCUUAUUUAUCGGC
uuCG, fun UUCGGCCGAUAAAUAAGAAGCAUCAAAG
start 646 Pseudoknots UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUACACUGGGAUC
GCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUAUACUU
UGGAGUUUUAAAAUGUCUCUAAGUACAGAAGCAUCAAAG
647 Scaffold GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCG
uuCG, stem ACUUCGGUCGUAUGGGUAAAGCGCUUAUUUAUCGGCUU
uuCG CGGCCGAUAAAUAAGAAGCAUCAAAG
648 Scaffold GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCA
uuCG, stem GCGACUUCGGUCGUAUGGGUAAAGCGCUUAUUUAUCGG
uuCG, no start CUUCGGCCGAUAAAUAAGAAGCAUCAAAG
649 Scaffold UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
uuCG
AGCGACUUCGGUCGUAUGGGUAAAGCGCUUAUUUAUCG
GAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
650 =+GCUC36 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUGCUC
CACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAU
CGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
651 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
te1omere AGCGACUAUGUCGUAUGGGUAAAGCGGGGUUAGGGUUA
basket+ ends GGGUUAGGGAAGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification 652 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
M3q AGCGACUAUGUCGUAUGGGUAAAGCGGAGGGAGGGAGG
GAGAGGGAAAGCAUCAAAG
653 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
telomere AGCGACUAUGUCGUAUGGGUAAAGCGUUGGGUUAGGGU
basket no ends UAGGGUUAGGGAAAAGCAUCAAAG
654 45,44 hairpin UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
(old version) AGCGACUAUGUCGUAUGGGUAAAGCGC-----AGGGCUUCGGCCG----GAAGCAUCAAAG
655 Sarcin-ricin UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
loop AGCGACUAUGUCGUAUGGGUAAAGCGCCUGCUCAGUAC
GAGAGGAACCGCAGGAAGCAUCAAAG
656 uvsX, C18G UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCC CUCUUCGGAG
GGAAGCAUCAAAG
657 truncated stem UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACC

loop, C18G, AGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUC
trip mut GGUCCGUAAGAAGCAUCAAAG
(U10C) 658 short phage UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACC
rep, C18G
AGCGACUAUGUCGUAUGGGUAAAGCGCGGACGACCUCU
CGGUCGUCCGAAGCAUCAAAG
659 phage rep UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACC
loop, C18G AGCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGAGGA
CCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
660 =+G18, UACUGGCGCCUUUAUCUGCAUUACUUUGAGAGCCAUCAC
stacked onto CAGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUC

661 truncated stem GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA

loop, C18G, - GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG

662 phage rep UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACC
loop, C18G, AGCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGACGA
trip mut CC UCUCGGUCGUCCUAUC UGAAGCAUCAAAG

(U10C) 663 short phage UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACC
rep, C18G, AGCGACUAUGUCGUAUGGGUAAAGCGCGGACGACCUCU
nip mut CGGUCGUCCGAAGCAUCAAAG
(U10C) SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification 664 uvsX, trip mut UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACC
(U1 OC) AGCGACUAUGUCGUAUGGGUAAAGCGCCCUCUUCGGAG
GGAAGCAUCAAAG
665 truncated stem UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
loop AGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUC
GGUCCGUAAGAAGCAUCAAAG
666 =+A17, UACUGGCGCCUUUAUCAUCAUUACUUUGAGAGCCAUCAC
stacked onto CAGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUC

667 3' HDV
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
genomic AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
ribozyme GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGCCGGCAU
GGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUU
CCGAGGGGACCGUCCCCUCGGUAAUGGCGAAUGGGACC
C
668 phage rep UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACC
loop, trip mut AGCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGAGGA
(U10C) CCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
669 -79:80 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAAAUCCGAUAAAUAAGAAGCAUCAAAG
670 short phage UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACC
rep, trip mut AGCGACUAUGUCGUAUGGGUAAAGCGCGGACGACCUCU
(U1 OC) CGGUCGUCCGAAGCAUCAAAG
671 extra UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
truncated stem AGCGACUAUGUCGUAUGGGUAAAGCGCCGGACUUCGGU
loop CCGGAAGCAUCAAAG
672 U17G, C18G UAC UGGCGC UUUUAUCGGAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
673 short phage UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
rep AGCGACUAUGUCGUAUGGGUAAAGCGCGGACGACCUC U
CGGUCGUCCGAAGCAUCAAAG
674 uvsX, C 18G, - GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA

GCGACUAUGUCGUAUGGGUAAAGCGCCCUCUUCGGAGG
GAAGCAUCAAAG
675 uvsX, Cl8G, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
trip mut GCGACUAUGUCGUAUGGGUAAAGCUCCCUCUUCGGAGG
(U10C), -1 GAGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification A2G, FIDV -676 3' HDV
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
antigenomic AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
ribozyme GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGGUCGGCA
UGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCC
GAAGGAGGACGCACGUCCACUCGGAUGGCUAAGGGAGA
GCCA
677 uvsX, Cl8G, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
trip mut GCGACUAUGUCGUAUGGGUAAAGCGCCCUCUUCGGAGG
(U10C), -1 GCGCAUCAAAG
A2G, HDV
AA(98:99)C
678 3' HDV
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
ribozyme AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
(Lior Nissim, GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGUUUUGGCCG
Timothy Lu) GCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAA
CAUGCUUCGGCAUGGCGAAUGGGACCCCGGG
679 TAC(1:3)GA, GAUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCA
stacked onto GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG

680 uvsX, -1 A2G GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCA
GCGACUAUGUCGUAUGGGUAAAGCGCCCUCUUCGGAGG
GAAGCAUCAAAG
681 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA

loop, C18G, GCGACUAUGUCGUAUGGGUAAAGCUCUUACGGACUUCG
trip mut GUCCGUAAGAGCAUCAAAG
(U10C), -1 AUG, HDV -682 short phage GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
rep, Cl 8G, GCGACUAUGUCGUAUGGGUAAAGCUCGGACGACCUCUC
trip mut GGUCGUCCGAGCAUCAAAG
(U10C), -1 A2G, HDV -683 3' sTRSV WT UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
viral AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
Hammerhead GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCCUGUCACC
ribozyme GGAUGUGCUUUCCGGUCUGAUGAGUCCGUGAGGACGAA
ACAGG

SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification 684 short phage GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
rep, C18G, -1 GCGACUAUGUCGUAUGGGUAAAGCGCGGACGACCUCUC

685 short phage GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
rep, C18G, GCGACUAUGUCGUAUGGGUAAAGCGCGGACGACCUCUC
trip mut GGUCGUCCGAAGCAUCAAAG
(-CHOC), -1 A2G, 3' genomic HDV
686 phage rep GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
loop, C18G, GCGACUAUGUCGUAUGGGUAAAGCUCAGGUGGGACGAC
trip mut CUCUCGGUCGUCCUAUCUGAGCAUCAAAG
(U10C), -1 A2G, HDV -687 3' HDV
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
ribozyme AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
(Owen Ryan, GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGAUGGCCOG
Jamie Cate) CAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAAC
ACC UUCGGGUGGCGAAUGGGAC
688 phage rep GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
loop, C18G, - GCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGACGAC

689 0.14 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUACUGGGUAAAGCGCUUAUUUAUCGG
AGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
690 -78, G77U
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGUG
AGAAAUCCGAUAAAUAAGAAGCAUCAAAG

GUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCAC
CAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGG
AGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
692 short phage GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCA
rep, -1 A2G GCGACUAUGUCGUAUGGGUAAAGCGCGGACGACCUCUC
GGUCGUCCGAAGCAUCAAAG
693 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA

loop, C18G, GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
trip mut GUCCGUAAGAAGCAUCAAAG
(U10C), -1 SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification 694 -1, A2G
GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCA
GCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
695 truncated stem GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCA

loop, trip mut GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
(U10C), -1 GUCCGUAAGAAGCAUCAAAG

696 uvsX, C 18G, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
trip mut GCGACUAUGUCGUAUGGGUAAAGCGCCCUCUUCGGAGG
(U10C), -1 GAAGCAUCAAAG

697 phage rep GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCA
loop, -1 MG GCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGACGAC
CUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
798 phage rep GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCA
loop, nip mut GCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGACGAC
(U10C), -1 CUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
MG
699 phage rep GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
loop, C18G, GCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGACGAC
hip mut CUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
(U10C), -1 MG
700 truncated stem UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACC

loop, C 18G AGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUC
GGUCCGUAAGAAGCAUCAAAG
701 uysX, trip mut GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCA
(U10C), -1 GCGACUAUGUCGUAUGGGUAAAGCGCCCUCUUCGGAGG
MG GAAGCAUCAAAG
702 truncated stem GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCA

loop, -1 MG GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
GUCCGUAAGAAGCAUCAAAG
703 short phage GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCA
rep, trip mut GCGACUAUGUCGUAUGGGUAAAGCGCGGACGACCUCUC
(U10C), -1 GGUCGUCCGAAGCAUCAAAG
MG

GAUGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGG
ribozyme CUGGGCAACACCUUCGGGUGGCGAAUGGGACUAC UGGC
(Owen Ryan, GCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACU
Jamie Cate) SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification AUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAU
CCGAUAAAUAAGAAGCAUCAAAG

GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUG
genomic GGCAACAUUCCGAGGGGACCGUCCCCUCGGUAAUGGCG
ribozyme AAUGGGACCCUACUGGCGCUUUUAUCUCAUUACUU UGAG
AGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUU
AU U UAU CGGAGAGAAAU CC GAUAAAUAAGAAGCAU CAAAG
706 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA

loop, C18G, GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
trip mut GUCCGUAAGCGCAUCAAAG
(U10C), -1 A2G, HDV
AA(98:99)C
707 5'env25 pistol CGUGGUUAGGGCCACGUUAAAUAGUUGCUUAAGCCCUAA
ribozyme GCOUUGAUC
UUCGGAUCAGGUGCAAUACUGGCGCUUUU
(with an added AUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCG
CUUCGG
UAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUA
loop) AAUAAGAAGCAUCAAAG

GGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACC
antigenomic UGGGCAUCCGAAGGAGGACGCACGUCCACUCGGAUGGC
ribozyme UAAGGGAGAGCCAUACUGGCGCUUUUAUCUCAUUACUUU
GAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCG
CUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUC
AAAG

UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
Hammerhead AGC GAC UAU GUCGUAUGGGUAAAGC GC U UAUU UAUCGGA
ribozyme GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCCAGUACUGA
(Lior Nissim, UGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUACUG
Timothy Lu) GCGCUUUUAUCUCAU
guide scaffold scar 710 =+A27, UACUGGCGCCUUUAUCUCAUUACUUUAGAGAGCCAUCAC
stacked onto CAGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACU UC

711 5'Hammerhea CGACUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCU
d ribozyme CGUCUAGUCGUACUGGCGCUUUUAUCUCAUUACUUUGAG
(Liar Nissim, AGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUU
Timothy Lu) AU U UAU C GGAGAGAAAU CC GAUAAAUAAGAAGCAU CAAAG
smaller scar SEQ
NUCLEOTIDE SEQUENCE
in NAME or NO: Modification 712 phage rep GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
loop, Cl8G, GCGACUAUGUCGUAUGGGUAAAGCGCAGGUGGGACGAC
nip mut CUCUCGGUCGUCCUAUCUGCGCAUCAAAG
(U10C), -1 A2G, 1-113V
AA(98:99)C
713 -27, stacked UACUGGCGCCUUUAUCUCAUUACUUUAGAGCCAUCACCA
onto 64 GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
GUCCGUAAGAAGCAUCAAAG
714 3' Hatchet UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCAUUCCUCAG
AAAAUGACAAACCUGUGGGGCGUAAGUAGAUCUUCGGAU
CUAUGAUCGUGCAGACGUUAAAAUCAGGU
715 3' UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
Hammerhead AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
ribozyme GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGACUACUGA
(Lior Nissim, UGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUC
Timothy Lu) GCGUGUAGCGAAGCA
716 5 Hatchet CAUUCCUCAGAAAAUGACAAACCUGUGGGGCGUAAGUAG
AUCUUCGGAUCUAUGAUCGUGCAGACGUUAAAAUCAGGU
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
717 5' HDV
UUUUGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCG
ribozyme GCUGGGCAACAUGCUUCGGCAUGGCGAAUGGGACCCCG
(Lior Nissim, GGUAGUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCA
Timothy Lu) CCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCG
GAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
718 5' CGACUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCU
Hammerhead CGUCUAGUCGCGUGUAGCGAAGCAUACUGGCGCUUUUA
ribozyme UCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGU
(Lior Nissim, AUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAA
Timothy Lu) AUAAGAAGCAUCAAAG
719 3' 111115 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
Minimal AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
Hammerhead GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGGAGCCCC
ribozyme GCUGAUGAGGUCGGGGAGACCGAAAGGGACUUCGGUCC
CUACGGGGCUCCC

SEQ
NUCLEOTIDE SEQUENCE
in NAME or NO: Modification 720 5' RB1VIX
CCACCCCCACCACCACCCCCACCCCCACCACCACCCUAC
recruiting UGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGC
motif GACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAG
AAAUCCGAUAAAUAAGAAGCAUCAAAG
721 3' UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
Hammerhead AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
ribozyme GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGACUACUGA
(Lior Nissim, UGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUC
Timothy Lu) G
smaller scar 722 3' env25 pistol UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
ribozyme AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
(with an added GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGUGGUUAG
CUUCGG
GGCCACGUUAAAUAGUUGCUUAAGCCCUAAGCGUUGAUC
loop) UUCGGAUCAGGUGCAA
723 3' Env-9 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
Twister AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGCAAUAAAG
CGGUUACAAGCCCGCAAAAAUAGCAGAGUAAUGUCGCGA
UAGCGCGGCAUUAAUGCAGCUUUAUUG
724 =-FAUUAUC UACUGGCGCUUUUAUCUCAUUACUAUUAUCUCAUUACUU
UCAUUACU UGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGC

GCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAU
CAAAG
725 5 Env-9 GGCAAUAAAGCGGUUACAAGCCCGCAAAAAUAGCAGAGU
Twister AAUGUCGCGAUAGCGCGGCAUUAAUGCAGCUUUAUUGUA
CUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAG
CGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGA
GAAAUCCGAUAAAUAAGAAGCAUCAAAG
726 3' Twisted UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
Sister 1 AGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA
GAGAAAUCCGAUAAAUAAGAAGCAUCAAAGACCCGCAAG
GCCGACGGCAUCCGCCGCCGCUGGUGCAAGUCCAGCCG
CCCCUUCGGGGGCGGGCGCUCAUGGGUAAC
727 no stem UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACC
AGCGACUAUGUCGUAUGGGUAAAG
728 5' HH15 GGGAGCCCCGCUGAUGAGGUCGGGGAGACCGAAAGGGA
Minimal CUUCGGUCCCUACGGGGCUCCCUACUGGCGCUUUUAUC
Hammerhead UCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU
ribozyme GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAU
AAGAAGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification 729 5' CCAGUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCU
Hammerhead CGUCUACUGGCGCUUUUAUCUCAUUACUGGCGCUUUUAU
ribozyme CUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA
(Lior Nissim, UGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAA
Timothy Lu) UAAGAAGCAUCAAAG
guide scaffold scar 730 5' Twisted ACCCGCAAGGCCGACGGCAUCCGCCGCCGCUGGUGCAA
Sister 1 GUCCAGCCGCCCCUUCGGGGGCGGGCGCUCAUGGGUAA
CUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCAC
CAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGG
AGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
731 5' sTRSV WT CCUGUCACCGGAUGUGCUUUCCGGUCUGAUGAGUCCGU
viral GAGGACGAAACAGGUACUGGCGCUUUUAUCUCAUUACUU
Hammerhead UGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGC
ribozyme GCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGGAU
CAAAG
732 148: =+G55, GUACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCAC
stacked onto CAGCGACUAUGUCGUAGUGGGUAAAGCGCUUACGGACU

733 158:
GUACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCAC
103+148(+65 CAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGG
5) -99, G65U AGGGAGCAUCAAAG
734 174: Uvsx ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
Extended stem GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
with [A99] GGAGCAUCAAAG
G65U), Cl 8G,AG55, [GU-1]
735 175: extended ACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCA
stem GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
truncation, GUCCGUAAGAAGCAUCAAAG
U10C, [GU-1]
736 176: 174 with GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
AlG
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
substitution GGAGCAUCAAAG
for T7 transcription 737 177: 174 with ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
bubble (+G55) GCGACUAUGUCGUAUGGGUAAAGCUCCCUCUUCGGAGG
removed GAGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
in NAME or NO: Modification 738 181: stem 42 ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
(truncated GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
stem loop); GUCCGUAAGAAGCAUCAAAG
U10C,C18G,[
GU-1]
(95+[GU-1]) 739 182: stem 42 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
(truncated GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
stem loop); GUCCGUAAGAAGCAUCAAAG
C18G,[GU-1]
740 183: stem 42 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
(truncated GCGACUAUGUCGUAGUGGGUAAAGCGCUUACGGACUUC
stem loop); GGUCCGUAAGAAGCAUCAAAG
C186,1\6-551 GU-1]
741 184: stem 48 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
(uvsx, -99 GCGACUAUGUCGUAUUGGGUAAAGCUCCCUCUUCGGAG
g650; GGAGCAUCAAAG
Cl 86,AT55,[
GU-1]
742 185: stem 42 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
(truncated GCGACUAUGUCGUAUUGGGUAAAGCGCUUACGGACUUC
stem loop); GGUCCGUAAGAAGCAUCAAAG
Cl8G,AU55,[
GU-1]
743 186: stem 42 ACUGGCGCCUUUAUCAUCAUUACUUUGAGAGCCAUCACC
(truncated AGCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUC
stem loop); GGUCCGUAAGAAGCAUCAAAG
U10C,AA17,[
GU-1]
744 187: stem 46 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
(uvsx);
GCGACUAUGUCGUAGUGGGUAAAGCGCCCUCUUCGGAG
Cl8G,AG55,[ GGAAGCAUCAAAG
GU-1]
745 188: stem 50 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
(ms2 Ul 5C, - GCGACUAUGUCGUAGUGGGUAAAGCUCACAUGAGGAUCA
99, g65t); CCCAUGUGAGCAUCAAAG
Cl8G,AG55,[
GU-1]

SEQ
NUCLEOTIDE SEQUENCE
in NAME or NO: Modification 746 189: 174 +
ACUGGCACUUUUACCUGAUUACUUUGAGAGCCAACACCA
G8A;U15C;U GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

ACUGGCACUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
190: 174+
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
GSA G GAG CAU CAAAG

ACUGGCCCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
191: 174 GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

UUUACCUGAUUACUUUGAGAGCCAUCACCA
192: 174+
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

UUUAUCUGAUUACUUUGAGAGCCAACACCA
193, 174+
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

751 195: 175+ AC U GGCACCU UUACC UGAUUAC U
UUGAGAGCCAACAC CA

GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
G8A;U15C;U GUCCGUAAGAAGCAUCAAAG

ACUGGCACCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
196: 175 +
GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
C18G + G8A GUCCGUAAGAAGCAUCAAAG

UGAGAGCCAUCACCA
197: 175 +
GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
C1 8G + G8C GUCCGUAAGAAGCAUCAAAG

ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAACACCA
198: 175 +
GCGACUAUGUCGUAUGGGUAAAGCGCUUACGGACUUCG
C18G + U35A GUCCGUAAGAAGCAUCAAAG
755 199: 174+
GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
A2G (test G GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
transcription GGAGCAUCAAAG
at start;
ccGCT...) 756 200: 174 + GACUGGCGCUUUUAUCUGAUUACUU
UGAGAGCCAUCACC

AGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGA
(ccGACU...) GGGAGCAUCAAAG

ACUGGCGCCUUUAUCUGAUUACUUUGGAGAGCCAUCACC
201: 174+
AGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGA
U1OC;AG28 GGGAGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification ACUGGCGCAUUUAUCUGAUUACUUUGUGAGCCAUCACCA
202: 174 +
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
U10A;A28U GGAGCAUCAAAG

ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
203: 174+
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
UlOC GGAGCAUCAAAG

ACUGGCGCUUUUAUCUGAUUACUUUGGAGAGCCAUCACC
204: 174 AGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGA

ACUGGCGCAUUUAUCUGAUUACUUUGAGAGCCAUCACCA
205: 174+
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
UlOA GGAGCAUCAAAG

ACUGGCGCUUUUAUCUGAUUACUUUGUGAGCCAUCACCA
206, 174+
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

UUUGAGAGCCAUCACC
207: 174 +
AGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGA

ACGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAG
208: 174 +
CGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGG
[U4] GAGCAUCAAAG

ACUGGCGCUUUUAUAUGAUUACUUUGAGAGCCAUCACCA
209: 174 +
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

UUUGAGAGCCAUCACC
210: 174+
AGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGA

767 211: 174+
ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAGCACCA

GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
(compare with GGAGCAUCAAAG
174 + U35A
above) 768 212: 174 ACUGGCGCUGUUAUCUGAUUACUUCGAGAGCCAUCACCA
+U11G, GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

(A86G), 769 213: 174 ACUGGCGCUCUUAUCUGAUUACUUCGAGAGCCAUCACCA
+1111C, GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

SEQ
NUCLEOTIDE SEQUENCE
ID NAME or NO: Modification (A86G), 770 214:
ACUGGCGCUUGUAUCUGAUUACUCUGAGAGCCAUCACCA
174+Ul2G; GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

(AS 7G), 771 215:
ACUGGCGCUUCUAUCUGAUUACUCUGAGAGCCAUCACCA
174+Ul2C; GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

(AS 7G), 772 216:
ACUGGCGCUUUGAUCUGAUUACCUUGAGAGCCAUCACCA
174_tx_11.G, GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
87.G,22.0 GGAGCAUCAAGG
773 217:
ACUGGCGCUUUCAUCUGAUUACCUUGAGAGCCAUCACCA
174 tx_11.C,8 GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
7.G,22.0 GGAGCAUCAAGG

ACUGGCGCUGUUAUCUGAUUACUUUGAGAGCCAUCACCA
218: 174 GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
+U11G GGAGCAUCAAAG
775 219: 174 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCA
+A105G
GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
(A86G) GGAGCAUCGAAG

ACUGGCGCUUUUAUCUGAUUACUUCGAGAGCCAUCACCA
220: 174 GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
+U26C GGAGCAUCAAAG
777 221: 182+
ACUGGCACUUCUAUCUGAUUACUCUGAGAGCCAUCACCA
GSA (196) GCGACUAUGUCGUAUGGGUAAAGCCGCUUACGGACUUC
+215 GGUCCGUAAGAGGCAUCAGAG
mutations +
AC63, A88G
778 222: 174 +
ACUGGCACUUCUAUCUGAUUACUCUGAGAGCCAUCACCA
GSA (196) GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
+215 GGAGCAUCAGAG
mutations 779 223: 181 +
ACUGGCACCUUUAUCUGAUUACUUUGAGAGCCAUCACCA
GSA (196) + GCGACUAUGUCGUAUGGGUAAAGCCGCUUACGGACUUC
AC63, A88G GGUCCGUAAGAGGCAUCAAAG

SEQ
NUCLEOTIDE SEQUENCE
1D NAME or NO: Modification 780 224: 182 +
ACUGGCACUUGUAUCUGAUUACUCUGAGAGCCAUCACCA
G8A (196) GCGACUAUGUCGUAUGGGUAAAGCCGCUUACGGACUUC
+214 GGUCCGUAAGAGGCAUCAGAG
mutations +
AC63, A88G
781 225: 174+
ACUGGCACUUGUAUCUGAUUACUCUGAGAGCCAUCACCA
G8A (196) GCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG
+214 GGAGCAUCAGAG
mutations [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 01 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.
1002711 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.
1002721 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.
1002731 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.
1002741 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.
1002751 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)), QI3 (UGCAUGUCUAAGACAGCA (SEQ ID NO: 205)), Ul hairpin II
(AAUCCAUUGCACUCCGGAUU (SEQ ID NO: 206)), Uvsx (CCUCUUCGGAGG (SEQ ID
NO: 207)), PP7 (AGGAGUUUCUAUGGAAACCCU (SEQ ID NO: 208)), Phage replication loop (AGGUGGGACGACCUCUCGGUCGUCCUAUCU (SEQ ID NO: 209)), Kissing loop_a (UGCUCGCUCCGUUCGAGCA (SEQ ID NO: 210)), Kissing loop_b1 (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 lID 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 >CDP is further facilitated.
1002801 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.
1002811 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 1D NO: 770, SEQ ID NO:771, SEQ ID NO: 775, or SEQ ID NO:
781.
1002821 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 1D NO: 662, SEQ ID NO: 599, SEQ ID NO: 663, SEQ ID NO: 601, SEQ
ID NO:
604, SEQ ID NO: 608, SEQ 1D NO: 656, SEQ ID NO: 666, SEQ 1D 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 Cl8G
substitution in the triplex loop; (b) a G55 insertion in the stem bubble; (c) a Ul deletion; (d) a modification of the extended stem loop wherein (i) a 6 nt loop and 13 loop-proximal base pain 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.
a 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 intemucleotide linkage other than a natural phosphodiester intemucleotide 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 2'43 ______________ C1-4a1ky1 such as 2'-0-methyl (2'-0Me), T-deoxy (T-H), 2'-0 __________________ C1-3alky1-0 ____ C1-3a1ky1 such as 2'-methoxyethyl ("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(SI (P(S)), phosphonocarboxylate (P(CH2)nCOOR) such as phosphonoacetate "PACE" (P(CH2C00-)), thiophosphonocarboxyl ate ((S)P(CH2)nCOOR) such as thiophosphonoacetate "thioPACE" ((S)P(CH2)nC00-)), 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, ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-ally1U"), 5-allylcytosine ("5-ally1C"), 5-aminoallyluracil ("5-aminoallyIU"), 5-aminoallyl-cytosine ("5-aminoallylC"), an abasic nucleotide, Z base, P base, Unstructured Nucleic Acid ("UNA"), isoguanine ("isoG"), isocytosine ("isoC"), 5-methyl-2-pytimidine, 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, MC, deuterium, 31-1, 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-1,3-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-fluorescent label, a tag (for 14C, example biotin, avidin, streptavidin, or moiety containing an isotopic label such as 15N, 13C, deuterium, 3H, 32P, 1251 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, S. (2013), the contents of which are incorporated herein by reference in its entirety.

Tropism Factors and Pseudotyping of XDP Systems 1002941 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 )(DP, 1002951 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.
1002961 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. Curt Opin Virol. 1:92 (2011)).
The envelope glycoproteins are typically observed as characteristic protein "spikes" on the surface of purified vitions 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 califomica 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 (HEWS), human foamy virus, human herpesvirus (HHV), human Herpesvirus 7, human herpesvirus type 6, human herpesvirus type 8, human immunodeficiency virus 1 (MV-O, 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 (IHIV3), 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
SEQ ID NO
SEQ ID NO
Virus Glycoprotein (DNA) (Protein) Vesicular Stomatitis Virus pGP2 Human Immunodeficiency pGP3 439 440 Virus Avian leukosis virus pGP4 Rous Sarcoma virus pGP5 Mouse mammary tumor virus pGP6 Human T-lymphotropic virus 1 pGP7 RD114 Endogenous Feline pGP8 449 450 Retrovirus Gibbon ape leukemia virus pGP9 Moloney Murine leukemia virus pGP10 Baboon Endogenous Virus pGP11 Human Foamy Virus pGP12 pGP13.1 Pseudorabies virus pGP13.2 pGP13.3 pGP13.4 pGP14.1 pGP14.2 Herpes simplex virus 1 (HHV1) pGP14.3 pGP14.4 Hepatitis C Virus pGP23 Rabies Virus pGP29 Mokola Virus pGP30 SEQ ID NO
SEQ ID NO
Virus Glycoprotein (DNA) (Protein) pGP32.1 Measles Virus pGP32.2 Ebola Zaire Virus pGP41 485 486 Dengue Virus pGP25 Zika virus pGP26 West Nile Virus pGP27 Japanese Encephalitis Virus pGP28 Hepatitis G Virus pGP24 495 496 Mumps Virus F pGP31.1 Mumps Virus I-IN pGP31.2 499 500 Sendai Virus F pGP33.1 Sendai Virus FIN pGP33.2 503 504 AcMNPV gp64 pGP59 Ross River Virus pGP54 507 508 Codon optimized rabies virus pGP29.2 Rabies virus (strain Nishigahara pGP29.3 RCEH) (RABV) Rabies virus (strain India) pGP29.4 (RABV) Rabies virus (strain CVS-11) pGP29.5 (RABV) Rabies virus (strain ERA) pGP29.6 (RABV) Rabies virus (strain SAD B19) pGP29.7 (RABV) Rabies virus (strain Vnukovo-pGP29.8 32) (RABV) Rabies virus (strain Pasteur pGP29.9 vaccins / PV) (RABV) SEQ ID NO
SEQ ID NO
Virus Glycoprotein (DNA) (Protein) Rabies virus (strain pGP29.1 PM1503/AVO1) (RABV) Rabies virus (strain China/DRY) pGP29.11 527 528 (BABY) Rabies virus (strain pGP29.12 529 530 China/MRV) (RABV) Rabies virus (isolate pGP29.13 531 532 Human/Algeria/1991) (RABV) Rabies virus (strain HEP-Flury) pGP29.14 533 534 (BABY) Rabies virus (strain silver-haired bat-associated) (RABV) pGP29.15 (SHBRV) HSV2 gB pGP15.1 HSV2 gD pGP15.2 HSV2 gH pGP15.3 HSV2 gL pGP15.4 Varicella gB pGP16.1 Varicella gK pGP16.2 Varicella gH pGP16.3 Varicella gL pGP16.4 Hepatitis B gL pGP22.1 Hepatitis B gM pGP22.2 Hepatitis B gS pGP22.3 Eastern equine encephalitis pGP65 virus (EEEV) Venezuelan equine encephalitis pGP66 viruses (VEEV) SEQ ID NO
SEQ ID NO
Virus Glycoprotein (DNA) (Protein) Western equine encephalitis pGP67 virus (WEEV) Semliki Forest virus pGP68 Sindbis virus pGP69 Chikungunya virus (CHIKV) pGP70 Bomavirus BoDV-I pGP58 Tick-borne encephalitis virus pGP71 (TBEV) Usutu virus pGP72 St. Louis encephalitis virus pGP73 Yellow fever virus pGP74 Dengue virus 2 pGP75 Dengue virus 3 pGP76 Dengue virus 4 pGP77 Murray Valley encephalitis pGP78 virus (MVEV) Powassan virus pGP79 Influenza A virus H5N1 pGP80 Influenza A virus H7N9 pGP81 Canine Distemper Virus pGP82 [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.
1002981 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, 501õ 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, 501õ 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 (CD10), membrane spanning 4-domains Al (CD20), CD22 molecule (CD22), TNF receptor superfamily member 8 (CD30), C-type lectin domain family 12 member A (CLL1), CD33 molecule (CD33), CD34 molecule (CD34), CD38 molecule (CD38), integrin subunit alpha 2b (CD41), CD44 molecule (Indian blood group) (CD44), CD47 molecule (CD47), integrin alpha 6 (CD49f), neural 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 I (HERI), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3)õ Integrin B7, intercellular cell-adhesion molecule-1 (ICAM-1), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor a2 (IL-13R-a2), K-light chain, Kinase insert domain receptor (KDR), Lewis-Y (LeY), chondromodulin-1 (LECT1), Ll 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 (MART!), glycoprotein 100 (GPI00), Proteinase3 (PRO, 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(TRBC1), TRBC2, and (T-cell immunoglobulin mucin-3) TTM-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, alc, ald, a2a, a2b, a2c, a2d, 131, 132, I33), Dopaminergic (e.g., D1, D2, D3, D4, D5), GABAergic (e.g., GABAA, GABA131a, GABAB15, GABAB2, GABAC), Glutaminergic (e.g., NMDA, AMPA, kainate, inGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7), Histaminergic (e.g., HI, H2, H3), Cholinergic (e.g., Muscarinic (e.g., MI, M2, M3, M4, M5; Nicotinic (e.g., muscle, neuronal (a-bungarotoxin-insensitive), neuronal (a-bungarotoxin-sensitive)), Opioid (e.g., i, 51, 52, ic), and Serotonergic (e.g., 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT IF, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5, 5-HT6, 5-HT7).
1003011 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, Epsdonreiroviruses, Spumaretrovirinae, or lentiviruses such as human immunodeficiency-1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (Sly), feline immunodeficiency virus (Hy), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CASEY) 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 p10 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), p2A, p2B, p10, 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, p24, p2B, p10, and NC), 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 Alphareirovirus-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 TENT, 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.
1003051 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/p12 spacer peptide; a capsid polypeptide (CA);
a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a capsid polypeptide (CA), pp21/24, p3-81p12, 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/p12 spacer, and NC), and optionally the protease cleavage site and protease, are derived from an Betareirovirus, 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.
1003061 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)õ 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.
1003071 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, and NC), and optionally the protease cleavage site and protease, are derived from an Epsilonreirovirus, 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-derived 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.
1003081 In some embodiments, the nucleic acids encoding the XDP viral components are derived from Garnmaretrovirus, including but not limited to murine leukemia virus (IVILV), 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 pp12 spacer peptide; a capsid polypeptide (CA); a nucleocapsid polypeptide (NC); a Gag polyprotein comprising a matrix polypeptide (MA), a pp12 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, pp12, CA, and NC), and optionally the protease cleavage site and protease, are derived from an Gammareirovirus, 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 Gannnaretrovirus-derived 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.
1003091 In some embodiments, the nucleic acids encoding the XDP viral components are derived from Lentivirus, including but not limited to HIV-1 and MV-2, and Simian immunodeficiency virus (SW). 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 p1/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 Lent/virus, including but not limited to HIV-1, HIV-2, and Simian immunodeficiency virus (SIV). In some embodiments, the encoding sequences for the Lent/virus-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.
1003101 In some embodiments, the nucleic acids encoding the XDP viral components are derived from Spumaretrovirinae, including but not limited to Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Similspumavirus, 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 Spumaretrovirthae including but not limited to Bovispuntavnws, Equispuntavirus, Felisputnavirus, Prosintlisputnavirus, Simiisputnavirus, and Sputnavirus. 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.
1003111 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 p211 spacer peptide; a p10 spacer peptide, a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a pp21/24 spacer peptide, a p3-p8 spacer peptide, a pp12 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 *hare/row/its, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lent/virus, Bovispumavirns, Equispumcrvirus, Felispumavirus, Prosimiispumcrvirus, Simiispumavirus, or Spumavirns.
1003121 In retroviral components derived from FITV-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 DNA Sequences Encoding Components Virus MATRIX P2A-P2B- CAPSID NUCLE
PROTEASE*

OCAPSI
D
ALV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
NO: 192) 193) NO: 195) NO: 198) 196) RSV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ (SEQ ID NO:
NO: 199) 200) NO: 782) ID NO: 234) 201) Virus MATRIX PP21/24 P3-P8/P12 CAPSID
NUCLEOCA Protease PSID
ENTV (SEQ ID (SEQ ID NO:
(SEQ ID (SEQ ID NO: (SEQ ID
NO: 235) 236) NO: 238) NO: 239) 237) MMT (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO: (SEQ ID
V NO: 240) 241) NO: 242) NO: 244) NO: 245) 243) MPM (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO: SEQ ID
V NO: 246) 247) NO: 248) NO: 250) NO: 251) 249) MPM (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO: (SEQ ID
V NO: 252) 253) NO: 254) NO: 256) NO: 257) 255) DNA Sequences Encoding Components Virus MATRIX P2A-P2B- CAPSID NUCLE
PROTEASE*

OCAPSI
D
Nativ e Virus MATRIX CAPSID NUCLEOC PROTE MA-CA-APSID ASE* CLEAVE
SITE*
BLV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
NO: 258) 259) NO: 260) NO: 262) 261) HTLV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
1 NO: 263) 264) NO: 265) NO: 267) 266) HTLV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
1 NO: 268) 269) NO: 270) NO: 272) Nativ 271) e Virus MATRIX P20 CAPSID NUCLE
PROTEASE*
OCAPSI
D
WDS (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
V NO: 273) 274) NO: 275) NO: 277) 276) Virus MATRIX P12 CAPSID NUCLE
PROTEASE*
OCAPSI
D
FLV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
NO: 278) 279) NO: 280) NO: 282) 281) MML (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
V NO: 283) 284) NO: 285) NO: 287) 286) Virus MATRIX CAPSID NUCLEOC PROTE MA-CA-APSID ASE' CLEAVE
SITE*
CAEV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
NO: 288) 289) NO: 290) NO: 292) 291) DNA Sequences Encoding Components Virus MATRIX P2A-P2B- CAPSID NUCLE
PROTEASE*

OCAPSI
D
EIAV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
NO: 293) 294) NO: 295) NO: 297) 296) Sly (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
NO: 298) 299) NO: 300) NO: 302) 301) Sly (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
Nativ NO: 303) 304) NO: 305) NO: 307) e 306) VMV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
NO: 308) 309) NO: 310) NO: 312) 311) Virus GAG PROTEASE* CLEAVAG
E SITE*
BFV (SEQ ID (SEQ ID NO: (SEQ ID
NO: 313) 314) NO: 315) BGPF (SEQ ID (SEQ ID NO: (SEQ ID
V NO: 316) 317) NO: 318) CCFV (SEQ ID (SEQ ID NO: (SEQ ID
NO: 319) 320) NO: 321) EFV (SEQ ID (SEQ ID NO: (SEQ ID
NO: 322) 323) NO: 324) FFV (SEQ ID (SEQ ID NO: (SEQ ID
NO: 325) 326) NO: 327) RHSF (SEQ ID (SEQ ID NO: (SEQ ID
V NO: 328) 329) NO: 330) SFV (SEQ ID (SEQ ID NO: (SEQ ID
NO: 331) 332) NO: 333) Virus MATRIX CAPSID P2 HIV (SEQ ID (SEQ ID NO: (SEQ ID
(SEQ ID (SEQ ID NO:
NO: 334) 335) NO: 336) NO: 338) 337) Protease*
(SEQ ID NO:
339) * Wild-type sequence (optionally incorporated, depending on configuration) 1003131 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.
1003141 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.
1003151 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.
1003161 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.
1003171 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).
1003181 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.
1003191 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.
1003201 In some embodiments, the disclosure provides nucleic acids comprising sequences encoding components of the )(DP 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.
1003211 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.
1003221 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.
1003231 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_ 1003241 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 HITI. 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 (1RES) 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 pot III, which synthesizes shRNAs), elongation factor 1 alpha (EF1-alpha), or Ill. In one embodiment, a constitutive promoter, such as the human cytomegalovirus immediate early (HCMV-1E) 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.
1003261 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 globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).
1003271 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 W094/016737, 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").
1003281 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 at. 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.
52A91 (1998)). In another embodiment, the HIV-based vector comprises a sequence encoding viral infectivity factor (Vii), 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 (Nee, an accessory protein essential for both evading host adaptive cell-mediated immunity as well as enhancing infectivity in the target cell (Basmaciogullari, S., et at. 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, Vii', Rev, Vpu, Nef, and VpR.
1003291 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 p1/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.
1003301 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.
1003311 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.
1003321 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, p1/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.
1003331 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, p1/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.
1003341 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, p1/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 gNAµ
[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, p1/p6, and CasX In some embodiments, the second nucleic acid comprises, from 5' to 3', sequences encoding MA, CA, NC, and p1/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.

1003371 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.
1003381 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 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, 1003391 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.
1003401 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 pi/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.
1003411 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 p1/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.
1003421 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 gNAµ
1003431 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, p1/p6, tev cleavage sequence (TCS), and CasX. In some embodiments, the second nucleic acid comprises, from 5' to 3', MA, CA, NC, p1/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.
1003451 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, p1/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.
1003461 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, p1/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, p1/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.
1003481 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, p1/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.
1003491 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 p1/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.
1003501 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, P1 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.
1003511 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 P1/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 p1/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.
1003521 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 p1/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.
1003531 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 P1/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.
1003541 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 P1/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.
1003551 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.
1003561 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
1003571 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 p1/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 gNAµ
[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.
1003641 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.
1003651 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, pp12, 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.
1003661 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, P213, 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.
1003751 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.
1003761 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, pp12, 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.
1003771 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, pp12, CA and CasX. In some embodiments, the second nucleic acid comprises, from 5' to 3', sequences encoding MA, pp12, 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.
1003781 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.
1003791 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 gNAµ
[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 gNAµ
[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, pp12, 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, p1/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
1003911 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 1003931 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, pot, 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 co/i.
Exemplary yeast cell types may include Saccharomyces cerevisiae. Also suitable for use as packaging cells are insect cell lines, such as Spodoptera ft-up-perdu 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, NSO cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, N1H3T3 cells, COS
cells, WI38 cells, MRCS 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 MEC receptors or that would otherwise trigger an immune response in a subject.
1003941 In the embodiments of the XDP system, vectors are introduced into the packaging cell that encode the particular therapeutic payload (e.g., a CasKgNA designed for editing target nucleic acid), as well as the other viral-derived structural components, detailed above, (e.g., the Gag polyprotein, the pot 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 Minis ho LLC, Lanza 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, Pr55gag.
This polyprotein is comprised of four major structural domains, which are cleaved by the viral protease into p17 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. Phannacol. 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 al., Assembly and release of HTV-1 precursor Pr55Gag virus-like particles from recombinant baculovirus-infected insect cells. Cell. 59(1):103 (1989)). In the context of the uncleaved Pr55Gag, 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 11IV-1 protease cleaves Pr5 stag into the mature Gag proteins p17 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 (EF1a), MMLV-Itr, internal ribosome entry site ORES) 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.
1004011 The packaging cell can be, for example, a mammalian cell (e.g., HEK293 cells, Lenti-X 293T cells, 131-IK 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, MRCS 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.
1004021 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 Minis Bio LLC, Lonza nucleofection, Maxagen electroporation and the like. Methods for transfection, transduction or infection are well known to those of skill in the art.
[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.
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 fiinctional protein.
1004051 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.
1004061 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.
1004071 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.
1004081 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 10" particles/kg, or at least about 1 x 10"

particles/kg, or at least about 1 x 1015 particles/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 10' 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, intracra.nial, 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.
VEIL 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 1004121 The following exemplary embodiments, are provided by way of example only.
1004131 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.
1004141 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.
1004151 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
1004161 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.
1004171 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.
1004181 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.
1004191 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 800/u 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 6613, 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, P28, 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. 53A. 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. 5911. The elements of the structural plasmid may be arranged as: MA, pp12, CA, and CasX. In an exemplary embodiment, the XDP system version 74a comprises elements of a structural plasmid arranged as: MA, pp12, 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, p1/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, P28, P10, CA, NC, Pro, and CasX. In an exemplary embodiment, the XDP system version 668 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 Oihoretrovirinae (Lentivirus, Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus), and Spurnaretrovirinae. 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 I
[00434] Embodiment I-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.
1004361 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.
1004371 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.

1004381 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, U1 A, phage replication loop, kissing loop_a, kissing loop b1, 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.
1004411 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 P1 protease, PreScission, b virus Ma 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, metalloproteinases MMP-2, -3, -7, -9, -10, and -11, and enterokinase.

[00443] Embodiment I-10. The XDP system of embodiment 1, wherein the poi polyprotein is a retroviral polyprotein.
[00444] Embodiment I-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.
1004471 Embodiment I-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 pl 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 pl polypeptide; and e. the pl 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), potyvirus HC protease, potyvirus PI protease, PreScission, b virus Ma 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, 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 MV-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.
1004591 Embodiment 1-26. The XDP system of any one of the preceding embodiments of Set 1, 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).

1004751 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 1, 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 (I HIV), 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-Coy), 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, NSO cells, SP2/0 cells, YO myeloma cells, A549 cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO, N11-13T3 cells, COS, W138, MRCS, 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 viva [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, intracerebroyentricular, 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 panicles, or at least about 1 x 109 particles, or at least about 1 x 1010 particles, or at least about 1 x 10" particles, or at least about 1 x 1012 particles, or at least about 1 x 10" particles, or at least about 1 x 10' particles, or at least about 1 x 10"
particles, or at least about 1 x 1016 particles.
Set II
1005091 Embodiment 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 11-2. 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.
1005111 Embodiment 11-3. The XDP system of Embodiment 11-2, 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.
1005121 Embodiment 11-4. 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 pseudo-typing viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.
[00513] Embodiment 11-5. The XDP system of Embodiment 11-4, 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 11-6. 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 11-7. The XDP system of Embodiment 11-6, wherein a. the components are encoded on a single nucleic acid;
ii 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.
1005161 Embodiment 11-8. The XDP system of Embodiment II-1, wherein the encoded components are selected from the gag-pal 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.
1005171 Embodiment 11-9. The XDP system of Embodiment 11-8, wherein a. the components are encoded on a single nucleic acid;
b. a first nucleic acid encodes the gag-pd polyproteinõ 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.
1005181 Embodiment II-10. The XDP system of Embodiment I1-1, 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 11-8-11, 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, U1 A, phage replication loop, kissing loop_a, kissing loop_b1, 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-pot 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-pot 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 I1-1, wherein the encoded components are selected from the MA, the CasX protein, the protease, the protease cleavage site, the 8NA, 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 II-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.
1005271 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-pot polyprotein [00528] Embodiment 11-20 The XDP system of Embodiment 11-2 or Embodiment 11-3, 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-TER-PR polyprotein, the gag polyprotein, and the gag-pol polyprotein are derived from a retrovirus.

1005301 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 (TITV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (Sly), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (B1V).
[00532] Embodiment 11-24. The XDP system of Embodiment 11-23, wherein the lentivirus is REV-1 or S1V.
1005331 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 p1 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 p1 polypeptide; and e. the pl polypeptide and the p6 polypeptide.
1005351 Embodiment 11-27. The XDP system of any one of the preceding embodiments of Set 1 of Set IT, 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 P1 protease, PreScission, b virus Ma 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), NIMP -3, MMP-7, 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.

1005371 Embodiment 11-29. The XDP system of any one of the preceding embodiments of Set I of Set 11, 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 (111V-1), human metapneumovirus, human T-Iymphotro pic virus 1, influenza A, influenza B, influenza C virus, Japanese encephalitis virus, Kaposirs sarcoma-associated herpesvirus (REM), 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 (1111V3), 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 11-31. The XDP system of any one of Embodiments 11-1-29, wherein the pseudotyping viral envelope glyc,oprotein 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.
1005401 Embodiment 11-32. The XDP system of any one of Embodiments 11-1-28, wherein the antibody fragment has binding affinity for a cell surface marker or receptor of a target cell.
1005411 Embodiment 11-33. The XDP system of Embodiment 11-32, wherein the antibody fragment is a scFv.
1005421 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.
1005431 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.
1005441 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 of Set IT, wherein the CasX protein further comprises one or more nuclear localization signals (NLS).
[00559] Embodiment 11-51. The XDP system of Embodiment 11-50, wherein the one or more NLS are selected from the group of sequences consisting of PKKKRKV, KRPAATKKAGQAKICKK, PAAKRVKLD, RQRRNELKRSP, NQSSNEGPMKGGNEGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZEKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, VSRICRPRP, PPICICARED, PQPKK1CPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RICLICKKIKKL, REKKICFLICRR, KRICGDEVDGVDEVAKICKSKK, RICCLQAGMNLEARKTICK, PRPRKIPR, PPRKICRTVV, NLSKICICKRKREK, RRPSRPFRKP, KRPRSPSS, KRGINDRNFWRGENERKTR, PRPPICMARYDN, KRSFSKAF, KLICIKRPVK, PKTRRRPRRSQRKRPPT, RRKKRRPRRICKRR, PKKKSRKPKICKSRK, THCICKHPDASVNFSEFSK, QRPGPYDRPQRPGPYDRP, LSPSLSPLLSPSLSPL, RGKGGKGLGKGGAKRFIRK, PICRGRGRPKRGRGR, and MSRRRKANPTICL,SENAICKLAICEVEN.
[00560] Embodiment 11-52. The CasX variant of Embodiment II-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 II-54, The CasX variant of Embodiment II-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 11-1-54, 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 11-1-54, 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, 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, W138, MRCS, A549, HeLa cells, CHO cells, and 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 11-1-56 are integrated into the genotne 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 H-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).
1005851 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.
1005901 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.
1005911 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 1I-58-62.
1005921 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.
1005931 Embodiment 11-85. The method of Embodiment 1I-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, intrarnedullary, intramuscular, intravenous, intracerebroventricular, intracistemal, intrathecal, intracranial, intralumbar, intratracheal, intraosseous, inhalatoty, intraeontralateral 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 108particles, or at least about 1 x 109 particles, oral least about 1 x 1010 particles, or at least about 1 x 10" particles, or at least about 1 x 1012 particles, or at least about 1 x 10E3 particles, or at least about 1 x 1014 particles, or at least about 1 x 1015 particles, or at least about 1 x 10' 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 viva [00608] Embodiment 11-100. The method of Embodiment 11-99, wherein the XDP is administered to a subject.
[00609] Embodiment H-101.The method of Embodiment 11-100, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.
[00610] Embodiment II-102.The method of any one of Embodiments 11-99-101, 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 iO3 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 10' particles.
Set In [00612] Embodiment I11-1. 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);
a gag-pol polyprotein; and (k) a protease capable of cleaving the protease cleavage sites.
[00613] Embodiment The XDP system of Embodiment 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 111-3. The XDP system of Embodiment 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-poi 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 wherein the encoded components are selected from the gag-TFR-PR polyprotein, the protease cleavage site, the CasX
protein, the gNA, and the pseudo-typing viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.
[00615] Embodiment The XDP system of Embodiment 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 111-6. 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 111-7. The XDP system of Embodiment 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 111-8. The XDP system of Embodiment In-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.
1006191 Embodiment 111-9. The XDP system of Embodiment 111-8, 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 II1-10. 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 I11-10, 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 8NA 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 111-12. The XDP system of any one of Embodiments In- 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 111-13. The XDP system of Embodiment 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 111-14. The XDP system of Embodiment 111-13, 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 111-15. 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.
1006261 Embodiment 111-16 The XDP system of Embodiment 111-15, 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 I11-15 or Embodiment 11I-16, further comprising the CA component linked between the MA and the CasX protein components with intervening protease cleavage sites.
[00628] Embodiment 111-18. The XDP system of Embodiment 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 111-19. The XDP system of Embodiment 111-18, 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 111-3, 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.
1006321 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 111-23, 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.
1006361 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 p1 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 p1 polypeptide; and (e) the p1 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 P1 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), WIMP -3, MMP-7, MMP-9, WIMP-b, MMP-11, and enterokinase.
1006381 Embodiment III-28. The XDP system of Embodiment III-27, wherein the protease capable of cleaving the protease cleavage site is HIV-1 protease.
1006391 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 califorrtica multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Bona 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 (FIFIV5), human foamy virus, human herpesvirus (HHV), human Herpesvirus 7, human herpesvirus type 6, human herpesvirus type 8, human immunodeficiency virus 1 (mV-1), human metapneumovirus, human T-lymphotro pic virus 1, influenza A, influenza B, influenza C virus, Japanese encephalitis virus, Kaposi's sarcoma-associated herpesvirus (HEIV8), 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 (HEIV3), 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 111-29, 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 III-Embodiments III-1-28, wherein the antibody fragment has binding affinity for a cell surface marker or receptor of a target cell.
[00643] Embodiment I11-33. The XDP system of Embodiment I11-32, wherein the antibody fragment is a scFv.
[00644] Embodiment 1I-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 111-36. The XDP system of Embodiment 111-29 or Embodiment I11-35, 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 111-36, wherein the targeting sequence of the guide RNA consists of 20 nucleotides.
[00648] Embodiment III-38. The XDP system of Embodiment 111-36, 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 111-42. 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 111-46. 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%, oral 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 Ill-Si. 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, NQSSNEGPMKGGNEGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILICRRNV, VSRICRPRP, PPKKARED, PQPICICKPL, SALIKKKKKMAP, DRLRR, PKQICICRIC, RICLICKKIICKL, REKKICFLICRR, ICRKGDEVDGVDEVAKKKSKK, RKCLQAGMNLEARKTICK, PRPRKIPR, PPRKICRTVV, NLSKICKKRKREK, RRPSRPFRKP, KRPRSPSS, KRGINDRNEWRGENERKTR, PRPPICMARYDN, KRSFSKAF, KLKIKRPVK, PKTRRRPRRSQRICRPPT, RRKICRRPRRICKRR, PKKKSRKPKICKSRK, HICICICHPDASVNESEFSK, QRPGPYDRPQRPGPYDRP, LSPSLSPLLSPSLSPL, RGKGGKGLGKGGAKRHRK, PKRGRGRPKRGRGR, and MSRRRKANPTKLSENAICKLAICEVEN.
[00662] Embodiment 1I-52. The CasX variant of Embodiment III-50 or Embodiment 1, 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 111-54. The CasX variant of Embodiment III-50 or Embodiment II1-5 1, 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 III-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 111-58, 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 111-59, 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 11I-59, wherein the donor template nucleic acid sequence comprises a mutation compared to the target nucleic acid.
[00672] Embodiment 111-62, The XDP system of Embodiment 111-61, 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 III-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 1I-64. The XDP system of Embodiment 111-63, 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 111-65. The XDP system of Embodiment 11I-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 111-66, wherein the cell is a packaging cell.
[00678] Embodiment 11I-68, The eukaryotic cell of any one of Embodiments III-Embodiments III-66 or Embodiment III-67, wherein the eukaryotic cell is selected from the group consisting of ITEK293 cells, Lenti-X 293T cells, BHK cells, 11epG2, Saos-2, flu117, 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, MRCS, A549, HeLa cells, CHO
cells, and HT1080 cells.

[00679] Embodiment 11I-69. The eukaryotic cell of Embodiment 111-67 or Embodiment 111-68, wherein the packaging cell comprises one or more mutations to reduce expression of a cell surface marker.
[00680] Embodiment 11I-70. The eukaryotic cell of any one of Embodiments III-Embodiments 11I-66-69, wherein all or a portion of the nucleic acids encoding the XDP
system of any one of Embodiments 111-1-56 are integrated into the genome of the eukaryotic cell.
[00681] Embodiment 11I-7 1. 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 111-67-70 under conditions such that XDPs are produced; and (b) harvesting the XDPs produced by the packaging cell.
[00682] Embodiment 11I-72. An XDP produced by the method of Embodiment HI-71.
[00683] Embodiment 11I-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);
(t) 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 11I-74. The XDP of Embodiment 11I-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 11I-75. The XDP of Embodiment 11I-74, further comprising the capsid polypeptide (CA).
[00686] Embodiment 11I-76. The XDP of Embodiment 111-74 or Embodiment further comprising the nucleocapsid polypeptide (NC).
[00687] Embodiment 11I-77. The XDP of any one of Embodiments 11I-74-76, further comprising an RNA binding domain.

[00688] Embodiment 111-78. 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, U1 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 111-74-78, 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 111-74-79, comprising the CasX of any one of Embodiments 111-47-65 and the guide RNA of any one of Embodiments III-34-46, [00691] Embodiment 111-81, The XDP of any one of Embodiments 111-74-80, 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.
1006921 Embodiment III-82. The XDP of any one of Embodiments 111-73-80, 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, Boma 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 (HEWS), human foamy virus, human herpesvirus (HMV), human Herpesvirus 7, human herpesvirus type 6, human herpesvirus type 8, human immunodeficiency virus 1 (I-11V-1), human metapneumovirus, human T-lymphotro pic virus 1, influenza A, influenza B, influenza C virus, Japanese encephalitis virus, Kaposi's sarcoma-associated herpesvirus (FI11V8), 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 (HEIV3), 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 111-83, The XDP of any one of Embodiments 111-73-82, further comprising the donor template nucleic acid sequence of any one of Embodiments 111-58-62.
[00694] Embodiment 111-84. 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 111-73-83, 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 111-85. The method of Embodiment 111-84, wherein the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence_ 1006961 Embodiment 111-86. The method of Embodiment 111-84, wherein the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence.
[00697] Embodiment 111-87 The method of any one of Embodiments 111-84-86, wherein the modification comprises insertion of the donor template into the target nucleic acid sequence.
[00698] Embodiment 111-88. The method of any one of Embodiments 111-84-87, wherein the cell is modified in vitro [00699] Embodiment 111-89. The method of any one of Embodiments 111-84-87, wherein the cell is modified in vivo [00700] Embodiment 111-90 The method of Embodiment 111-89, wherein the XDP is administered to a subject.
[00701] Embodiment 111-91. The method of Embodiment 111-90, wherein the subject is the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

1007021 Embodiment 111-92. The method of Embodiment 111-90 or Embodiment 111-91, 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 111-90-92, 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 panicles, 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.
1007051 Embodiment I11-95. 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 111-79-83, such that the RNP enters the cell.
[00706] Embodiment III-96. The method of Embodiment 111-95, wherein the RNP
binds to the target nucleic acid.
1007071 Embodiment 111-97. The method of Embodiment 111-96, wherein the target nucleic acid is cleaved by the CasX
[00708] Embodiment 111-98. The method of any one of Embodiments 111-95-97, wherein the cell is modified in vitro [00709] Embodiment 111-99. The method of any one of Embodiments 111-95-97, wherein the cell is modified in viva [00710] Embodiment III-100. The method of Embodiment 111-99, 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 II1-103.
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 103 particles, or at least about 1 x 109 particles, oral 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 10" 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 pl 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 p1 spacer, and a p6 spacer;
(h) a CasX protein;
(i) a guide nucleic acid (gNA);
(i) 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;
(I) a protease cleavage site;
(m) a gag-transframe region-pol protease polyprotein (gag-TFR-PR);
(n) a gag-pal 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.

1007161 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.
1007171 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.
1007181 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.
1007191 Embodiment IV-6. The XDP system of Embodiment IV-I, wherein the encoded components are selected from the gag polyprotein, the protease cleavage site, the protease, the CasX protein, the 8NA and the pseudotyping viral envelope glycoprotein or antibody fragment, wherein the components are encoded on one, two, or three individual nucleic acids.
1007201 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-poi 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, 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.
[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 g,lycoprotein 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-poi 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 I1v-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.

1007341 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.
1007351 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.
1007361 Embodiment IV-23. The XDP system of Embodiment IV-22, wherein the lentivirus is selected from the group consisting of human immunodeficiency-1 (H1V-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FLY), and bovine immunodeficiency virus (BIV).
1007371 Embodiment IV-24, The XDP system of Embodiment IV-23, wherein the lentivirus is HEY-1 or SIN.
1007381 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.
1007391 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 p1 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 p1 polypeptide; and (e) the pl polypeptide and the p6 polypeptide.
1007401 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 P1 protease, PreScission, b virus NIa protease, B virus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A 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, metalloproteinase-2 (MMP-2), MMP -3, MMP-7, MMP-9, MMP-10, MMP-11, and enterokinase.

1007411 Embodiment 1V-28. The XDP system of Embodiment 1V-27, wherein the protease capable of cleaving the protease cleavage site is HIV-1 protease.
1007421 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, Boma 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 (HEWS), human foamy virus, human herpesvirus (HMV), human Herpesvirus 7, human herpesvirus type 6, human herpesvirus -type 8, human immunodeficiency virus 1 (HIV-1), human metapneumovirus, human T-lymphotro pie 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 (1111V3), varicella zoster virus (HEIV3), 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.
1007431 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 glyc,oprotein 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 D/-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 D/-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 D/-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, KRPAATKKAG-QAKICKK, PAAKRVKLD, RQRRNELKRSP, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFICNKGKDTAELRRRRVEVSVELRKAKICDEQILKRRNV, VSRICRPRP, PPIUCARED, PQPKKICPL, SALLKKKKKMAP, DRLRR, PKQKKRK, RICLICKKIKKL, REKKKFLICRR, ICRKGDEVDGVDEVAKICKSKK, RKCLQAGMNLEARKTICK, PRPRKIPR, PPRK1CRTVV, NLSKICICKRKREK, RRPSRPFRKP, KRPRSPSS, KRGINDRNFWRGENERKTR, PRPPICMARYDN, KRSFSKAF, KLKIKRPVK, PKTRRRPRRSQRICRPPT, RRKKRRPRRKKRR, PKKKSRKPKICKSRK, HICICKUPDASVNFSEFSK, QRPGPYDRPQRPGPYDRP, LSPSLSPLLSPSLSPL, RGKGGKGLGKGGAKRHRK, PICRGRGRPKRGRGR, and MSRRRKANPTICLSENAICKLAKEVEN.
[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.
1007691 Embodiment IV-56. The XDP system of any one of Embodiments IV-I-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 W-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, 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, MRCS, 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 IV-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 IV-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, U1 A, phage replication loop, kissing loop a, kissing loop_b I, 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.
1007951 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, Boma 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 (HEWS), human foamy virus, human herpesvirus (HMV), human Herpesvirus 7, human herpesvirus type 6, human herpesvirus -type 8, human immunodeficiency virus 1 (HIV-1), human metapneumovirus, human T-Iymphotro pie 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 (HEIV3), varicella zoster virus (HIIV3), 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.
1007961 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 W-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 W-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 viva [00803] Embodiment IV-90. The method of Embodiment D/-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 W-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 W-90-92, wherein the XDP is administered to the subject using a therapeutically effective dose [00807] Embodiment IV-94 The method of Embodiment W-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 10 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 10" particles, or at least about 1 x 1012 particles, or at least about 1 x 10" 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 B1-95-97, 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 viva [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, oral 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 10E3 particles, or at least about 1 x 1014 particles, or at least about 1 x 1015 particles, or at least about 1 x 10' particles.
Set V
[00817] Embodiment V-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 [00818] Embodiment V-2. The XDP system of Embodiment V-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.
[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 1D
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, 501õ
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 CR1SPR
protein is a Class 1 or Class 2 CRISPR protein.

1008241 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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-11. 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 (AS0s), 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-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.
[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-15. 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-pot 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 P28 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 pl 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, 501õ
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 Cas12a, Cas12b, Cas12c, Caslal (CasY), Cas12j and CasX.
1008561 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.
1008571 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), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 134), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKICDEQILKRRNV (SEQ ID NO: 135), VSR1CRPRP (SEQ ID NO: 136), PPICKARED (SEQ ID NO: 137), PQPICKICPL (SEQ ID NO:

138), SALIICKICICKMAP (SEQ ID NO: 139), DRLRR (SEQ ID NO: 140), PKQKKRK (SEQ
ID NO: 141), RICLKKKIKKL (SEQ ID NO: 142), REKKICFLKRR (SEQ ID NO: 143), KRKGDEVDGVDEVAICKKSICIC (SEQ ID NO: 144), RKCLQAGMNLEARKTKK (SEQ ID
NO: 145), PRPRK1PR (SEQ ID NO: 146), PPRKICRTVV (SEQ ID NO: 147), NLSKICKICRICREK (SEQ ID NO: 148), RRPSRPFRKP (SEQ ID NO: 149), KRPRSPSS (SEQ
ID NO: 150), ICR.GINDRNFWRGENERKTR (SEQ ID NO: 151), PRPPICMARYDN (SEQ ID
NO: 152), KRSFSKAF (SEQ ID NO: 153), KLKIKRPVK (SEQ ID NO: 154), PKTRRRPRRSQRICRPPT (SEQ ID NO: 156), RRKKRRPRRICICRR (SEQ ID NO: 159), P1CKKSRKPKICKSRK (SEQ ID NO: 160), HICICKHPDASVNFSEFSK (SEQ ID NO: 161), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 162), LSPSLSPLLSPSLSPL (SEQ ID NO: 163), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 164), PKRGRGRPKRGRGR (SEQ ID NO: 165), MSRRRKANPTICLSENAICKLAKEVEN (SEQ ID NO: 157), PKKICRKVPPPPAA1CRVKLD
(SEQ ID NO: 155), and PICKKRKVPPPPICKKRKV (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 (AS0s), 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.
1008651 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, 501õ
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, 501õ 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).
1008801 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.
1008811 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 (AS0s), a double-stranded RNA interference (RNA 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 p1 peptide;
(b) an Inv p6 peptide;
a Gag-Pol polyprotein;
(d) one or more protease cleavage sites;
(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and (0 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, 501õ
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, 501õ 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.

1009131 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 Cas12a, Cas12b, Cas12c, Caslal (CasY), Cas12j 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 (AS0s), 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-110. The XDP of Embodiment V-109, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.
[00927] Embodiment V-111. The XDP system of Embodiment V-110, 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-112. 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-113. The XDP system of Embodiment V-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 [00930] Embodiment V-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.
[00931] Embodiment V-115. The XDP system of Embodiment V-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).
[00932] Embodiment V-116. The XDP system of Embodiment V-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).
[00933] Embodiment V-117. The XDP system of any one of Embodiments V-114-116, wherein the nucleic acids encode one or more components selected from (a) an HIV pl peptide;
(b) an HIV p6 peptide;
(c) a Gag-Pal polyprotein;
(d) one or more protease cleavage sites;
(e) a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and (0 a gag-transframe region-pol protease polyprotein.
[00934] Embodiment V-118. The XDP system of any one of Embodiments V-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.
1009351 Embodiment V-119. The XDP system of Embodiment V-118, wherein the tropism factor is a g,lycoprotein 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, 501õ 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.
1009361 Embodiment V-120. The XDP system of Embodiment V-118, wherein the tropism factor is a g,lycoprotein 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, 501õ
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.
1009371 Embodiment V-121. The XDP system of Embodiment V-120, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).
1009381 Embodiment V-122. The XDP system of any one of Embodiments V-114-121, wherein the therapeutic payload comprises a protein, a nucleic acid, or comprises both a protein and a nucleic acid.
1009391 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 V128. 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 (AS0s), 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 lot 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-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.
1009521 Embodiment V136. 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.
1009531 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.
1009541 Embodiment V-138. The XDP system of any one of Embodiments V-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.
1009551 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.
1009571 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.
1009581 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 p12 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 p12 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 p 1 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 (0 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 g,lycoprotein 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, 501õ 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 g,lycoprotein having a sequence selected from the group consisting of SEQ lD
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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 (AS0s), 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 p1 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 pl peptide, and a p6 peptide.
1009911 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-Pal 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.
1009921 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 (111V-2), simian immunodeficiency virus (Sty), feline immunodeficiency virus (Hy), and bovine immunodeficiency virus (BIV).
1009931 Embodiment V-177. The XDP system of Embodiment V-176, wherein the lentivirus is HIV-1 1009941 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.
1009951 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, 501õ 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.
1009961 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.
1009971 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.
1009991 Embodiment V-183. The XDP system of Embodiment V-182, wherein the protein payload is selected from the group consisting of a cytolcine, an interleulcin, 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.
[0010011Embodiment 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 (AS0s), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.
[0010071Embodiment 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.
10010081 Embodiment V-192. The XDP system of Embodiment V-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.
10010091Embodiment 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.
10010101Embodiment 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.
[0010111Embodiment 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.
10010121 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.
10010131Embodiment 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.
[0010141Embodiment 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.
10010151Embodiment V-199. The XDP of Embodiment V-198, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.
10010161Embodiment 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.
10010171Embodiment V-201. The XDP of Embodiment V-198, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.
10010181Embodiment 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.
100101.91Embodiment 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.
10010201Embodiment 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.
10010211Embodiment 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.
10010221Embodiment 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 pl 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 (1) a gag-transframe region-pol protease polyprotein.
10010231Embodiment 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.
10010241Embodiment V-208. The XDP system of Embodiment V-207, wherein the tropism factor is a g,lycoprotein 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, 501õ 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.
10010251Embodiment 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, 501õ
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.
10010261 Embodiment V-210. The XDP system of Embodiment V-209, wherein the tropism factor is glycoprotein G from vesicular stomatitis virus (VSV-G).
10010271Embodiment 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.

[0010291Embodiment V-213. The XDP system of Embodiment V-212, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.
[0010301Embodiment 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.
[0010311Embodiment V-215. The XDP system of Embodiment V-214, wherein the CRISPR protein is a Type V protein selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j and CasX.
[0010321Embodiment 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.
[0010331Embodiment 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.
[0010341Embodiment 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.
[0010351Embodiment 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 (AS0s), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.
[0010361Embodiment 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.
[0010371Embodiment 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.
10010381 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.
10010391 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.
10010401 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.
10010411Embodiment 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.
10010421 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.
10010431Embodiment V-227. 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.
10010441Embodiment V-228. The XDP of Embodiment V-227, wherein the therapeutic payload is encapsidated within the XDP upon self-assembly of the XDP.
10010451 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.

10010461Embodiment V-230. The XDP of Embodiment V-227, wherein the tropism factor is incorporated on the XDP surface upon self-assembly of the XDP.
10010471Embodiment 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 10010481Embodiment 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.
10010491Embodiment 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.
10010501Embodiment 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.
10010511Embodiment 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.
10010521Embodiment 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-Pal polyprotein, the protease of the gag-transframe region-pal protease polyprotein, or the non-retroviral, heterologous protease.
10010531Embodiment 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.
10010541 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-Pot polyprotein 0010551 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 P1 protease, PreScission (HRV3C protease), b virus Ma protease, B virus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A
protease, rhinovirus 2A 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.
10010561Embodiment V-240. The XDP system of Embodiment V-239, wherein the non-retroviral, heterologous protease is PreScission (IIRV3C protease).
10010571Embodiment V-241. The XDP system of Embodiment V-239, wherein the non-retroviral, heterologous protease is tobacco etch virus protease (TEV).
10010581Embodiment 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.
10010591Embodiment 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.
10010601Embodiment 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.
10010611Embodiment 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.
10010621Embodiment 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, Ul A, and phage I&-loop.
10010631Embodiment V-247. A eukaryotic cell comprising the XDP system of any one of the preceding embodiments of Set V.
10010641Embodiment V-248. The eukaryotic cell of Embodiment V-247, wherein the cell is a packaging cell 10010651Embodiment V-249. The eukaryotic cell of Embodiment V-247 or Embodiment V-248, wherein the eukaryotic cell is selected from the group consisting of 11EIC293 cells, Lenti-X 293T cells, MIK 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, NIFI3T3 cells, COS, W138, MRCS, A549, HeLa cells, CHO cells, and HT1080 cells.
10010661 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.
10010671 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.
10010681Embodiment 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.
10010691Embodiment V-253. An XDP produced by the method of Embodiment V-252.
10010701 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.
[0010711Embodiment 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.
10010721 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.
10010731Embodiment 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.
10010741Embodiment 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.
10010751 Embodiment V-259. The method of any one of Embodiments V-255-258, wherein the cell is modified in vitro or ex vivo.

10010761Embodiment V-260. The method of any one of Embodiments V-255-258, wherein the cell is modified in viva 10010771 Embodiment V-261. The method of Embodiment V-260, wherein the XDP is administered to a subject.
10010781Embodiment 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.
10010791 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.
10010801 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.
10010811 Embodiment V-265. The method of Embodiment V-264, wherein the XDP is administered at a dose of at least about 1 x 10A5 particles/kg, or at least about 1 x 10^6 particles/kg, or at least about 1 x 101\7 particles/kg, or at least about 1 x HY'S particles/kg, or at least about 1 x 10.'9 particles/kg, or at least about 1 x 1090 particles/kg, or at least about 1 x 1091 particles/kg, or at least about 1 x 1092 particles/kg, or at least about 1 x 1093 particles/kg, or at least about 1 x 1094 particles/kg, or at least about 1 x 101\15 particles/kg, or at least about 1 x 10^16 particles/kg.
10010821Embodiment 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.
10010831 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.

10010841Embodiment 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.
10010851Embodiment V-269. The method of Embodiment V-268, wherein the RNP
binds to the target nucleic acid.
10010861 Embodiment V-270. The method of Embodiment V-269, wherein the target nucleic acid is cleaved by the CasX.
10010871 Embodiment V-271. The method of any one of Embodiments V-268-270, wherein the cell is modified in vitro.
10010881 Embodiment V-272. The method of any one of Embodiments V-268-270, wherein the cell is modified in vivo.
10010891Embodiment V-273. The method of Embodiment V-272, wherein the XDP is administered to a subject.
10010901Embodiment 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.
[0010911Embodiment 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.
10010921Embodiment V-276. The method of Embodiment V-275, wherein the XDP is administered at a dose of at least about 1 x 10^5 particles/kg, or at least about 1 x 101\6 particles/kg, or at least about 1 x 10'7 particles/kg, or at least about 1 x 10^8 particles/kg, or at least about 1 x 10^9 particles/kg, or at least about 1 x 101\10 particles/kg, or at least about 1 x 10^11 particles/kg, or at least about 1 x 1092 particles/kg, or at least about 1 x 1093 particles/kg, or at least about 1 x 1094 particles/kg, or at least about 1 x 101\15 particles/kg, or at least about 1 x 1096 particles/kg.
10010931 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.
10010941Embodiment 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 p12 peptide (f) a PP21/24 peptide;
(8) a P12/P3/P8 peptide;
(h) a P20 peptide;
(1) A pl peptide; and (i) a p6 peptide.
10010951 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.
10010961 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 97A, at least about 98%, or at least about 99% sequence identity thereto.
10010971Embodiment 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_ 10010981Embodiment 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.

10010991 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 tlu-ombolytic protein, a CRISPR protein, and an anti-cancer modality.
10011001 Embodiment V-284. The XDP particle of Embodiment V-283, wherein the CRISPR protein is a Class 1 or Class 2 CRISPR protein.
[001101jEmbodiment 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.
10011021 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j and CasX.
10011031Embodiment 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.
10011041 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 (AS0s), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.
10011051 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.
10011061Embodiment 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 haying 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.

10011071Embodiment V-291. The XDP particle of Embodiment V-290, wherein the scaffold sequence comprises a sequence of SEQ ID NOS: 597-781.
10011081Embodiment 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.
10011091Embodiment 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.
10011101Embodiment 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, [0011111Embodiment 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.
10011121Embodiment 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.
10011131The 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 10011141An 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.coll. 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. coil 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 13-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 REPES-NaOH, 250 m1VI NaC1, 5 mM MgCl2, 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 Construct DNA Sequence SV40 NLS-CasX-SV40 NLS-TEV cleavage site ¨
(SEQ ID NO: 354) TwinStrep tag 2. Purification 10011151Frozen 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 m/vl HEPES-NaOH, 250 mM NaCl, 5 mM
MgCl2, 1 mM TCEP, 10% glycerol, pH 8), then with 5 CV of Heparin Buffer B
(Buffer A with the NaC1 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 m114 NaC1, 5 mM MgCl2, 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 NaC1, 1 mM TCEP, 10% glycerol, pH 7.25) operated by an AKTA 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 10011161 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 Thai: 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 Thai: 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 10011171 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 Planctomyeetes CasX SEQ Wi 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 oIC88 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, oIC688 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 XbaI and SpeI 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 E coil 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. coh (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).
10011181 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.
10011191CasX 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.
10011201 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.
10011211 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 Construct DNA Amino Acid Sequence Sequence CasX 119 (SEQ ID
QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPE
NO: 340) NI PQPI SNTSRAN LN KLLTDYTEM KKAI LHVYWEEFQKDPVG LMSRVAQP
APKN I DQR KLI PVKDG N ER LTSSG FACSQCCQPLYVYKLEQVN DKGKPHTN
YFGRCNVSEHERLI LLSPH KPEAN D ELVTYSLG KFGQRALD FYSI HVTR ESN H

KRLANLKDIASANG LAFPKITLPPQPHTKEGIEAYNNVVAQIVIWVNLNLW
QKLKIG RDEAKPLQRLKGEPSFPLVERQAN EVDWWDMVCNVKKLINEKKE
DGKVFWQN LAGYKRQEALRPYLSSE EDRKKG KKFARYQFGDLLLH LE KKH
G EDWG KVYDEAWERI DKKVEGLSKH I KLEEE RRSEDAQSKAALTDWLRAK
ASFVIEG LKEADKDEFCRCELKLQKWYG DLRG KPFAI EAENSI LDISG FSKQY
NCAFI WQKDGVKKLN LYLI I NYFKGG KLRFKKI KPEAFEAN RFYTVI N KKSG El VPMEVNINFDDPNLIILPLAFGKRO.GREFIWNDLLSLETGSLKLANGRVIEK
TLYN RRTRQDEPALEVALTFE RREVLDSSN I KPM N LI GI DRGENI PAVIALTD
PEGCPLSRFKDSLG N PTH I LRI GESYKEKQRTI QAKKEVEQRRAGGYSRKYAS
KAKN LADDMVRNTARDLLYYAVTQDAM LI FEN LSRGFG RQG KRTFMAER
QYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGITITSADYDRVLE
KLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESV
NN DI SSWTKGRSGEALSLLKKR FSH RPVQEKFVCLNCGFETHADEQAALN I
ARSWLFLRSQEYKKYQTN KTTG NTDKRAFVETWQSFYRKKLKEVWKPAV
(SEQ ID NO: 343) CasX 457 (SEQ ID
QEIKRINKIRRRLVKDSNTKKAGICGPMKTLLVRVMTPDLRERLENLRKKPE
NO: 341) NI PQPI SNTSRAN LN KLLTDYTEM KKAI LHVYWEEFQKDPVG LMSRVAQP
APKN I DQR KLI PVKDG N ER LTSSG FACSQCCOPLYVYKLEQVN DKGKPHTN
YFGRCNVSEHERLI LLSPH KPEAN D ELVTYSLG KFGQRALD FYSI HVTR ESN H
PVKPLECII GGNSCASG PVGKALSDACMGAVASFLTKYQDII LEHKKVIKKNE
KRLANLKDIASANG LAFPKITLPPQPHTKEGI EAYN NVVAQI VI WVNLNLW
QKLKIG RDEAKPLQRLKGEPSFPLVERQAN EVDWWDMVCNVKKLINEKKE
DGKVFWQN LAGYKRQEALRPYLSSPEDRKKGKKFARYQLGDLLLHLEKKH
G EDWG KVYDEAWERI DKKVEGLSKH I KLEEE RRSEDAQSKAALTDWLRAK
ASEVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGESKQY
NCAFI WQKDGVKKLN LYLI I NYFKGG KLRFKKI KPEAFEAN RFYTVI N KKSG El VPMEVNENFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEK
PLYNRRTRQDEPALEVALTFERREVLDSSN I KPM N LIGVDRG EN I PAVIALT
DPEGCPLSRFKDSLG N PTH I LRIG ESYKEKQRTIQAKKEVEQRRAGGYSRKY
ASKAKN LAD DMVRNTAR DLLYYAVTQDAM LI FEN LSRG FG RQG KRTFMA
E RQYTR ME DW LTAKLAYEGLSKTYLSKTLAQYTSKTCSNCG FTITSA DYD RV
LEKLKKTATGWMTTINGKELKVEGQITYYNRRKRQNVVKDLSVELDRLSEE
SVN ND ISSWTKGRSGEALSLLKKRFSH RPVQEKFVCLN CG FETHADEQAAL
NIARSWLFLRSQEYKKYQTN KTTGNTDKRAFVENVQSFYRKKLKEVWKPA
V (SEQ ID NO: 344) CasX 438 (SEQ ID ..
QEIKRINKIRRRLVKDSNIKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPE
NO: 342) NI PQPI SNTSRAN LN KLLTDYTEM KKAI LHVYWEEFQKDPVG LMSRVAQP
APKN I DQR KLI PVKDG N ER LTSSG FACSOCCOPLYVYKLEQVN DKGKPHTN

YFGRCNVSEHERLI LLSPHKPEANDELVTYSLGKFGQRALDFYSI HVTR ESN H

KRLANLKDIASANG LAFPKITLPPQPHTKEGI EAYN NVVAQI VI WVNLNLW
QKLKIGRDEAKPLQRLKGFPSFPLVERQAN EVDWWDMVCNVKKLINEKKE
DGKVFWQN LAGYKRQEALRPYLSSEEDRKKG KKFARYQLGDLLKHLEKKH
GEDWGKVYDEAWERI DKKVEGLSKH I KLEEE RRSEDAQSKAALTDW LRAK
ASFVI EGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSI LDISGFSKQY
NCAF I WQKDGVKKLN LYLI I NYFKGG KLRFKKI KPEAFEAN RFYTVI N KKSG El VP M EVN FN FD DPN LI I LPLAFG KRQG REF IWN DLLSLETGSLKLA NG RVI EK
TLYN RRTRQDEPALFVALTFE RREVLDSSN I KPM N LI GVDRGENI PAVIALTD
PEGCPLSRFKDSLG N PTH I LRIGESYKEKORTIQAKKEVEQRRAGGYSRKYAS
KAKN LADDMVRNTARDLLYYAVTQDAM LI F EN LSRGFG RQG KRTFMAER
QYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLE
KLKKTATGWMTTI NGKELKVEGQITYYNRRKRQNVVKDLSVELDRLSEESV
N N DI SSWTKGRSGEALSLLKKR FSH RPVQEKFVCLNCGF ETHADEQAALN I
ARSWLFLRSQEYKKYQTNKTTG NTDKRAFVETVVQSFYRKKLKEVWKPAV
(SEQ ID NO: 345) Example 3: CasX construct 438, 491, 515 and 527 10011221 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 II 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 lD
NO: 2, with a A708K substitution, a L379R 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 Rio 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 pStx1 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 pStx1 were digested with XbaI and BamHIE
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.
10011231To 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 XbaI and SpeI
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-1ot promoter for the protein as well as a selection marker for both puromycin and carbenicillin. pStX56 includes an EF-1ot 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:
10011241CasX 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.
0011251 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.
10011261CasX variant 515: following the same expression and purification scheme for WT
CasX SEQ 113 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 Construct DNA Amino Acid Sequence Segue nce CasX 488 (SEQ QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRER
ID NO: LENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWE
346) EFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGF
ACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILL
AOLKPEKDSDEAVTYSLGKFGORALDFYSIHVTKESTHPVKPL
AQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVK
GNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVI
ARVRMVVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQA
NEVDVVVVDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALR
PYLSSEEDRKKGKKFARYQFGDLLLHLEKKHGEDWGKVYDE
AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDVVLRAKAS
FVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSILDI
SGFSKQYNCAFIVVQKDGVKKLNLYLIINYFKGGKLRFKKIKPE
AFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQG
REFIVVNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVA
LTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCPLSR
FKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKY
ASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQ
GKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSK
TCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQI
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGE
ALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSWL
FLRSQEYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEVVVK
PAV (SEQ ID NO: 350) CasX 491 (SEQ QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRER
ID NO: LENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWE
347) EFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGF
ACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILL
AQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPL
AQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVK

GNQ KR LESLR ELAGKENLEYPSVTLPPQ PHTKEGVDAYNEVI
ARVRMVVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQA
NEVDVVVVDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALR
PYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGIONDE
AWER I DKKVEGLSKH IKLEE ERRSE DAQSKAALTDVVLRAKAS
FVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSILDI
SGFS KQYNCAF IWQ KDGVKKLN LYL I INYF KGG KLRFKKI KPE
AFEANRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQG
REF IWNDLLSLETGS LKLANG RVI EKTLYN RRTRQ DE PALFVA
LTFERREVLDSSN IKPM NLIGVDRGEN I PAVIALTDP EGC PLSR
F KDSLGNPTHILR IGESYKEKQ RTIQAKKEVEQR RAGGYSRKY
ASKAKN LADDMVRNTARD LLYYAVTQ DAML I FENLSRGFGRQ
GKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSK
TCS N CGFTITSADYD RVLEKLKKTATGVVMTTI NGKE LKVEGQ I
TYYN RYKRQ NVVKD LSVELDRLS E ESVN ND I SSWTKGRSGE
ALS LLKKRFS H RPVQ EKFVC LNCGFETHADEQAALN IARSWL
FLRSQEYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEVVVK
PAV (SEQ ID NO: 351) CasX 515 (SEQ QEIKRIN KIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRER
ID NO: LENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWE
348) EFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGF
ACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILL
AOLKP EKDSDEAVTYSLGKFGQ RALD FYS I HVTKESTH PVKPL
AQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVK
GNQKRLESLRELAGKENLEYPSVTLPPQ PHTKEGVDAYNEVI
ARVRMVVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQA
NEVDVVVVDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALR
PYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE
AWER I DKKVEGLSKH IKLEE ERRSE DAQSKAALTDVVLRAKAS
FVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSILDI
SGFS KQYNCAF IVVQ KDGVKKLN LYL I INYF KGG KLRFKKI KPE
AFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQG
REF IVVNDLLSLETGS LKLANG RVI EKTLYN RRTRQ DE PALFVA
LTFERREVLDSSN IKPM NLIGVDRGEN I PAVIALTDP EGGPLSR
F KDSLGNPTHILR IGESYKEKQ RTIQAKKEVEQR RAGGYSRKY
AS KAKN LADDMVRNTARD LLYYAVTQ DAM L I FE N LS RGFG RQ
GKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEG
IDITYYNRYKRONVVKDLSVELDRLSEESVNNDISSVVTKGRSG
EALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALN IARSW
LFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVW
KPAV (SEQ ID NO: 352) CasX 527 (SEQ Q EIKR IN KIR RR LVKDSNTKKAGKTRGPM KTLLVRVMTP DLR E
ID NO: RLEN LRKKPEN IPQP ISNTSRANLNKLLTDYTEMKKAILHVYW
349) EEFQ KD PVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAG
FACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLIL
LAQ LKP E KDS DEAVTYSLGKFGQ RALD FYS I HVTKE STH PVKP
LAO IAGN RYASG PVGKALS DACMGTIAS FLS KYQ DI II EH QKVV

KGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEV
IARVRMVVVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQA
NEVDVVVVDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALR
PYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGIONDE
AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDVVLRAKAS
FVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENSILDI
SGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPE
AFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQG
REF IWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVA
LTFERREVLDSSNIKPMNLIGVDRGENIPAVIALTDPEGCPLSR
FKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKY
ASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQ
GKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTSK
TCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQI
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVIKGRSGE
ALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSWL
FLRSQEYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEVVVK
PAV (SEQ ID NO: 353) Example 4: Design and Generation of CasX Constructs 278-280, 285-288, 290, 291, 293, 300, 492, and 493 10011271ln 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 pStx.34 were transformed into chemically-competent Turbo Competent E. colt 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. cob (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.
10011281 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 SV4ONLS-CasX119.
Construct 279 has the configuration CasX119-SV4ONLS. Construct 280 has the configuration CasX119-SV4ONLS-PPP linker-SV4ONLS. Construct 285 has the configuration CasX119-SV4ONLS-PPP linker-SynthNLS3. Construct 286 has the configuration CasX119-PPP linker-SynthNLS4. Construct 287 has the configuration CasX119-SV4ONLS-PPP
linker-SynthNLS5. Construct 288 has the configuration CasX119-SV4ONLS-PPP linker-SynthNLS6.
Constrict 290 has the configuration CasX119-SV4ONLS-PPP linker-EGL-13 NLS.
Construct 291 has the configuration CasX119-SV4ONLS-PPP linker-c-Myc NLS. Construct 293 has the configuration CasX119-SV4ONLS-PPP linker-Nucleolar RNA Helicase II NLS.
Construct 300 has the configuration CasX119-SV4ONLS-PPP linker-Influenza A protein NLS.
Construct 492 has the configuration SV4ONLS-CasX119- SV4ONLS-PPP linker-SV4ONLS. Construct 493 has the configuration SV4ONLS-CasX119- SV4ONLS-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 E. coil 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 E. cold (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.
10011291 In order to generate constructs 492 and 493, constructs 280 and 291 were digested using XbaI and Banaill (NEB# R01455 and NEB# R31365) 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# M02025) according to the manufacturer's protocol into the digested and purified pSbc34,119.174,NT using XbaI and BamHE and Zymoclean Gel DNA Recovery Kit.
Assembled products in the pStx34 were transformed into chemically-competent Turbo Competent E call 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 E cold (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 Construct Amino Acid Sequence DLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEF
QKDPVGLMSRVAQPAPKNIDORKLIPVKDGNERLTSSGFACSOCCOPLY
VYKLEQVNDKGKPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLG
KFGQRALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGA

EGIEAYNNVVAQIVIVVVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQ
ANEVDWVVDMVCNVKKLINEKKEDGKVFWONLAGYKRQEALRPYLSSE
EDRKKGKKFARYQFGDLLLHLEKKHGEDWGIWYDEAWERIDKKVEGLS
KHIKLEEERRSEDAQSKAALTDVVLRAKASFVIEGLKEADKDEFCRCELKL
QKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIIN
YFKGGKLRFKKIKPEAFEANRFYIVINKKSGEIVPMEVNFNFDDPNLIILPL
AFGKROGREFIVVNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVA
LTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGN
PTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVR
NTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRMEDWLT
AKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATG
WMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISS
WTKGRSGEALSLLKKRFSHRPVQEKFVGLNCGFETHADEQAALNIARS
WLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVVVKPAV
(SEQ ID NO: 355) KPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRV
AQPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKG
KPHTNYFGRCNVSEHERLILLSPHKPEANDELVTY'SLGKFGQRALDFYSI

EHQKVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQI
VIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWVVDMVC
NVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
FGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDA
OSKAALTDV'VLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKP FA
lEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRF'YTVINKKSGEIVPMEVNENFDDPNLIILPLAFGKROGREFIVVN
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNI
KPMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA

Construct] Amino Acid Sequence M L I FE N LS RGFGRQ GKRTFMAERQYTRM EDVVLTAKLAYEG LS KTYLS KT
LAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQ I
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKR
FS H RPVQ E KFVC LN CGFETHADEQAALN IARSVVLF LRSQ EYKKYQTN KT
TGNTDKRAFVETVVQSFYRKKLKEVWKPAVTSPKKKRKV (SEQ ID NO:
356) KP EN IPQ P IS NTSRANLNKLLTDYTEMKKAILHVYWE EFQ KDPVGLMSRV
AQ PAP KN IDQ RKLIPVKDGNE RLTSSGFACSQ CCQ PLYVYKLEQVNDKG
KPHTNYFGRCNVSEH ERLI LLS PH KPEAN DELVTYSLGKFGQ RALDFYS I
HVTRESNH PVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQDIIL
EHQKVI KKN E KRLANLKD IASANGLAFPKITLP PQ PHTKEG I EAYNNVVAQ I
VIVVVNLNLWOKLKIGRDEAKPLQRLKGFPSFPLVEROANEVDVVVVDMVC
NVKKL I N E KKE DGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
FG DLLLH LEKKHGEDWG KVYD EAWE RIDKKVEG LSKH IKLEE ERRSE DA
QS KAALTDVVLRAKAS FVI E GLKEAD KD E FCRC E LKLQ KVVYGDLRGKP FA
I EAENS ILD ISG FSKQYN CAF IWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRFYTVINKKSGE IVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVN
D LLS LETGSLKLAN G RVI E KTLYN R RTRQ D E PALFVALTFE RREVLDS SN I
KPM NLIG ID RGE N I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA
M L I FE N LS RGFGRQ GKRTFMAERQ'YTRM EDVVLTAKLAYEG LS KTYLS KT
LAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ I
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR
FS H RPVQ E KFVC LN CGFETHADEQAALN IARSVVLF LRSQ EYKKYQTN KT
TGNTDKRAFVETWQSFYRKKLKEVWKPAVTSPKKKRKVPPPPKKKRKV
(SEQ ID NO: 357) KP EN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYWE EFQ KDPVGLMSRV
AQ PAP KN IDQ RKLIPVKDGNE RLTSSGFAC SQ CCQ PLYVYKLEQVNDKG
KPHTNYFGRCNVSEH ERLI LLS PH KPEAN DELVTYSLGKFGQ RALDFYS I
HVTRESNH PVKPLEQ IGGNSCASGPVGKALS DACMGAVASF LTKYQD It EHQKVI KKN E KRLANLKD IASANGLAFPKITLP PQ PHTKEG I EAYN NVVAQ I
VIVVVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVWDMVC
NVKKL I N E KKE DGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
FG DLLLH LEKKHGEDWGKVYD EAWE RIDKKVEG LSKH IKLEE ERRSE DA
QS KAALTDVVLRAKAS FVI E GLKEAD KD E FCRC E LKLQ KVVYGDLRGKP FA
I EAENS ILD ISG FSKQYN CAF IWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRFYTVINKKSGE IVPMEVNFNFDDPNLIILPLAFGKRQGREFIWN
D LLS LETGSLKLAN G RVI E KTLYN RRTRQ D E PALFVALTFE RREVLDS SN I
KPM NLIG ID RGE N I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA
M L I FE N LS RGFGRQ GKRTFMAERQ'YTRM EDWLTAKLAYEG LS KTYLS KT
LAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ I
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKR
FS H RPVQ E KFVCLN CGFETHADEQAALN IARSVVLF LRSQ EYKKYQTN KT

Construct] Amino Acid Sequence TGNTDKRAFVETVVQSFYRKKLKEVVVKPAVISPKKKRKVPPPHKKKHPD
ASVNFSEFSK (SEQ ID NO: 358) KP EN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYVVE EFQ KDPVGLMSRV
AQ PAP KNIDQRKLIPVKDGNERLTSSGFACSQCCQ PLYVYKLEQVNDKG
KPHTNYFGRCNVSEH ER LILLSPHKPEANDELVTYSLGKFGQ RALDFYS I
HVTRESNH PVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIIL
EHQKVI KKN E KR LANLKD IASANGLAFPKITLP PQ PHTKEG I EAYNNVVAQ I
VIVVVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVWDMVC
NVKKLINEKKE DGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
FGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKH IKLEEERRSEDA
QS KAALTDWLRAKAS FVI E GLKEAD KD E FC RC E LKLQ KVVYGDLRGKP FA
I EAENS ILD ISGFSKQYNCAF IWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRF'YTVINKKSGE IVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVN
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNI
KPMNLIGIDRGEN I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA
M L I FE N LS RGFGRQ GKRTFMAEROYTRM EDVVLTAKLAYEG LS KTYLS KT
LAQYTS KTCSNCGFTITSADYD RVLEKLKKTATGVVMTTI NGKELKVEGQ I
TYYNRYKRONVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKR
FS H RPVQ E KFVCLN CGFETHADEQAALN IARSWLF LRSQ EYKKYQTN KT
TGNTDKRAFVETWQSFYRKKLKEVINKPAVISPKKKRKVPPPQRPGPYD
RPQRPGPYDRP (SEQ ID NO: 359) KP EN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYWE EFQ KDPVGLMSRV
AQ PAP KNIDQRKLIPVKDGNERLTSSGFACSQCCQ PLYVYKLEQVNDKG
KPHTNYFGRCNVSEH ER LILLSPHKPEANDELVTYSLGKFGQ RALDFYS I
HVTRESNH PVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIIL
EHQKVI KKN E KRLANLKD IASANGLAFPKITLP PQ PHTKEG I EAYNNVVAQ I
VIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVWDMVC
NVKKLINEKKE DGKVFWQ N LAGYKRO EALRPYLSS EE D RKKGKKFARYQ
FGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKH IKLEEERRSEDA
QS KAALTDVVLRAKAS FVI E GLKEAD KD E FC RC E LKLQ KVVYGDLRGKP FA
lEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRFYTVINKKSGE IVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVN
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNI
KPMNLIGIDRGEN I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA
M L I FE N LS RGFGRQ GKRTFMAERQYTRM EDVVLTAKLAYEG LS KTYLS KT
LAQYTS KTCSNCGFTITSADYD RVLEKLKKTATGVVMTTI NGKELKVEGQ I
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKR
FS H RPVQ E KFVCLN CGFETHADEQAALN IARSWLF LRSQ EYKKYQTN KT
TGNTDKRAFVETINQSFYRKKLKEVVVKPAVTSPKKKRKVPPPLSPSLSPL
LSPSLSPL (SEQ ID NO: 360) KP EN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYVVE EFQ KDPVGLMSRV

Construct] Amino Acid Sequence AQ PAP KN IDQ RKLIPVKDGNERLTMSSGFACS Q CC Q PLYVYKLEQVNDK
GKPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFY
S IHVTR ESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVAS FLTKYQ DI
ILEHQKVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVA
QIVIVVVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMV
C NVKKL I N E KKE DGKVFWQ N LAGYKRQEALRPYLSS EE DRKKG KKFARY
QFGDLLLHLEKKHGEDWGIWYDEAVVERIDKKVEGLSKH IKLEEERRSED
AQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPF
AlEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIK
PEAFEANRFYTVI NKKSGEIVPMEVN FN FDDP N LI ILPLAFGKRQGREFIW
NDLLSLETGSLKLANGRVI EKTLYNRRTRQDEPALFVALTFERREVLDSS
NIKPMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKE
KQRTIQAKKEVEQ RRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVT
Q DAM L I F EN LSRGFG RQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTY
LSKTLAQYTSKTCSNCOFTITSADYDRVLEKLKKTATGVVMTTINGKELKV
EGQITYYN RYKRQ NVVKD LSVE LD R LS E ESVN N D ISSWTKG RSG EALS L
LKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVLFLRSQEYKKYQ

GGKGLGKGGAKRHRK (SEQ ID NO: 361) KP EN IPQ P ISNTSRANLNKLLTDYTEMKKAILHVYWEEFQ KDPVGLMSRV
AC:213AP KN IDQ RKLIPVKDGNERLTSSGFAC SQCCQ PLYVYKLEQVNDKG
KPHTNYFGRCNVSEH ERLI LLS PH KPEAN DELVTYSLGKFGQ RALDFYS I
HVTRESNH PVKP LEO IGGNSCASGPVGKALSDACMGAVASF LTKYQDIIL
EHQKVI KKN E KRLANLKD IASANGLAFPKITLP PQ PHTKEG I EAYNNVVAQ I
VIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWVVDMVC
NVKKLINEKKE DGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
FGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKH IKLEEERRSEDA
QS KAALTDVVLRAKAS FVI E GLKEAD KD E FCRC E LKLQ KVVYGDLRGKP FA
lEAENSILDISGFSKQYNCAF IWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRFYTVINKKSGE IVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVN
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNI
KPMNLIGIDRGEN I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA

LAQYTS KTCSNCGFTITSADYD RVLEKLKKTATGVVMTTI NGKELKVEGQ I
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKR
FS H RPVQ E KFVC LN CGFETHADEQAALN IARSVVLF LRSQ EYKKYQTN KT
TGNTDKRAFVERNQSFYRKKLKEVVVKPAVISPKKKRKVPPPSRRRKAN
PTKLSENAKKLAKEVEN (SEQ ID NO: 362) KP EN IPQ P ISNTSRANLNKLLTDYTEMKKAILHVYWEEFQ KDPVGLMSRV
AQ PAP KN IDQ RKLIPVKDGNERLTSSGFAC SOCCQ PLYVYKLEQVNDKG
KPHTNYFGRCNVSEH ER LILLSPHKPEANDELVTYSLGKFGQ RALDFYS I

EHQKVI KKN E KRLANLKD IASANGLAFPKITLP PQ PHTKEG I EAYNNVVAQ I

Construct] Amino Acid Sequence VIVVVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDVVVVDMVC
NVKKLINEKKE DGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
FGDLLLHLEKKHGEDWGIWYDEAWERIDKKVEGLSKHIKLEEERRSEDA
QS KAALTDVVLRAKAS FVI E GLKEAD KD E FC RC E LKLQ KVVYGDLRGKP FA
I EAENS ILD ISGFSKQYNCAF IWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRFYTVINKKSGE IVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVN
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNI
KPMNLIGIDRGEN I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA
M L I FE N LS RGFGRQ GKRTFMAERQYTRM EDVVLTAKLAYEG LS KTYLS KT
LAQYTS KTCSNCGFTITSADYD RVLEKLKKTATGVVMTTI NGKELKVEGQ I
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKR
FS H RPVQ E KFVCLN CGFETHADEQAALN IARSVVLF LRSQ EYKKYQTN KT
TGNTDKRAFVETWQSFYRKKLKEVVVKPAVISPKKKRKVPPPPAAKRVK
LD (SEQ ID NO: 363) KP EN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYVVE EFQ KDPVGLMSRV
AQ PAP KNIDQRKLIPVKDGNERLTSSGFACSOCCQ PLYVYKLEQVNDKG
KPHTNYFGRCNVSEH ER LILLSPHKPEANDELVTYSLGKFGQ RALDFYS I
HVTRESNH PVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIIL
EHQKVI KKN E KR LANLKD IASANGLAFPKITLP PQ PHTKEG I EAYNNVVAQ I
VIWVNLNLWOKLKIGRDEAKPLORLKGFPSFPLVEROANEVDWWDMVC
NVKKLINEKKE DGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
FGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKH IKLEEERRSEDA
QS KAALTDVVLRAKAS FVI E GLKEAD KD E FC RC E LKLQ KVVYGDLRGKP FA
I EAENS ILD ISGFSKQYNCAF IWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRF'YTVINKKSGE IVPMEVNFNFDDPNLIILPLAFGKRQGREFIVVN
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNI
KPMNLIGIDRGEN I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA
M L I FE N LS RGFGRQ GKRTFMAERQYTRM EDVVLTAKLAYEG LS KTYLS KT
LAQYTS KTCSNCGFTITSADYD RVLEKLKKTATGVVMTTI NGKELKVEGQ I
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKR
FS H RPVQ E KFVCLN CGFETHADEQAALN IARSWLF LRSQ EYKKYQTN KT
TG NTD KRAFVETVVQ S FYRKKLKEVVVKPAVTS PKKKRKVPPP KRS FS KA
F (SEQ ID NO: 364) KP EN I PQ P IS NTSRAN LN KLLTDYTEM KKAI LHVYVVE EFQ KDPVGLMSRV
AQ PAP KNIDQRKLIPVKDGNERLTSSGFACSQCCQ PLYVYKLEQVNDKG
KPHTNYFGRC NVSEH ERLI LLS PH KPEAN DELVTYSLGKFGQ RALDFYS I
HVTRESNH PVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIIL
EHQKVI KKN E KR LANLKD IASANGLAFPKITLP PQ PHTKEG I EAYNNVVAQ I
VIWVNLNLWOKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMVC
NVKKLINEKKE DGKVFWQ NLAGYKRQEALRPYLSSEEDRKKGKKFARYQ
FGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKH IKLEEERRSEDA
QS KAALTDWLRAKAS FVI E GLKEAD KD E FCRC E LKLQ KVVYGDLRGKP FA

Construct] Amino Acid Sequence I EAENS ILD ISG FSKQYN CAF IWQKDGVKKLNLYLIINYFKGGKLRFKKIKP
EAFEANRF'YTVINKKSGE IVPM EVN F NFDD P N LI ILPLAFGKRQ GRE F IVVN
DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNI
KPM N LI G ID RGE N I PAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQ
RTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA
M L I FE N LS RGFGRQ GKRTFMAERQYTRM EDVVLTAKLAYEG LSKTYLS la LAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ I
TYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRSGEALSLLKKR
FS H RPVQ E KFVC LN CGFETHADEQAALN IARSVVLF LRSQ EYKKYQTN KT
TGNTDKRAFVETVVQSFYRKKLKEV'VVKPAVTSPKKKRKVPPPKRGINDR
NFVVRGENERKTR (SEQ ID NO: 365) KTLLVRVMTP
DLRERLENLRKKP EN I PQ PISNTSRANLNKLLTDYTEMKKAILHVYVVEEFQ
KD PVG LMSRVAQ PAP KN I DQ RKLIPVKDGN E RLTSSGFACSQ CCQ PLYV
YKLEOVNDKGKP HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKF
GQRALDFYS I HVTRESN H PVKPLEQ I GGNSCASG PVG KALS DACM CAVA
SFLTKYQDI I LEHQ KVI KKN EKRLAN LKD IASAN GLAFP KITLPPQPHTKE G I
EAYNNVVAQ IVIWVN LN LWQ KLKI GR DEAKP LQ RLKG FPSF PLVE ROAN E
VDVVVVDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRK
KGKKFARYQ FGDLLLH LE KKHG EDWG KVYDEAVVERI DKKVEG LS KH I KL
EEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVY
G DLRGKP FAIEAE NS ILD ISGFSKQYNCAFIWOKDGVKKLN LYLIINYFKG
GKLRFKKIKPEAFEANRFYTVI NKKSG EIVPM EVNFNF D DP NL I ILPLAFGK
RQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFE
RREVLDS SNIKPM N LIGIDRG EN IPAVIALTDPE GC P LSRFKDS LG NPTH IL
RIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARD
LLYYAVTQ DAML I FENLSRGFGRQGKRTFMAERQYTRMEDVVLTAKLAYE
GLSKTYLS KTLAQYTS KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTIN
GKELKVEGQ ITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRS
GEALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVLFLRSQ
EYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEVVVKPAVTSPKKKRKV
PPPPKKKRKV (SEQ ID NO: 366) KTLLVRVMTP
DLRERLENLRKKP EN I PQ PISNTSRANLNKLLTDYTEMKKAILHVYWEEFQ
KD PVG LMSRVAQ PAP KN I DQ R KLIPVKDGN E RLTSSGFACSQ CCQ PLYV
YKLEQVNDKGKP HTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKF
GQRALDFYS I HVTRESN H PVKPLEQ I GGNSCASG PVG KALS DACM CAVA
SFLTKYQDIILEHQKVIKKN EKRLAN LKD IASAN GLAFP KITLPPQPHTKE G I
EAYNNVVAQ IVIVVVNLNLWQKLKIGRDEAKPLQ RLKG FPSF PLVE ROAN E
VDVVVVDMVCNVKKLINEKKEDGKVFWONLAGYKRQEALRPYLSSEEDRK
KGKKFARYQ FGDLLLH LE KKHG EDWG KVYDEAVVERI DKKVEG LS KH I KL
EEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKVVY
G DLRGKP FAIEAE NS ILD ISGFSKOYNCAFIWQKDGVKKLN LYLIINYFKG
GKLRFKKIKPEAFEANRFYTVI NKKSG EIVPM EVNFNF D DP NL I ILPLAFGK
ROGREFIVVNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFE
RREVLDS SNIKPM N LIGIDRG EN IPAVIALTDPE GC P LSRFKDS LG NPTH IL

Construct] Amino Acid Sequence RIGESYKEKQRTIOAKKEVEORRAGGYSRKYASKAKNLADDMVRNTARD
LLYYAVTODAMLIFENLSRGFGRQGKRTFIVIAERQYTRMEDVVLTAKLAYE
GLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGVVMTTIN
GKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSVVTKGRS
GEALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQ
EYKKYQTNKTTGNTDKRAFVETVVOSFYRKKLKEVVVKPAVTSPKKKRKV
PPPPAAKRVKLD (SEQ ID NO: 367) Table 10: Nuclear localization sequence list CasX NLS DNA Sequence Amino Acid Sequence 278, 279, SV40 CCAAAGAAGAAGCGGAAGG
PKKKRKV (SEQ ID
280, 492, TC (SEQ ID NO: 368) NO: 130) 285 SynthNL S3 CACAAGAAGAAACATCCAGA
HICKICHPDASVNFSE
CGCATCAGTCAACTTTAGCG
FSK (SEQ ID NO:
AGTTCAGTAAA (SEQ ID NO:
369) 376) 286 SynthNLS4 CAGCGCCCTGGGCCTTACGA
QRPGPYDRPQRPGP
TAGGCCGCAAAGACCCGGAC
YDRP (SEQ ID NO:
CGTATGATCGCCCT (SEQ ID
NO: 370) 162) 287 SynthNLS5 CTCAGCCCGAGTCTTAGTCC LSPSLSPLLSPSLSPL
ACTGCTTTCCCCGTCCCTGTC
(SEQ ID NO: 163) TCCACTG (SEQ ID NO: 371) 288 SynthNLS6 CGGGGCAAGGGTGGCAAGG RGKGGKGLGKGGA
GGCTTGGCAA
A
KRIIRK (SEQ ID NO:
AAGAGGCACAGGAAG (SEQ
ID NO: 372) 164) SRRRKANPTKLSEN
TCCTACAAAACTGTCAGAAA
AICICLAKEVEN (SEQ
ATGCGAAAAAACTTGCTAAG
GAGGTGGAAAAC (SEQ ID
ID NO: 157) NO: 373) 291 c-Myc CCTGCCGCAAAGCGAGTGAA
PAAICRVKLD (SEQ
ATTGGAC (SEQ ID NO: 374) ID NO: 132) CasX NLS DNA Sequence Amino Acid Sequence 293 Nucleolar RNA AAGCGGTCCTTCAGTAAGGC KRSFSKAF
(SEQ ID
Helicase II CTTT (SEQ ID NO: 375) NO: 153) 300 Influenza A AAACGGGGAATAAACGACC
KRGINDRNFIATRGEN
protein GGAACTTCTGGCGCGGGGAA
ERKTR (SEQ ID NO:
AACGAGCGCAAAACCCGA
(SEQ ID NO: 376) 151) Example 5: Design and Generation of CasX Constructs 387, 395, 485-491, and 494 10011301ln 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, colE1 ori, and CasX with fused NLS (pStx1) 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 pStx1 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 pStx1 staging vector were transformed into chemically-competent Turbo Competent E:
coil 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.
10011311 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.
10011321 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 CasX119 construct of Example 2) were cloned respectively into a 4kb staging vector that was made up of a KanR marker, colE1 ori, and STX with fused NLS (pStx1) using standard cloning methods. Gibson primers were designed to amplify the CasX Stx1 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 pStx1 respectively. The NTSB and Helical I domain from CasX SEQ ID NO: I 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 pStx1 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. coil (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.
10011331ln 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, colE1 ori, and STX with fused NLS (pStx1) using standard cloning methods. Gibson primers were designed to amplify the CasX Stx1 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 pStx1 respectively. The NTSB domain from CasX Stx1 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 o1C762 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 pStx1 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 (LW 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 DNA
Construct Amino Acid Sequence Sequence CasX 387 (SEQ ID MAP KKKRKVSRQ E IKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRERLEN LRKKP EN IP QP
ISNTSRAN LN KLLTDYTE
378) M KKAILHVYVVEEFQKDPVGLMSRVAQ
PASKKIDQ NKLKPEMD
EKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCN
VAE H E KLI LLAQ LKP E KDS DEAVTYSLGKFGQ RALD FYS I HVTR
ESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQ
DI I LEHQ KV IKKNEKRLANLKDIASANGLAFP KITLP PQ PHTKEGI
EAYNNVVAQ IVIWVN LN LWQ KLKI GR D EAKP LQ RLKG FPS FPL
VERQANEVDVWVDMVCNVKKL IN E KKEDG KVFWQ N LAGYKR
QEALRPYLSSEEDRKKGKKFARYQFGDLLLHLEKKHGED WOK
VYDEAINERIDKKVEGLSKH IKLEEERRSEDAQSKAALTDVVLRA
KASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENS
ILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIK
PEAFEANRFYIVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKR
QGREFIINNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALF
VALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCPLS
RFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRK

DNA
Construct Amino Acid Sequence Sequence YASKAKN LADDMVR NTARD LLYYAVTQ DAM L I FE NLSRG FG R
QGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ
ITYYN RYKRQ NVVKD LSVE LDRLS E ESVN N D I SSWTKGRSG EA
LSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVLFL
RSQEYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEVVVKPA
VTSPKKKRKV (SEQ ID NO: 388) CasX 395 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRE RLEN LRKKP E N IP QP
ISNTSRAN LN KLLTDYTE
379) M KKAILHVYVVEEFQKDPVGLMSRVAQ PAPKN I DQ RKL I PVKDG
NERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYFGRCN
VSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTKE

IEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGV
DAYNEVIARVRMVVVNLNLWQKLKLSRDDAKPLLRLKGFPSFP
LVERQAN EVDVVVVDMVC NVKKL I N EKKE DG KVFWQ N LAGYKR
QEALRPYLSSEEDRKKGKKFARYQFGDLLLHLEKKHGEDWGK
VYDEAWER I D KKVE GLSKH I KLEE E R R SE DAQ SKAA LTDINLRA
KASFVIEGLKEADKDEFCRCELKLQKVVY'GDLRGKPFAIEAENS
ILDISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIK
PEAFEAN RFYTVINKKSGEIVPMEVNFNFDDPNLI ILPLAFGKR
QGREFIVVNDLLSLETGSLKLANGRVIEKTLYNRRTRQ DEPALF
VALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCPLS
RFKDSLGNPTH I LRIG ESYKE KQ RTIQAKKEVEQ RRAGGYS RK
YASKAKN LADDMVR NTARD LLYYAVTQ DAM L I FE NLSRG FG R
QGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ
ITYYN RYKRQ NVVKD LSVE LDRLS E ESVN N D I SSVVTKGRSG EA
LSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVLFL
RSOEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVVIMPA
VTSPKKKRKVTSPKKKRKV (SEQ ID NO: 389) CasX 485 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRE RLEN LRKKP E N IP QP
ISNTSRAN LN KLLTDYTE
380) M KKAILHVYVVEEFQKDPVGLMSRVAQ PAPKN I DQ RKL I PVKDG
NERLTSSGFACSOCCOPLYVYKLEQVNDKGKPHTNYFGRCN
VSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTKE

IEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGV
DAYNEVIARVRMVVVNLNLVVQKLKLSRDDAKPLLRLKGFPSFP
LVERQAN EVDWVVDMVC NVKKL I N EKKE DG KVFWQ N LAGYKR
QEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGK
VYDEAINE R I DKKVEGLSKH I KLEE ERRSE DAQ SKAALTDVVLRA
KAS FV I EGLKEAD KDE FC RCE LKLQ KVVYGDLRG KP FAIEAE N S
ILDISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIK
PEAFEAN RF'YTVINKKSGEIVPMEVNFNFDDPNLI ILPLAFGKR
Q GR E F IINN DLLS LETGS LKLANG RVI E KTLYN RRTRQ DEPALF

DNA
Construct Amino Acid Sequence Sequence VALTFERREVLDSSNIKPMNLIGVDRGEN IPAVIALTDPEGCPL
SRFKDSLGN PTH I LR IGESYKEKQ RTI QAKKEVEQ R RAGGYSR
KYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFG
RQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGVVIVITTINGKELKVEG

EALS LLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVL
FLRSQ EYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEVVVKP
AVTSPKKKRKV (SEQ ID NO: 390) CasX 486 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRER LEN LRKKP EN IP QP
ISNTSRAN LN KLLTDYTE
381) M KKAILHVYVVEEFQKDPVGLMSRVAQ PAPKN I DQ RKL I PVKDG
NERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYFGRCN
VSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTKE
STHPVKPLAQ IAGN RYASG PVG KALSDACMGTIASFLSKYQDI I
IEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGV
DAYNEVIARVRMVVVNLNLWQKLKLSRDDAKPLLRLKGFPSFP
LVERQANEVDIANVDMVCNVKKLINEKKEDGKVFWQNLAGYKR
QEALRPYLSSEEDRKKGKKFARYQLGDLLKHLEKKHGEDWGK
VYDEAWER I DKKVEGLSKH I KLEE ER RSE DAQ SKAALTDVVLRA
KASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENS
ILDISGFSKQYNCAFIVVQKDGVKKLNLYLI INYFKGGKLRFKKIK
PEAFEAN RFYTVINKKSGEIVPMEVNFNFDDPNLI ILPLAFGKR
QGREFIVVNDLLSLETGSLKLANGRVIEKTLYNRRTRQ DEPALF
VALTFERREVLDSSNIKPMNLIGVDRGEN IPAVIALTDPEGCPL
SRFKDSLGN PTH I LR IGESYKEKQ RTI QAKKEVEQ RRAGGYSR
KYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFG
RQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEG
Q ITYYNRRKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSG
EALS LLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVL
FLRSQ EYKKYQTNKTTGNTD KRAFVETWQ S FYRKKLKEVWKP
AVTSPKKKRKV (SEQ ID NO: 391) CasX 487 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRERLEN LRKKP EN IP QP
ISNTSRAN LN KLLTDYTE
382) M KKAILHVYWEEFQKDPVGLMSRVAQ PAPKN I DQ RKL I PVKDG
NERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYFGRCN
VSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTKE

IEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGV
DAYNEVIARVRMVVVNLNLWQKLKLSRDDAKPLLRLKGFPSFP
LVEROAN EVDVVVVDMVC NVKKL I N EKKE DG KVFWQ N LAGYKR
QEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGK
VYDEAINE R I DKKVEGLSKH I KLEE ERRSE DAQ SKAALTDINLRA
KASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAENS
ILDISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIK

DNA
Construct Amino Acid Sequence Sequence PEAFEAN RFYIVINKKSGEIVPMEVNENFDDPNLI ILPLAFGKR
Q GR E F IVVN DLLS LETGS LKLANG RVI E KTLYN RRTRQ DEPALF
VALTFERREVLDSSNIKPMNLIGVDRGEN IPAVIALTDPEGCPL
SRFKDSLGN PTH I LR IGESYKEKQ RTI QAKKEVEQ RRAGGYSR
KYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFG
RQGKRTFMAERQ'YTRMEDVVLTAKLAYEGLSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGINMTTINGKELKVEG
Q ITYYN RYKRQ NVVKDLSVE LD RLS EESVN N D I SSVVTKGRSG
EALS LLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVL
FLRSQ EYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEVWKP
AVTSPKKKRKV (SEQ ID NO: 392) CasX 488 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGICTGPMKTLL
NO: VRVMTPDLRE R LEN LRKKP E N IP QP
ISNTSRAN LN KLLTDYTE
383) M KKAILHVYWEEFQKDPVGLMSRVAQ PASKKIDQ NKLKPEMD
EKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRGN
VAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQ RALD FYS I HVTK
ESTHPVKPLAQ IAGNRYASGPVGKALSDACMGTIASFLSKYQD
III EHQ KVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEG
VDAYNEVIARVRMVVVNLNLWQKLKLSRDDAKPLLRLKGFPSF
PLVE ROAN EVDVVVVDMVCNVKKLI NE KKE DGKVFWQ N LAGYK
RQEALRPYLSSEEDRKKGKKFARYQFGDLLLHLEKKHGEDWG
KVYDEAVVE RI DKKVEG LS KH I KLE EE RRS EDAQS KAALTDVVLR
AKASFVIEGLKEADKDEFCRCELKLQKVVYGDLRGKPFAIEAEN
SILD ISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKI
KPEAFEANRFYTVIN KKSGEIVPMEVNFNFDDPNLIILPLAFGKR
Q GRE F IVVN DLLS LETGS LKLANG RVI E KTLYN RRTRQ DEPALF
VALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCPLS
RFKDSLGNPTH I LR IG ESYKEKQ RTIQAKKEVEQ RRAGGYSR K
YASKAKN LADDMVR NTARD LLYYAVTQ DAM L I FE NLSRG FG R
QGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTS
KTCSNCGFTITSADYDRVLEKLKKTATGVVMTTINGKELKVEGQ
ITYYN RYKRQ NVVKD LSVE LDRLS E ESVN N D I SSWTKGRSG EA
LSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVLFL
RSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVVVKPA
VTSPKKKRKV (SEQ ID NO: 393) CasX 489 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRE RLEN LRKKP E N IP QP
ISNTSRAN LN KLLTDYTE
384) M KKAILHVYVVEEFQKDPVGLMSRVAQ PASKKIDQ NKLKPEMD
EKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCN
VAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQ RALD FYS I HVTK
ESTHPVKPLAQ IAGNRYASGPVGKALSDACMGTIASFLSICYQD
III EHQ KVVKGNQKRLESLRELAGKENLEYPSVTLPPQ PHTKEG
VDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSF
PLVE RQAN EVDWVVDMVC NVKKLI NE KKE DGKVFWQ N LAGYK
RQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWG
KVYDEAWE R I DKKVEG LS KH I KLE EE RRS EDAQS KAALTDWLR

DNA
Construct Amino Acid Sequence Sequence AKASFVIEG LKEADKDEFC RC ELKLQ KVVYG DLRGKP FAI EAE N
SILD ISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKI
KPEAFEANRFYTVIN KKSGEIVPMEVNFNFDDPNLIILPLAFGKR
QGREFIINNDLLSLETGSLKLANGRVIEKTLYNRRTRQ DEPALF
VALTFERREVLDSSNIKPMNLIGVDRGENIPAVIALTDPEGCPL
SRFKDSLGN PTH I LR IGESYKEKQ RTI QAKKEVEQ RRAGGYSR
KYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFG
RQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEG
Q ITYYN RRKRQNVVKDLSVE LD RLS EESVN N D ISSVVTKG RS G
EALS LLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSVVL
FLRSQ EYKKYQTNKTTGNTD KRAFVETWQ S FYRKKLKEVWKP
AVTSPKKKRKV (SEQ ID NO: 394) CasX 490 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRERLEN LRKKP EN IP QP
ISNTSRAN LN KLLTDYTE
385) M KKAILHVYWEEFQKDPVGLMSRVAQ PASKKIDQ NKLKPEMD
EKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCN
VAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQ RALD FYS I HVTK
ESTHPVKPLAQ IAGNRYASGPVGKALSDACMGTIASFLSKYQD
III EHQ KVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEG
VDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSF
PLVE RQAN EVDVVVVDMVCNVKKLI NE KKE DGKVFWQ N LAGYK
RQEALRPYLSSEEDRKKGKKFARYQLGDLLKHLEKKHGEDW
GKVYDEAVVE RI D KKVEG LSKH IKLE E E RRS E DAQSKAALTDW
LRAKAS FVI EG LKEADKD EFCRCE LKLQ KVVYG DLRGKPFAI EA
ENSI LD ISG FSKQYN CAF IVVQ KDGVKKLN LYLI I NYFKGGKLRFK
KIKP EAFEANRFYTVI NKKS GE IVP MEVNF NFDDP NLIILP LAFG
KROGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPA
LFVALTFERREVLDSSNIKPMNLIGVDRGENIPAVIALTDPEGC
PLSR FKDSLGNPTH I LR I GESYKE KQ RTIQAKKEVEQ R RAGGY
SRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGF
GRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQY
TS KTCS N CGFTITSADYD RVLE KLKKTATGINMTTI NC KE LKVE
GQ ITYYNRRKRQ NVVKDLSVELDRLSEESVNND I SSVVTKGRS
GEALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARS
VVLFLRSQ EYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEV
VVKPAVTSPKKKRKV (SEQ ID NO: 395) CasX 491 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRER LEN LRKKP EN IP QP
ISNTSRAN LN KLLTDYTE
386) M KKAILHVYWEEFQKDPVGLMSRVAQ PASKKIDQ NKLKPEMD
EKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCN
VAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQ RALD FYS I HVTK
ESTHPVKPLAQ IAGNRYASGPVGKALSDACMGTIASFLSKYQD
III EHQ KVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEG
VDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSF
PLVE RQAN EVDVVVVDMVCNVKKLI NE KKE DGKVFWQ N LAGYK

DNA
Construct Amino Acid Sequence Sequence RQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWG
KVYDEAWE R I DKKVEG LS KH I KLE EE RRS EDAQS KAALTDWLR
AKASFVIEGLKEADKDEFC RC ELKLQ KVVYG DLRGKP FAI EAE N
SILD ISGFSKIDYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKI
KPEAFEANRFYTVIN KKSGE IVP M EVN FN F DDP NLI I LP LAFGKR
Q GRE F IINN DLLS LETGS LKLANG RVI E KTLYN RRTRQ DEPALF
VALTFERREVLDSSNIKPMNLIGVDRGEN IPAVIALTDPEGCPL
SRFKDSLGN PTH I LR IGESYKEKQ RTI QAKKEVEQ RRAGGYSR
KYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFG
RQGKRTFMAERQYTRMEDVVLTAKLAYEGLSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEG
Q ITYYN RYKRQ NVVKDLSVE LD R LS EESVN N D I SSWTKGRSG
EALS LLKKRFSH RPVQEKFVCLNCGFETHADEQAALNIARSVVL
FLRSQ EYKKYQTNKTTGNTDKRAFVETVVQSFYRKKLKEVVVKP
AVTSPKKKRKV (SEQ ID NO: 396) CasX 494 (SEQ ID MAPKKKRKVSRQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLL
NO: VRVMTPDLRE RL EN L RKKP E N IP QP I
SNTS RAN LN KLLTDYTE
387) M KKAILHVYINEEFQKDPVGLMSRVAQ
PASKKIDQ NKLKPEMD
EKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRC N
VAE H E KL I LLAQ LKP E KDS DEAVTYSLGKFGQ RAL D FYS I HVTR
ESNHPVKPLEQ IGGNSCASGPVGKALSDACMGAVASFLTKYQ
D I I LEHQ KV IKKN E KRLAN LKD IASANG LAFP KITLP PQ P HTKEGI
EAYNNVVAQ IVIVVVN LN LWQ KLKI GR D EAKP LQ RLKG FPS FPL
VERQANEVDVWVDMVCNVKKLINEKKEDGKVFVVQNLAGYKR
QEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGK
VYDEAVVE R I DKKVEGLSKH I KLEE ERRSE DAQ SKAALTDINLRA
KAS FV I EGLKEAD KDE FC RCE LKLQ KVVYGDLRGKPFAIEAENS
ILDISGFSKQYNCAFIWQKDGVKKLNLYLI INYFKGGKLRFKKIK
PEAFEAN RFYTVINKKSGEIVPMEVNFNFDDPNLI ILPLAFGKR
Q GR E F IINN DLLS LETGS LKLANG RVI E KTLYN RRTRQ DEPALF
VALTFERREVLDSSNIKPMNLIGVDRGENIPAVIALTDPEGCPL
SRFKDSLGN PTH I LR IGESYKEKQ RTI QAKKEVEQ RRAGGYSR
KYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFG
RQGKRTFMAERQ'YTRMEDVVLTAKLAYEGLSKTYLSKTLAQYT
SKTCSNCGFTITSADYDRVLEKLKKTATGINMTTINGKELKVEG
Q ITYYN RYKRQ NVVKDLSVE LD RLS EESVN N D I SSVVTKGRSG
EALS LLKKRFSH RPVQEKFVCLNCGFETHADEQAALNIARSVVL
FLRSQ EYKKYQTNKTTGNTD KRAFVETWQ S FYRKKLKEVVVKP
AVTSPKKKRKV (SEQ ID NO: 397) Example 6: Generation of RNA guides 10011341For 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 rtM each, and the amplification primers (T7 promoter and the unique spacer primer) were included at a final concentration of 1 gM each. The sg2, sg32, sg64, and sg174 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.
10011351ln vitro transcriptions were carried out in buffer containing 50 mM
Ttis pH 8.0, 30 mM
MgCl2, 0.01% Triton X-100, 2 mM spermidine, 20 mM DTT, 5 mM N'TPs, 0.5 ttM
template, and 100 gg/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 lx phosphate buffered saline. To fold the sgRNAs, samples were heated to 700 C for 5 min and then cooled to room temperature. The reactions were supplemented to 1 mM
final MgCl2 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
Primer Primer sequence RNA product T7 promoter GAAATTAATACGACTCACTATA (SEQ ID NO: Used for all primer 398) sg2 backbone GAAATTAATACGACTCACTATAGGTACTGG GGUACUGGCGCU
fwd CGCTTTTATCTCATTACTTTGAGAGCCATC UUUAUCUCAUUAC
ACCAGCGACTATGTCGTATGGGTAAAG
UUUGAGAGCCAU
(SEQ ID NO: 399) CACCAGCGACUAU
sg2 backbone CTTTGATGCTTCTTATTTATCGGATTTCTCT GUCGUAUGGGUA
rev CCGATAAATAAGCGCTTTACCCATACGACA AAGCGCUUAUUUA

TAGTCGCTGGTGATGGC (SEQ ID NO: 400) UCGGAGAGAAAU
sg2.7.37 CGGAGCGAGACATCTCGGCCCTTTGATGC CCGAUAAAUAAGA
spacer primer TTCTTATTTATCGGATTTCTCTCCG (SEQ ID AGCAUCAAAGGG
NO: 401) CCGAGAUGUCUC
GCUCCG (SEQ ID
NO: 411) Primer Primer sequence RNA product sg32 GAAATTAATACGACTCACTATAGGTACTGG GGUACUGGCGCU
backbone fwd CGCTTTTATCTCATTACTTTGAGAGCCATC UUUAUCUCAUUAC
ACCAGCGACTATGTCGTATGGGTAAAGCG UUUGAGAGCCAU
C (SEQ ID NO: 402) CACCAGCGACUAU
sg32 CTTTGATGCTTCCCTCCGAAGAGGGCGCT GUCGUAUGGGUA
backbone rev TTACCCATACGACATAG (SEQ ID NO: 403) AAGCGCCCUCUU
CGGAGGGAAGCA
sg32.7.37 CGGAGCGAGACATCTCGGCCCTTTGATGC UCAAAGGGCCGA
spacer primer TTCCCTCCGAAGAG (SEQ ID NO: 404) GAUGUCUCG
(SEQ ID NO: 412) sg64 GAAATTAATACGACTCACTATAGGTACTGG GGUACUGGCGCC
backbone fwd CGCCTTTATCTCATTACTTTGAGAGCCATC UUUAUCUCAUUAC
ACCAGCGACTATGTCGTATGGGTAAAGCG UUUGAGAGCCAU
C (SEQ ID NO: 405) CACCAGCGACUAU
sg64 CTTTGATGCTTCTTACGGACCGAAGTCCGT GUCGUAUGGGUA
backbone rev AAGCGCTTTACCCATACGACATAG (SEQ ID AAGCGCUUACGG
NO: 406) ACUUCGGUCCGU
sg64.7.37 CGGAGCGAGACATCTCGGCCCTTTGATGC AAGAAGCAUCAAA
spacer primer TTCTTACGGACCGAAG (SEQ ID NO: 407) GGGCCGAGAUGU
CUCGCUCCG
(SEQ ID NO: 413) sg174 GAAATTAATACGACTCACTATAACTGGCGC ACUGGCGCUUUU
backbone fwd TTTTATCTGATTACTTTGAGAGCCATCACCA AUCUgAUUACUUU
GCGACTATGTCGTAGTGGGTAAAGCT
GAGAGCCAUCAC
(SEQ ID NO: 408) CAGCGACUAUGU
sg174 CTTT
TT T GATGCTCCCTCCGAAGAGGGAGC
CGUAgUGGGUAAA
backbone rev ACCCACTACGACATAGTCGC (SEQ ID NO: GCUCCCUCUUCG
409) GAGGGAGCAUCA
sg174.7,37 CGGAGCGAGACATCTCGGCCCTTTGATGC AAGGGCCGAGAU
spacer primer TCCCTCC (SEQ ID NO: 410) GUCUCGCUCCG
(SEQ ID NO: 414) Example 7: Assessing binding affinity to the guide RNA
10011361Purified 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 ELM 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
10011371Purified 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 n.M, while the RNP will be titrated from 1 pM to 100 M 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: CasX:gNA In Vitro Cleavage Assays 1. Determining cleavage-competent fractions for protein variants compared to wild-type reference CasX
10011381The 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 TGAACCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTCGCCTTAGCTGTGCTOGC
GCT (target strand, TS (SEQ ED 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 NaC1, 1 mM TCEP, 5%
glycerol, mM MgC12), heating to 95 C for 10 minutes, and allowing the solution to cool to room temperature.
10011391 CasX RNPs were reconstituted with the indicated CasX and guides (see graphs) at a final concentration of 1 rtM with 1.5-fold excess of the indicated guide unless otherwise specified in lx cleavage buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM
TCEP, 5%
glycerol, 10 mM MgCl2) 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.
10011401 Cleavage reactions were prepared with final RNP concentrations of 100 nM and a final target concentration of 100 nM. Reactions were carried out at 370 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 950 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 1Q11. 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.
10011411 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 fanned when CasX457, CasX488, or CasX491 was added to the sgRNA compared to CasX2.
2. In vitro Cleavage Assays ¨ Determining kcieave for CasX variants compared to wild-type reference CasX
10011421Cleavage-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.
10011431 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 M for the guide and 1,5 jiM
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.
10011441 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.
10011451 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.
10011461 CasX RNPs were reconstituted with the indicated CasX (see FIG. 18) at a final concentration of 1 uM with 1.5-fold excess of the indicated guide in lx cleavage buffer (20 tnivl Tris HC1 pH 7.5, 150 in.M NaC1, 1 mM TCEP, 5% glycerol, 10 mM MgCl2) 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 nIvl and a final target concentration of 10 WO. Reactions were carried out at 37 C except where otherwise noted and initiated by the addition of the target DNA. Aliquots were taken at 025, 0.5, 1, 2, 5, and 10 minutes and quenched by adding to 95%
formamide, 20 mM EDTA. Samples were denatured by heating at 950 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 (lccieave) 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.
10011471 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 kthave should be taken as a lower bound.
10011481 The data indicate that the CasX variants have a higher level of activity, with ndeave 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 10011491 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.
10011501 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 n.M/min, 7.8 1.8 aM/min, and 49.3 1.4 n.M/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.
10011511 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 100 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.
10011521The 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 RNP Construct likleave*
Initial velocity* Competent fraction 2.2.7.37 20.4 1.4 nM/min 16 3%
2.32.7.37 18.4 2.4 nM/min 13 3%
1641.37 7.8 1.8 nM/min 5 2%
2.174.7.37 0.51 0.01 min" 49.3 1.4 nM/min 22 5%
119.174.7.37 6.29 1 2.11 min"
35 6%
457.174.7.37 3.01 0.90 min"
53 7%
488.1747.37 15.19 min-1 67%
16.59 min" / 0.447 83% / 79 9%
491.174.7.37 0.031 min" (10 C) (guide-limited) 491.175.7.37 0.089 min" (10 C) 5% (guide-limited) 491 185737 0.2828 0.014 min"
91 4% (guide-. ..
(10 C) limited) 491 186.7.37 0.118 0.010 min"
32 2% (guide-.
(10 C) limited) 491 196737 0.439 0.067 min"
91 1% (guide-. ..
C) limited) 491 214.7.37 0.413 0.059 min"
73 8% (guide-.
(10 C) limited) 491.215.7.37 0.668 0.165 min"
81 6% (guide-(10 limited) *Mean and standard deviation Example 10: Assessing differential PAM recognition in vitro 10011531ln vitro cleavage assays were performed essentially as described in Example 9, using CasX2, CasX119, and CasX438 complexed with sg174.7.37. Fluorescently labeled dsDNA
targets with a 7.37 spacer and either a TIC, 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 (kcleave) 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.
10011541For 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 keleave is shown. For all non-NTC PAMs, the relative cleavage rate as compared to the ITC 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 nic 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 lc, ¨leaves are comparable to or greater than the kcleave 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.
Guide* DNA Sequence 7.37 TIC AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGAA
PAM TS TGCTGTCAGCTTCA (SEQ ID NO: 417) 7.37 TIC TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAG
PAM NTS CTGTGCTCGCGCT (SEQ ID NO: 418) 7.37 CTC AGCGCGAGCACAGCTAAGGCCAGGGAGCGAGACATCTCGGCCCGAG
PAM IS TGCTGTCAGCTTCA (SEQ ID NO: 419) 7.37 CTC TGAAGCTGACAGCACTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAG
PAM NTS CTGTGCTCGCGCT (SEQ ID NO: 420) 7.37 GTC AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGAC
PAM IS TGCTGTCAGCTTCA (SEQ ID NO: 421) 7.37 GTC TGAAGCTGACAGCAGTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAG
PAM NTS CTGTGCTCGCGCT (SEC) ID NO: 422) 7.37 ATC AGCGCGAGCACAGCTAAGGCCAGGGAGCGAGACATCTCGGCCCGAT
PAM IS TGCTGTCAGCTTCA (SEQ ID NO: 423) 7.37 ATC TGAAGCTGACAGCAATCGGGCCGAGATGTCTCGCTCCGTGGCCTTAG
PAM NTS CTGTGCTCGCGCT (SEQ ID NO: 424) *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.
Variant TTC CTC
GTC ATC
2 0.267 min-1 9.29E-3 min-1 3.75E-3 min-1 1.87E-2 min-1 (0.035) (0.014) (0.070) 119 8.33 min-1 0.303 min-1 8.64E-2 min-1 0.540 m1n-1 (0.036) (0.010) (0.065) 438 4.94 min-1 0.212 m1n-1 1.31E-2 min-1 0.408 m1n-1 (0.043) (0.013) (0.083) 491 16.42 m1n-1 8.605 m1n-1 2.447 min4 11.33 min-1 (0.524) (0.149) (0.690) Example 11: Identification of nicking variants 10011551Purified modified CasX variants will be complexed with single-guide RNA bearing a fixed targeting sequence. The RNP complexes will be added to buffer containing MgCl2 at a final concentration of 100 n1V1 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 tiM. 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 10011561Wild-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 10011571CasX delivery particles (XDPs) containing RNP of CasX, CasX 119, CasX
438, or CasX 457 protein and single guide RNA 174 with spacer sequence 12/ (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 (pXID10 encodes CasX 119; pXD11 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-Pot (psPax2) proteins were also used. MI 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 Construct DNA
SEQUENCE
pStx42.174. ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACTAT

12.7 GTCGTAGTGGGTAAAGCTCCCTCTTCGGAGGGAGCATCAAAGCTGC
ATTCTAGTTGTGGTTT (SEQ ID NO: 426) pXDP10 ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGAT
GGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTA
AAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTA
ATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGG
ACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCAT
TATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAG
ATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAA
ACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACA
CAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAG
GGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATG
GGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCC
ATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACAC
CATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTA
AAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATC
CAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAA
GGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAAT
AGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATA
AAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGC
CCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTA
GAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCT
TCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAA
TGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGGAG
CGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGAC
CCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAAC
AAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAGGAACCAAA
GAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGC
CAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGA

Construct DNA
SEQUENCE
AAGGAAGGAGACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTT
TTTAG GGAAGATCTGGCCTTCC CACAAGGGAAG GC CAGG GAATTTT
CTTCAGAGCAGACCAGAGCCAACAGC CCCACCAGAAGAGAGCTTCA
GGTTTG GGGAAGAGACAACAACTCCCTCTCAGAAG GAG GAGC C GAT
AGACAAGGAACTGTATC CTTTAGCTTCC CTCAGATCACTCTTTGGCA
GC GAC CC CTCGTCACAAAAC TTTAGC CAGAACTATC CGATTGTG CA
GACCG GTG C CC CAAAGAAGAAG CGGAAG GTCTCTAGACAAGAGAT
CAAGAGAATCAACAAGATCAGAAGGAGACTGGTCAAGGACAGCAAC
ACAAAGAAG GC C GG CAAGACAG GCCC CATGAAAACCCTGCTC GTCA
GAGTGATGACCCCTGACCTGAGAGAGCGGCTGGAAAACCTGAGAA
AGAAG CC CGAGAACATCCCTCAGC CTATCAG CAACACCAG CAGGGC
CAACCTGAACAAGCTGCTGACCGACTACACCGAGATGAAGAAAGCC
ATC CTG CAC GTGTACTG GGAAGAGTTC CAGAAAGACCC CGTGGGCC
TGATGAG CAGAGTTG CTCAG C CC G CTCCTAAGAACATCGACCAGAG
AAAGCTGATCC CCGTGAAGGACGGCAACGAGAGACTGACCTCTAGC
GGCTTTGCCTGCAGCCAGTGTTGCCAGCCTCTGTAC GTGTACAAGC
TGGAACAAGTGAACGACAAGG GCAAGC CC CACAC CAACTACTTC GG
CAGATGCAAC GTGTCC GAG CAC GAGAG GCTGATC CTG CTGTCTC CT
CACAAGC CC GAGGCCAACGATGAGCTG GTCACATACAGC CTGG GC
AAGTTCGGACAGAGAG CC CTG GACTTCTACAGCATC CACGTGAC CA
GG GAGAGCAATCAC CCTGTGAAGC C CCTG GAACAGATCG GC GG CA
ATAGCTGTGCCTCTGGACCTGTGGGAAAAGCCCTGAGCGACGCCT
GTATGGGAGCCGTGGCATC CTTCCTGACCAAGTAC CAC GACATCAT
CCTG GAACACCAGAAAGTGATCAAGAAGAACGAGAAAAGACTGG CC
AACCTCAAGGATATC GC CAGC GCTAAC GGCCTGGC CTTTCCTAAGA
TCACCCTGCCTCCACAGCCTCACAC CAAAGAGGGCATCGAGGCCTA
CAACAACGTGGTGGCCCAGATCGTGATTTGGGTCAACCTGAATCTG
TGGCAGAAGCTGAAGATCGGCAGGGACGAAGC CAAGC CACTGCAG
AGACTGAAG G G CTTCC CTAGCTTCC CTCTGGTGGAAAGACAG GC CA
ATGAAGTGGATTGGTGGGACATG GTCTGCAACGTGAAGAAGCTGAT
CAACGAGAAGAAAGAGGATGGCAAGGTTTTCTGGCAGAACCTGGCC
GG CTACAAGAGACAAGAAG CC CTGAGGCCTTACCTGAGCAGCGAA
GAG GAC C GGAAGAAG GGCAAGAAGTTCG C CAGATAC CAGTTCGGC
GACCTGCTG CTG CAC CTG GAAAAGAAGCACGG C GAG GACTG G GG C
AAAGTGTACGATGAGGCCTGGGAGAGAATCGACAAGAAGGTGGAA
GGC CTGAGCAAGCACATTAAG CTG GAAGAG GAAAGAAG GAG C GAG
GACGCCCAATCTAAAGCCGCTCTGACCGATTGGCTGAGAGCCAAGG
CCAGCTTTGTGATC GAG G GC CTGAAAGAG GCC GACAAGGAC GAGT
TCTGCAGATGCGAGCTGAAGCTGCAGAAGTG GTACG GC GATCTGA
GAG G CAAG C CCTTC GC CATTGAG G CCGAGAACAGCATCCTGGACAT
CAGCGGCTTCAGCAAGCAGTACAACTGCGC CTTCATTTGGCAGAAA
GACG GC GTCAAGAAACTGAAC CTGTACCTGATCATCAATTACTTCAA
AG GC GG CAAGCTGC G GTTCAAGAAGATCAAAC CC GAG G C CTTC GA
GG CTAACAGATTCTACACCGTGATCAACAAAAAGTCCG GCGAGATC
GTG CC CATG GAAGTGAACTTGAACTTC GAC GAC CC CAACCTGATTAT

Construct DNA
SEQUENCE
CCTGCCTCTGGC CTTCGGCAAGAGACAGGGCAGAGAGTTCATCTG
GAAC GATCTGCTGAGC CTGGAAAC C G G CTCTCTGAAG CTG GC CAAT
GGCAGAGTGATCGAGAAAACC CTGTACAACAGGAGAACCAGACAG
GACGAGGCTGCTCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGG
TGCTGGACAGCAGCAACATCAAGCCCATGAACCTGATC GGCATC GA
CC GGGGCGAGAATATCC CTGCTGTGATCGCCCTGACAGACCCTGAA
GGATGCCCACTGAGCAGATTCAAGGACTCCCTGGGCAACCCTACAC
ACATC CTGAGAATC GGC GAGAGCTACAAAGAGAAGCAGAGGACAAT
CCAGGCCAAGAAAGAGGTGGAAGAGAGAAGAGCCGGCGGATACTC
TAGGAAGTAC GC CAG CAAG GC CAAGAATCTG G CC GAC GACATGGT
CC GAAACAC C G CCAGAGATCTGCTGTACTAC GC CGTGACACAGGAC
GC CATGCTGATCTTCGAGAATCTGAG CAGAGG CTTCG GC C GG CAG
GGCAAGAGAACCTTTATGGC C GAGAGGCAGTACAC CAGAATGGAAG
ATTGGCTCACAGCTAAACTGGC CTACGAGGGACTGAGCAAGACCTA
CCTGTCCAAAACACTGGC CCAGTATAC C TCCAAGACCTGCAGCAAT
TGCGGCTTCACCATCAC CAGC GC C GACTACGACAGAGTGCTGGAAA
AGCTCAAGAAAACCGCCACCGGCTGGATGACCACCATCAACGGCAA
AGAGCTGAAGGTTGAGGGC CAGATCAC C TACTACAACAGGTACAAG
AG G CAGAAC GTCGTGAAGGATCTGAGC GTGGAACTGGACAGACTG
AG C GAAGAGAG C GTGAACAAC GACATCAG CAG CTG GACAAAG G G C
AGATCAG GC GAGGCTCTGAGCCTGCTGAAGAAGAGGTTTAGCCACA
GACCTGTGCAAGAGAAGTTCGTGTGCCTGAACTGCGGCTTCGAGAC
ACAC GCC GATGAACAGGCTGCC CTGAACATTGCCAGAAGCTGGCTG
TTCCTGAGAAGCCAAGAGTACAAGAAGTACCAGACCAACAAGACCA
CC GG CAACAC C GACAAGAG G GC CTTTGTG GAAAC CTG G CAGAG CT
TCTACAGAAAAAAGCTGAAAGAAGTCTGGAAGCC C GC C GTGACTAG
TCCAAAAAAGAAGAGAAAGGTAGCC CTCGAGTACC CATATGATGTC
CCTGACTACGCT (SEQ ID NO: 427) pXDP 11 ATGGGTGC GAGAGC GTCAGTATTAAG CGG G
GGAGAATTAGATC GAT
GGGAAAAAATTC GGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTA
AAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTA
ATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGG
ACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCAT
TATATAATACAGTAGCAACC CTCTATTGTGTGCATCAAAGGATAGAG
ATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAA
ACAAAAGTAAGAAAAAAG CACAG CAAG CAG CAG CTGACACAG GACA
CAGCAATCAGGTCAGC CAAAATTACCCTATAGTGCAGAACATC CAG
GGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATG
GGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCC CAGAAGTGATACC C
ATGTTTTCAG CATTATCAGAAG GAG C CACC CCACAAGATTTAAACAC
CATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTA
AAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATC
CAGTGCATGCAGGG CCTATTG CAC CAGG C CAGATGAGAGAAC CAA
GGGGAAGTGACATAGCAGGAACTACTAGTAC CCTTCAGGAACAAAT

Construct DNA
SEQUENCE
AGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATA
AAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGC
CCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTA
GAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCT
TCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAA
TGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGGAG
CGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGAC
CCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAAC
AAATC CAGCTAC CATAATGATACAGAAAGGCAATTTTAGGAAC CAAA
GAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGC
CAAAAATTGCAGGGCC C CTAG GAAAAAG GG CTGTTG GAAATGTG GA
AAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTT
TTTAGGGAAGATCTGGCCTTCC CACAAGGGAAG GC CAGG GAATTTT
CTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCA
GGTTTGGGGAAGAGACAACAACTC CCTCTCAGAAGCAGGAGCC GAT
AGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCA
GCGACCCCTCGTCACAAAACTTTAGCCAGAACTATCCGATTGTGCA
GAC CGGTGCCCCAAAGAAGAAGC GGAAGGTCTCTAGACAAGAGAT
CAAGAGAATCAACAAGATCAGAAGGAGACTGGTCAAGGACAGCAAC
ACAAAGAAGGCCGGCAAGACAGGCCCCATGAAAACCCTGCTCGTCA
GAGTGATGACCCCTGACCTGAGAGAGCGGCTGGAAAACCTGAGAA
AGAAGCCCGAGAACATCCCTCAGCCTATCAGCAACACCAGCAGGGC
CAACCTGAACAAGCTGCTGAC CGACTACACC GAGATGAAGAAAGCC
ATCCTG CAC GTGTACTG GGAAGAGTTC CAGAAAGACC CCGTGGGCC
TGATGAGCAGAGTTGCTCAGCCCGCTCCTAAGAACATCGACCAGAG
AAAGCTGATCC CCGTGAAGGACGGCAACGAGAGACTGACCTCTAGC
GGCTTTGCCTGCAGCCAGTGTTGCCAGCCTCTGTACGTGTACAAGC
TGGAACAAGTGAACGACAAGGGCAAGC C CCACAC CAACTACTTC GG
CAGATGCAACGTGTCC GAG CACGAGAG G CTGATC CTG CTGTCTC CT
CACAAGCCCGAGGCCAACGATGAGCTGGTCACATACAGCCTGGGC
AAGTTCGGACAGAGAGCCCTGGACTTCTACAGCATCCACGTGACCA
GGGAGAGCAATCACCCTGTGAAGCCCCTGGAACAGATCGG C GG CA
ATAGCTGTGCCTCTGGACCTGTGGGAAAAGC CCTGAGCGACGC CT
GTATG GGAG CC GTG GCATC CTTCCTGACCAAGTAC CAGGACATCAT
CCTGGAACACCAGAAAGTGATCAAGAAGAACGAGAAAAGACTGGCC
AACCTCAAGGATATC GC CAGC GCTAAC GGCCTGGC CTTTCCTAAGA
TCACCCTGC CTCCACAGCCTCACAC CAAAGAGGGCATC GAG G CCTA
CAACAAC GTG GTG GC CCAGATC GTGATTTGGGTCAACCTGAATCTG
TGGCAGAAGCTGAAGATC G GCAG G CAC GAAG C CAAGC CACTGCAG
AGACTGAAGGGCTTCC CTAGCTTCC CTCTG GTG GAAAGACAG GC CA
ATGAAGTGGATTGGTGGGACATGGTCTGCAACGTGAAGAAGCTGAT
CAACGAGAAGAAAGAGGATGGCAAGGTTTTCTGGCAGAACCTGGCC
GG CTACAAGAGACAAGAAG CC CTGAGGCCTTACCTGAGCAGCGAA
GAGGACCGGAAGAAGGGCAAGAAGTTCGCCAGATACCAGCTGGGC
GACCTGCTGAAGCACCTGGAAAAGAAGCACGGCGAGGACTGGGGC

Construct DNA
SEQUENCE
AAAGTGTACGATGAGGCCTGGGAGAGAATCGACAAGAAGGTGGAA
GG C CTGAG CAAG CACATTAAG CTG GAAGAG GAAAGAAG GAG C GAG
GACGC CCAATCTAAAGCCGCTCTGACCGATTGGCTGAGAGCCAAGG
CCAGCTTTGTGATC GAG G GC CTGAAAGAG G CC GACAAGGAC GAGT
TCTGCAGATGCGAGCTGAAGCTGCAGAAGTGGTACGGCGATCTGA
GAG G CAAG C CCTTC GC CATTGAG G C CGAGAACAGCATC CTGGACAT
CAGCGGCTTC AGCAAGCAGTACAACTGCGCCTTCATTTGGCAGAAA
GAC G GC GTCAAGAAACTGAAC CTGTAC CTGATCATCAATTACTTCAA
AG G C GG CAAGCTGC G GTTCAAGAAGATCAAAC CC GAG G C CTTC GA
GGCTAACAGATTCTACACCGTGATCAACAAAAAGTCCGGCGAGATC
GTGC CCATGGAAGTGAACTTCAACTTC GAC GAC CC CAACCTGATTAT
CCTGCCTCTGGC CTTCGGCAAGAGACAGGGCAGAGAGTTCATCTG
GAAC GATCTGCTGAGC CTGGAAAC C G G CTCTCTGAAG CTG GC CAAT
GGCAGAGTGATCGAGAAAACC CTGTACAACAGGAGAACCAGACAG
GAC GAG C CTGCTCTGTTTGTG GC C CTGAC CTTC GAGAGAAGAGAGG
TGCTGGACAGCAGCAACATCAAGCCCATGAACCTGATC GGC GTG GA
CCGGGGCGAGAATATCCCTGCTGTGATCGCCCTGACAGACCCTGAA
GGATGC C CACTGAGCAGATTCAAGGACTCCCTGGGCAAC C CTACAC
ACATCCTGAGAATC GGCGAGAGCTACAAAGAGAAGCAGAGGACAAT
CCAGGCCAAGAAAGAGGTGGAACAGAGAAGAGCCGGCGGATACTC
TAGGAAGTAC GC CAG CAAG GC CAAGAATCTG G CC GAC GACATGGT
CCGAAACACCGCCAGAGATCTGCTGTACTACGCCGTGACACAGGAC
GC CATGCTGATCTTC GAGAATCTGAG CAGAGG CTTC G GC C GG CAG
GGCAAGAGAACCTTTATGGCCGAGAGGCAGTACACCAGAATGGAAG
ATTGGCTCACAGCTAAACTGGC CTACGAGGGACTGAGCAAGAC CTA
CCTGTCCAAAACACTGGC CCAGTATAC C TCCAAGACCTGCAGCAAT
TGCGGCTTCACCATCACCAGCGCCGACTACGACAGAGTGCTGGAAA
AG CTCAAGAAAAC CGC CAC C G GCTGGATGAC CAC CATCAAC GGCAA
AGAGCTGAAGGTTGAGGGCCAGATCAC CTACTACAACAG GAG GAAG
AG G CAGAAC GTCGTGAAGGATCTGAGC GTGGAACTGGACAGACTG
AG C GAAGAGAG C GTGAACAAC GACATCAG GAG CTG GACAAAG G G C
AGATCAG GC GAGGCTCTGAGCCTGCTGAAGAAGAGGTTTAGCCACA
GAC CTGTGCAAGAGAAGTTC GTGTGC CTGAACTGC GGCTTC GAGAC
ACAC G CC GATGAACAGGCTGCC CTGAACATTGCCAGAAGCTGGCTG
TTCCTG AGAAGCCAAGAGTACAAGAAGTACCAGAC CAACAAGACCA
CC GG CAACAC C GACAAGAG G GC C TTTGTG GAAAC CTG G CAGAG CT
TCTACAGAAAAAAGCTGAAAGAAGTCTGGAAGCC C GC CGTGACTAG
TC CAAAAAAGAAGAGAAAGGTAGCC CTC GAGTACC CATATGATGTC
CCTGACTACGCT (SEQ ID NO: 428) pXDP1 2 ATGG GTG C GAGAG C GTCAGTATTAAG CG G G GGAGAATTAGATCGAT
GGGAAAAAATTC GGTTAAGGC CAGGGGGAAAGAAAAAATATAAATTA
AAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTA
ATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGG
ACAGCTACAACCATC CCTTCAGACAGGATCAGAAGAACTTAGATCAT

Construct DNA
SEQUENCE
TATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAG
ATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAA
ACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACA
CAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAG
GG GCAAATG GTACATCAGG C CATATCAC CTAGAACTTTAAATGCATG
GGTAAAAGTAGTAGAAGAGAAGGCTTTCAGGCCAGAAGTGATACCC
ATGTTTTCAG CATTATCAGAAG GAG CCACC CCACAAGATTTAAACAC
CATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTA
AAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATC
CAGTGCATG CAC GG CCTATTG CAC CAGG C CAGATGAGAGAAC CAA
GGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAAT
AGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATA
AAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGC
CCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTA
GAGACTATGTAGACC GATTCTATAAAACTCTAAGAGC C GAG CAAGCT
TCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAA
TGCGAAC CCAGATTGTAAGACTATTTTAAAAGCATTGGG AC CAGGAG
CGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGAC
CC GGCCATAAAG CAAGAGTTTTGGCTGAAG CAATGAG CCAAGTAAC
AAATC CAGCTAC CATAATGATACAGAAAGG CAATTTTAGGAAC CAAA
GAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGC
CAAAAATTG CAG GGCC C CTAGGAAAAAGGGCTGTTGGAAATGTG GA
AAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTT
TTTAG GGAAGATCTGGCCTTCC CACAAGGGAAG GC CAGG GAATTTT
CTTCAGAGCAGACCAGAGC CAACAGC C C CAC CAGAAGAGAG CTTCA
GGTTTG GGGAAGAGACAACAACTC C CTCTCAGAAG CAG GAGC C GAT
AGACAAG GAACTGTATC CTTTAGCTTCC CTCAGATCACTCTTTGGCA
GC GAC C C CTC GTCACAAAACTTTAGCCAGAACTATC C GATTGTG CA
GACCG GTG C CC CAAAGAAGAAG CGGAAG GTCTCTAGACAAGAGAT
CAAGAGAATCAACAAGATCAGAAGGAGACTGGTCAAGGACAGCAAC
ACAAAGAAGGCCGGCAAGACAGGCCCCATGAAAACCCTGCTCGTCA
GAGTGATGACC C CTGACCTGAGAGAG CGOCTOGAAAACCTGAGAA
AGAAGCC C GAGAACATC CCTCAGC CTATCAGCAACACCAG CAGGGC
CAAC CTGAACAAGCTGCTGACCGACTACAC CGAGATGAAGAAAG C C
ATCCTG CAC GTGTACTG GGAAGAGTTC CAGAAAGACC CCGTG GGCC
TGATGAGCAGAGTTGCTCAGC CCGCTCCTAAGAACATC GAC CAGAG
AAAG CTGATCCCCGTGAAGGACGG CAACGAGAGACTGACCTCTAGC
GG CTTTGCCTGCAG CCAGTGTTG CCAGCCTCTGTAC GTGTACAAG C
TGGAACAAGTGAACGACAAGG GCAAGC C C CACAC CAACTACTTC GG
CAGATGCAACGTGTCC GAG CACGAGAG GCTGATC CTG CTGTCTC CT
CACAAGC C C GAGGC CAAC GATGAGCTG GTCACATACAGC CTGG GC
AAGTTCGGACAGAGAG CC CTG GACTTCTACAGCATC CACGTGAC CA
GG GAGAGCAATCAC CCTGTGAAGC CCCTG GAACAGATCG GC GG CA
ATAGCTGTGCCTCTGGACCTGTGGGAAAAGCCCTGAGCGACG C CT
GTATGGGAG CC GTG GCATC CTTCCTGACCAAGTAC GAG GACATCAT

Construct DNA
SEQUENCE
CCTG GAACACAAGAAAGTGATCAAGAAGAACGAGAAAAGACTGGC C
AACCTCAAGGATATCGC CAGCGCTAAC G GC CTG G C CTTTC CTAAGA
TCACCCTGC CTCCACAGCCTCACAC CAAAGAG GGCATC GAG G CCTA
CAACAACGTGGTGGCCCAGATCGTGATTTGGGTCAACCTGAATCTG
TGGCAGAAGCTGAAGATCGGCAGGGACGAAGC CAAGC CACTGCAG
AGACTGAAG G G CFTC C CTAGCTTCC CTCTG GTGGAAAGACAG GC CA
ATGAAGTGGATTGGTGGGACATG GTCTGCAACGTGAAGAAGCTGAT
CAACGAGAAGAAAGAGGATGGCAAGGTTTTCTGGCAGAAC C TGGCC
GG CTACAAGAGACAAGAAG CC CTGAGG GOTTA CCTGAGCAGC CC C
GAG GAC CG GAAGAAG GGCAAGAAGTTCGCCAGATACCAGCTGG GC
GAC CTGCTG CTG CAC CTG GAAAAGAAGCACGG C GAG GACTG G GG C
AAAGTGTACGATGAGGCCTGGGAGAGAATCGACAAGAAGGTGGAA
GGC CTGAG CAAG CACATTAAG CTG GAAGAG GAAAGAAG GAG C GAG
GACGCCCAATCTAAAGCCGCTCTGACCGATTGGCTGAGAGCCAAGG
CCAGCTTTGTGATC GAG G GC CTGAAAGAG GCC GACAAGGAC GAGT
TCTGCAGATGCGAGCTGAAGCTGCAGAAGTGGTACGGCGATCTGA
GAG G CAAG C CCTTC GC CATTGAG G C CGAGAACAGCATC CTGGACAT
CAGCGGCTTCAGCAAGCAGTACAACTGCGC CTTCATTTGGCAGAAA
GACG GC GTCAAGAAACTGAAC CTGTACCTGATCATCAATTACTTCAA
AG GC GG CAAGCTGC G GTTCAAGAAGATCAAAC CC GAG G C CTTC GA
GGCTAACAGATTCTACACCGTGATCAACAAAAAGTCCG GCGAGATC
GTGC CCATG GAAGTGAACTTCAACTTC GAC GAC CC CAACCTGATTAT
CCTGC CTCTGGCC TTCGGCAAGAGACAGGGCAGAGAGTTCATCTG
GAAC GATCTGCTGAGC CTGGAAACCG G CTCTCTGAAG CTG GC CAAT
GGCAGAGTGATCGAGAAAC CCCTGTACAACAGGAGAACCAGACAG
GAC GAG C CTGCTCTGTTTGTG GC C CTGAC CTTCGAGAGAAGAGAGG
TG CTG GACAG CAG CAACATCAAG CC CATGAACCTGATC GG C GTG GA
CC GGGGC GAGAATATCC CTGCTGTGATCGC CCTGACAGAC CCTGAA
GGATGCCCACTGAGCAGATTCAAGGACTCCCTGGGCAACCCTACAC
ACATCCTGAGAATC GGCGAGAGCTACAAAGAGAAGCAGAGGACAAT
CCAGGCCAAGAAAGAGGTGGAACAGAGAAGAGCCGGCGGATACTC
TAGGAAGTAC GC CAGCAAG GC CAAGAATCTG G CC GAC GACATGGT
CC GAAACAC C GC CAGAGATCTGCTGTACTAC GC C GTGACACAGGAC
GC CATGCTGATCTTCGAGAATCTGAGCAGAGGCTTCG GC C GGCAG
GGCAAGAGAACCTTTATGGCCGAGAG GCAGTACACCAGAATGGAAG
ATTGGCTCACAGCTAAACTGGC CTACGAGGGACTGAGCAAGACCTA
CCTGTCCAAAACACTG G CC CAGTATAC CTCCAAGACCTGCAGCAAT
TGCGGCTTCACCATCACCAGC GC C GACTACGACAGAGTGCTGGAAA
AG CTCAAGAAAAC CGC CAC C G GCTGGATGAC CAC CATCAAC GGCAA
AGAGCTGAAGGTTGAG GGCCAGATCAC CTACTACAACAG GAG GAAG
AG GCAGAAC GTCGTGAAGGATCTGAGC GTGGAACTGGACAGAC TG
AG C GAAGAGAG C GTGAACAAC GACATCAG CAG CTG GACAAAG GGC
AGATCAG GC GAGGCTCTGAGCCTGCTGAAGAAGAGGTTTAGCCACA
GACCTGTGCAAGAGAAGTTCGTGTGCCTGAACTGC GGCTTCGAGAC
ACACGCCGATGAACAGGCTGCCCTGAACATTGCCAGAAGCTGGCTG

Construct DNA
SEQUENCE
TTCCTGAGAAGCCAAGAGTACAAGAAGTACCAGAC CAACAAGACCA
CCGGCAACACCGACAAGAGGGCCTTTGTGGAAACCTGGCAGAGCT
TCTACAGAAAAAAG CT GAAAGAAGTCTG GAAGCC C GC C GT GACTA G
TCCAAAAAAGAAGAGAAAG GTAGCC CTCGAGTACC CATATGATGTC
CCTGACTACGCT (SEG ID NO: 429) psPax2 AT GG GTG C GAGA G C GTCAGTATTAAG CG G
GGGAGAATTAGATCGAT
GO GAAAAAATTC GGTTAAGG CCAGGG GGAAAGAAAAAATATAAATTA
AAACATATAGTATGGG CAA GC A GG GA GC TAGAACGATTCG CAGTTA
AT C CTG G C C TGTTA GAAAC AT CAGAAG GC TGTAGACAAATAC T GG G
ACAG CTACAAC CAT CC CTTCAGACAG GAT CAGAA GAACTTAGATCAT
TATATAATACAGTAG CAACC CTCTATTGTGTGCATC AAAGGATAGAG
ATAAAAGACAC CAAGGAAG CTTTAGACAAGATAGAGGAAGAGCAAA
AC AAAA GTAAGAAAAAAG CAC AG CAAG C A G CAG CT GACAC AG GAC A
CAGCAATCAGGTCAGC CAAAATTAC CCTATAGTGCAGAACATC C AG
GO GCAAATG GTACATCAGG CC ATATCAC CTA GAAC TTTAAATG CATG
GGTAAAAGTAGTAGAAGAGAAGG CTTT CA G CC CAGAAGTGATACC C
AT GTTTTCAG CATTATCAGAAG GAG CCACC CCACAAGATTTAAACAC
CATG CTAAACACAGTG G G GG GAC ATCAA GC A G CC AT G CAAATGTTA
AAAGAGACCATCAATGAGGAAGCTG CAGAATG GGATAGAGTG CATC
CAGTGCATG CAC GO CC TATTG CAC CA GG C CAGATGAGAGAAC CAA
GG GGAAGTGACATAGCAGGAACTACTAGTAC C C TTC AG GAACAAAT
AG GATGGATGACACATAATC CAC CTATC C CAGTAGGAGAAATC TATA
AAAGATGGATAATC CTG GGATTAAATAAAATAGTAAGAATGTATAG C
CCTAC CAGCATTCTGGACATAAGACAAGGACCAAAGGAAC CCTTTA
GAGACTATGTAGAC C GATT CTATAAAACTCTAAGA G C C GAG CAA GC T
TCACAA GAG GTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAA
TGCGAAC C CA GATTGTAAGAC TATTTTAAAAG CATTG GGAC CAGGAG
CGACACTAGAAGAAATGAT GACAG C ATGT CAC GGAGTGGGGG GAG
CCGGCCATAAAG CAAGAGTTTTGGCTGAAG CAATGAG C CAAGTAAC
AAATC CAGCTAC CATAATGATACAGAAAGG CAATTTTAGGAAC CAAA
GAAAGACTGTTAAGTGTTTCAATTGTGG CAAAGAAGGG C AC ATAG C
CAAAAATTG CAG GGCCCC TAG GAAAAAG GG C TGTTGGAAATGTG GA
AA GGAAG GACACC AAATGAAA GATTGTACTGA GA GAC A GG CTAATTT
TTTAG GGAAGATCTGGC CTTCC CACAAGGGAAG GC C AGG GAATTTT
CTTCAGAGCAGACCAGAGC CAACAGC CCCACCAGAAGAGAG CTTCA
GGTTTG GG GAA GAGAC AACAACTC C CTC TC A GAA G CAG GAGC C GAT
AGACAAG GAACTGTATC CTTTAGCTTCC CT CAGATCACTCTTTG GCA
GCGACCCCTCGTCACAATAAAGATAGGGGGGCAATTAAAGGAAGCT
CTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAATTT
GC CAG GAAGATGGAAAC CAAAAATGATAGGGGGAATTG GAGGTTTT
AT CAAAGTAAGAC AGTATGATCAGATAC TC ATAGAAATC TG CC GACA
TAAAGCTATAGGTACAGTATTAGTAGGACCTACAC CT GTC AACATAA
TTGGAAGAAATCTGTTGACTCAGATTGG CTGCACTTTAAATTTTCC C
ATTAGTC CTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGA

Construct DNA
SEQUENCE
TGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAG
CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCA
AAAATTG GGCCTGAAAATC CATACAATACTCCAGTATTTGC CATAAA
GAAAAAAGACAGTAGTAAATGGAGAAAATTAGTAGATTTCAGAGAAC
TTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCA
CATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGT
GG GCGATGCATATTTTTCAGTTC CCTTAGATAAAGACTTCAGGAAGT
ATAC TGCATTTAC CATAC CTAGTATAAACAATGAGACAC CAGG GATT
AGATATCAGTACAATGTGCTTCCACAGG GATGGAAAGGATCA C CAG
CAATATTCCAGTG TAG CATGACAAAAATCTTAGAGC CTTTTAGAAAA
CAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTA
GGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAAC
TGAGACAACATCTGTTGAG GTG GGGATTTACCACACCAGACAAAAA
ACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATC
CTGATAAATG GACAGTACAGCCTATAGTGCTGCCAGAAAAG GACAG
CTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGG
CAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTT
CTTAGGG GAAC CAAAG CACTAACAGAAGTAGTAC CACTAACAGAAG
AAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACC
GGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAA
TACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGA
GCCATTTAAAAATCTGAAAACAGGAAAGTATGCAAGAATGAAGGGTG
CCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATA
GCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATT
ACC CATACAAAAGGAAACATGG GAAGCATGGTGGACAGAGTATTGG
CAAG C CAC CTG GATTCCTGAGTGGGAGTTTGTCAATAC C C CTC C CT
TAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCA
GAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGG
AAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCC
CTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCT
AGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCAC
AATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCA
GAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGT
CTACCTGGCATGG GTAC CAGCACACAAAGGAATTG GAG GAAATGAA
CAAGTAGATAAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTT
AGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTA
ATTGGAGAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCA
AAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAG
CCATGCATG GACAAGTAGACTGTAGCC CAGGAATATG GCAGCTAGA
TTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAG
CCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCA
AGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAA
AAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTT
AAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATT
CCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGA

Construct DNA
SEQUENCE
ATTAAAGAAAATTATAG GACAG GTAAGAGATCAGG CTGAACATCTTA
AGA C A GC A GTAC AAATG G CAGTATTC ATC CA CAATTTTAAAAGAAAA
GG GGGGATTGG GGGGTACAGTGCAGGGGAAAGAATAGTAGACATA
ATAGCAAGAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAA
ATTCAAAATTTTC GG GTTTATTACAG GGACAGCAGAGATC CAGTTTG
GAAAGGACCAGCAAAG CTC CT CT GGAAA GGTGAA GGG GCAGTAGT
AATA C AA GATAATA GTGA C ATAAAAGTA GTG C CAAGAA GAAAAG CAA
AGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGT
GG CAA GTA GA CAG GAT GAG GATTAACACATGGAATTCTGCAACAAC
TG CTGTTTATC CATTTC A GAATTG G GT GTC GACATAG CAGAATA GG C
GTTACTC GACAGAGGAGAG CAAGAAATG GAG CCAGTAGATC CTA GA
CTAGA GC C CT G GAAGCATC C A GGAA GTC A G CC TAAAAC TG CTTGTA
C CAATT G C TATTGTAAAAAGTGTT GC TTTCATTG C CAAGTTT GTTT CA
TGACAAAAG C CTTAGG CAT CT CC TATGG CAG GAAGAAG CG GAGAGA
GC GAC GAAGAG CTC AT CAGAA CAGTC AGAC T CATCAAG C TTCTC TAT
CAAAGCAGTAAG (SEQ ID NO: 430) pGP2 AT GAAGTGC CTTTTGTACTTAG CC TTTTTATT CATTG GG
GTGAATTGC
AA GTTC A CC ATA GTTTTT C CA CAC AA C CAAAAA G GAAA CTG GAAAAA
TGTTC C TT C TAATTAC C ATTATTGCC C GTCAAG CTCAGATTTAAATTG
GCATAATGACTTAATAGGCACAGC CTTACAAGTCAAAATGC CCAAGA
GT CAC AA GG C TATTCAAGCAGAC GGTTGGATGTGTCATGCTTC CAA
AT GG GTCAC TAC TT GTGATTT C C GCTGGTATGGACC GAAGTATATAA
CAC ATT C CATCC GATC CTTC A C TCC ATCTGTA GAA CAATG C AAG GAA
AG CATTGAACAAAC GAAACAAGGAACTTGGCTGAATCCAGGCTTC C
CT CC TCAAAGTTGT GGATATG CAA CTGTGA C GGATG CCGAAGCAGT
GATTGTCCAGGTGACTCCTCAC CATGT GC TG GTT GATGAATA C A CA
GGAGAATGG GTTGATTCACAGTTCATCAACGGAAAATG CAGCAATTA
CATATG C CC CA CTGTC CATAACTCTAC AA C CTG GCATTCTGACTATA
AG GTCAAAG GG C TATGT GATTC TAAC C TC ATTTC C AT G GAC AT CAC C
TTCTTCTCAGAG GACG GAGAG CTATCATC CC TG G GAAAGGAG G G CA
CAGG GTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGG CAAGG C
CTGCAAAATGCAATAC TG C AA GCATT GG G GAGTCAGACTC C CATC A
GGTGTCTGGTTCGAGATG GC TGATAA GGATCTCTTT G C TG CA GC CA
GATTC C CTGAATGC C CAGAAGGGTCAAGTATC TCTGC TC CATCTCA
GAC CT CA GTG GATGTAAGTCTAATTCAG GACGTTGAGAGGATCTTG
GATTATTC CC TCTGC CAAGAAAC CTG GAG CAAAATCAGA G CG GGTC
TTC CAATC TC TC CAGTG GATCTCAG C TATCTT GC TC CTAAAAAC CCA
GGAACC GGTCC TG C TTT CA C CATAATCAATG GTA CC CTAAAATAC TT
TGAGACCAGATACATCAGAGTC GATATTG C T GC TC CAATC CTCTCAA
GAATG GT CG GAATGATCAGTGGAACTACCACAGAAAG GGAACTGTG
GGATGACTG G G CAC CATATGAAGACGTGGAAATTG GAC C CAATG GA
GTTCTGAGGACCAGTTCAGGATATAAGTTTC CTTTATACATGATTG G
AC ATG GTAT GTTG GAC TC C GATC TTC AT C TTA GC TC AAA GG C TC A GG
TGTTC GAACATC C T CAC ATTC AAGAC GC TGC TTC GCAACTTC C TGAT

Construct DNA
SEQUENCE

CGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCT
CTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCC
GAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGA
CAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTAA (SEQ
ID NO: 431) Transfection 10011581 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 8th 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 jig of pXDP10, pXDP11, or pXDP12.5 pig of pStx42.174.12.7, 3.3 jig of psPax2, and 1 pg of pGP2 in 680 gl of Opti-MEM
media. 87.5 gl 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 10011591Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 i.tM 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 N'TE, as described below.
Example 14: Purification of XDP
10011601 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 jaM
PES filters before being stored at 4 C until purification.
Centrifugation Protocol [0011611Filtered supernatant was divided evenly into an appropriate number of centrifuge tubes or bottles and 115th of the supernatant volume of Sucrose Buffer (50mM Tris-HCL, 100mM
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 NaC1, 15% Trehalose dihydrate, pH 8) or an appropriate media by gentle trituration and vortexing.
Column Protocol 10011621Filtered 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-HCI, pH
7.5).
Bound material was eluted using a gradient elution from 0% - 100% Buffer B
(100 mM Tris-HCI, 1M NaCI, 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 mlkil Tris-HC1, 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 10011631Samples 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 an NP150 nanopore.
10011641FIG. 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 pM 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.
10011651On 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 10011661 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; CasX119, 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 FIEK293T cells (Takara Biosciences) using one or more structural plasmids (derived from one or more components of the Gag-Pol 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 Versio Design XDP plasmid DNA SEQUENCE
n& number CasX
V1 MA-CA-NC-Pl/P6-X pXDP17 (SEQ ID NO: 432) MA-CA-NC-PI/P6-(-1)- 491 POL
pXDP4 (SEQ ID NO: 433) V2 MA-CA-NC-P1/P6-X pXDP17 (SEQ ID NO: 434) 491 MA-CA-NC-P1/P6-X-PR pXDP22 (SEQ ID NO: 435) V3 MA-CA-NC-P1/P6-X pXDP17 (SEQ ID NO: 436) 491 MA-CA-NC-P1/P6-(-1)-pXDP13 (SEQ ID NO: 783) X-PR

MA-CA-NC-Pl/P6-(-1)-pX0P13 (SEQ ID NO: 784) X-PR

MA-CA-NC-P1/P6-X-PR pXDP22 (SEQ ID NO: 785) MA-CA-NC-P1/P6-X-PR pXDP22 (SEQ ID NO: 786) MA-CA-NC-P1/P6-X pXDP17 (SEQ ID NO:787) MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 788) MA-CA-NC-P1/P6-X pXDP17 SEQ ID NO: 789) V8 MA-CA-NC-P1/P6-X pXDP17 (SEQ ID NO: 790) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 791) MA-CA-NC-X (no p1/p6) pXDP0023 (SEQ ID NO: 792) MA-CA-NC-P1-X pXDP0024 (SEQ ID NO: 793) V11 MA-CA-NC-X-(-1)-PR pXDP0025 (SEQ ID NO: 794) Versio Design XDP plasm id DNA SEQUENCE
n& number CasX

MA-CA-X-(-1)-PR
pXDP0026 (SEQ ID NO: 795) MA-X-NC-(-1)-PR
pXDP0027 (SEQ ID NO: 796) MA-X-(-1)-PR
pXDP0028 (SEQ ID NO: 797) MA-X-PR
pXDP0029 (SEQ ID NO: 798) MA-CA-X-PR
pXDP0030 (SEQ ID NO: 799) MA-X
pXDP0031 (SEQ ID NO: 800) MA-CA-X
pXDP0032 (SEQ ID NO: 801) MA-X-X-(-1)-PR
pXDP0033 (SEQ ID NO: 802) MA-CA-X-X-(-1)-PR
pXDP0034 (SEQ ID NO: 803) MA-CA-NC-X-X-(-1)-PR pXDP0035 (SEQ ID NO: 804) V22 Gag-1%TCS-STx-HA
pXDP0036 (SEQ ID NO: 805) 491 Gag-PCS-HRV3c pXDP0039 (SEQ ID NO: 806) V23 Gag-TCS-STx-HA
pXDP0037 (SEQ ID NO: 807) 491 Gag-PCS-HRV3c pXDP0039 (SEQ ID NO: 808) V24 Gag-PCS-STx-HA
pXDP0038 (SEQ ID NO: 809) 491 Gag-PCS-IIRV3c pXDP0039 (SEQ ID NO: 810) Transfection 10011671 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 tug of the guide plasmid, and 0.1 Kg of pMD2.G in 680 Eil of Opti-MEM media. 87.5 gl 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 10011681 Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 KM 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
10011691tdTomato 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 10x 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.
[0011701Results: 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 CasX119 with VSV-g;
Bald VLP is XDP CasX119 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 Group % TDT+ cells (% editing) VSV-G XDP 20%
CasX119 No Glycoprotein <0.5%
XDP CasX119 Buffer control <0.5%
Example 17: Construction of XDP with incorporated glycoproteins to evaluate tropism and editing capabilities 10011711 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.
10011721Experiments 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 fig of the VSV-G plasmid was used for the 100% VSV-G group, 0.3 mg was used for the 30% VSV-G group, 0.1 pig was used for the 10% VSV-G group, 0.03 lig was used for the 3% VSV-G
group, 0.01 gg was used for the 1% VSV-G group, and 0.003 gg 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.
10011731 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. 2613 10011741 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 lig (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 g,lycoprotein(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 028 pg of pGP14.1, 022 pg of pGP14.2, 0.27 pg of pGP14.3, and 020 pg of pGP14.4;
RD114 used 0.96 jig of pGP8; HCV used 0.97 ug of pGP23; EBOV used 1.02 pg of pGP41;
Mokola used 1.02 jig 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.
10011751The 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.
10011761We 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 lx 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 jig of psPax2, 1.8 itg of pGP2, and 7.2 mg 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
1X. 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. 28A. 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.
Glycoprotein SEQUENCE
pGP2 (VSV-G) (SEQ ID NO: 811) pGP29 (Rabies) (SEQ ID NO: 812) pGP60 (FUG E) (SEQ ID NO: 813) pGP14.1 (HSV-1 gB) (SEQ ID NO: 814) pGP14.2 (HSV-1 gD) (SEQ ID NO: 815) pGP14.3 (HSV-1 gH) (SEQ ID NO: 816) pGP14.4 (HSV-1 gL) (SEQ ID NO: 817) pGP8 (RD114) (SEQ ID NO: 818) pGP23 (HCV) (SEQ ID NO: 819) pGP41 (EBOV) (SEQ ID NO: 820) pGP30 (Mokola) (SEQ ID NO: 821) Example 18: Construction and evaluation of XDP with RNP comprising CasX with enhanced editing capabilities 10011771ln 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 jig of pStx43.119, RNP 438 was transfected using 19.8 pig of pStx43.438, and RNP 119 was transfected using 19.8 jig 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 10011781 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.
10011791 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 transfeetion, 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)11IV-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 H1V-1 protease (PR) and lacks the HIV-1 reverse transcriptase (RT), p15, 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-Pot 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 pug of pGP2, 5 pg of pStx42 (guide 174) with spacer 12.7, and 19.8 pig of pStx43.119 (CasX 119). Additional constructs used the following plasmid amounts:
100% Gag-Pol used 3.3 mg of psPax2; the 50% Gag-Pol + 50% Gag-CasX construct used 1.65 1.tg of psPax2 and 1.48 pg of Gag-CasX-PR; the 30% Gag-Pot + 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 tug of Gag-CasX-PR, and the 100% Gag-CasX
construct used 3.00 lug of Gag-CasX-PR. Sequences are provided in Table 20.
10011801Editing 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-Pd. 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 Construct Sequence pMRG103 (Gag-CasX119-PR) (SEQ ID NO:
822) pM1RG103 (Gag-CasX438-PR) (SEQ ID NO:
823) Example 20: Construction and evaluation of XDP targeting human cells 10011811 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 (GTGTAGTACAAGAGATAGAA, SEQ ID NO: 824) and 7.37 (GGCCGAGATGTCTCGCTCCG, SEQ ID NO: 826) target the human B2M locus and spacer 12.7 (CTG-CATTCTAGTTGTGGTTT, 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 RPM1 media supplemented with 10% FBS, sodium pyruvate, and GlutalvIAX. 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:
10011821The 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.
10011831The 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 HRT-1 capsid designed specifically for RNP delivery, we created thirty-five different versions of HINT-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 10011841 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 10011851 In order to generate pXDP3, pXDP17, pXDP23-32, pXDP98-100, pXDP102 and pXDP103, pXDP1 (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 UD Cloning Kit from Takara (Cat# 639650) according to the manufacturer's protocols. Assembled products were transformed into chemically-competent Turbo Competent E. tole 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 10011861The 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 Tak.ara (Cat 639650) according to manufacturer protocols. Assembled products were transformed into chemically-competent Turbo Competent E. coil bacterial cells, plated on LB-Agar plates (LB: Teknova Cat# L9315, Agar: Quartzy Cat's/
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
10011871 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 (CTGCATTCTAGTTGTGGITT, 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. call (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 10011881 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 coil bacterial cells, plated on LB-Agar plates containing kanarnycin and incubated at 37t.
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 10011891HEK293T 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 pig. Each transfection also received 13 pig of p42.174.12.7 and 0.25 rig 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 10011901 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, 100mM NaCl, 10% Sucrose, pH 7.4). XDPs were resuspended in 300 Ed, of DMEM/

supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
Resuspension and transduction [0011911tdTomato 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 10011921 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 X_X where 122 (MA-CA-pl/p6) has no p2 and achieves 444%
editing and versions 128 (MA-CA-p2-pl/p6) includes p2 and archives only 29.2%
editing. In addition, constructs with multiple p 1 /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.

10011931Overall, 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.
XDP Structural Structural Structural Structural Version Plasmid 1 Plasmid 2 Plasmid 1 plasmid 2 CasX % Editing 1 Gag-stx gag-pol pXDP17 pXDP1 491 95.4 gag(-1) gag(-1)-PR-4 Stx pXDP88 491 2.78 7 Gag-Stx pXDP17 491 92.2 8 Gag-Stx gag pXDP17 pXDP3 491 87.5 Ma-CA-NC-9 X pXDP23 491 14.20 MA-CA-NC-P1-X pXDP24 491 7.19 MA-CA-NC-X(-1)-11 Pro pXDP25 491 35.8 MA-CA-X(-12 1)-Pro pXDP26 491 28.5 MA-Stx-

13 NC-(-1)-PR pXDP27 491 17.3 MA-X-(-1)-

14 Pro pXDP28 491 32.6 MA-X-Pro pXDP29 491 MA-CA-16 Stx-Pro OCDP30 491 1.86 17 MA-X pXDP31 491 37.9 18 MA-CA-X pXDP32 491 18.3 31 MA-CA- Gag pXDP23 pXDP3 491 17.0 XDP Structural Structural Structural Structural Version Plasmid 1 Plasmid 2 Plasmid 1 plasmid 2 CasX % Editing NC-X
MA-CA-32 NC-P1-X Gag pXDP24 pXDP3 491 13.40 MA-CA-NC-X(-1)-33 PR Gag pXDP25 pXDP3 491 90.1 MA-CA-X(-34 1)-PR Gag pXDP26 pXDP3 491 95.3 MA-X-NC-pl/p6-(-1)-35 Pro Gag pXDP27 pXDP3 491 11.6 MA-X-(-1)-36 Pro Gag pXDP28 pXDP3 491 25.10 MA-CA-38 STx-Pro Gag pXDP30 pXDP3 491 8.5 39 MA-X Gag pXDP31 pXDP3 491 30.7 40 MA-CA-X Gag pXDP32 pXDP3 491 843 MA-CA-122 p1/p6-X pXDP98 491 44.4 MA-CA-p 1 /p6-pl/p6-123 X pXDP99 491 91.5 MA-CA-NC-p1/136-124 p1/p6-X pXDP100 491 73.2 MA-CA-126 NC-X-pl/p6 pXDP102 491 44.8 MA-CA-p2-128 pl/p6-x pXDP104 491 29.2 *% Editing was calculated by taking the maximum editing percentage of the 5 dilutions' averaged replicates.

Table 22: Encoding sequences for guides and glycoproteins Plasmid DNA
sequence p42.174,12.7 A CTG G C G CTTTTATC TgATTA CTTT G A GAG C CATC AC CAC C GA CT
ATGTC GTAgTG GGTAAAG CT C C CTC TTC G GAG G GAG CAT CAAAG
CTGCATTCTAGTTGTGGTTT (SEQ ID NO: 828) A C TG G C GC TTTTATC TgATTA C TTT G A GAG C C ATC AC C AG C GA C T
ATGTC GTAgTG GGTAAAG CTC CCTCTTC G GAG G GAG CATCAAAG
p42.174,NT CGAGACGTAATTACGTCTCG (SEQ ID NO: 829) pGP2 ATG AA GTG CCTTTTGTACTTAGCCTTTTTATTCATTG G G
GT GAATT
GCAAGTTCAC CATAGTTTTTC CACACAAC CAAAAAG GAAAC TG GA
AAAATGTTCCTTCTAATTAC CATTATTG C CC GTC AAG CTCAGATTT
AAATTGGCATAATGACTTAATAGG C AC AG C CTTACAAGTCAAAAT
GCCCAAGAGTCACAAGGCTATTCAAGCAGACG GTTGGATGTGTC
ATG CTTCCAAATG G GTCA CTA C TTGTG A TTT C C G CTGGTATGGAC
C GAAGTATATAACACATTC CATCC GATCCTTCACTC CATCTGTAG
AAC AATGC AAG GAAAG CATTG AA CAAAC GAAACAAGGAACTTG G
C TG AATCC AG GCTTCCCTCCTCAAAGTTGTGGATATG CAA CT GTG
ACG GATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGT
GCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCAT
CAACG GAAAATG GAG CAATTACATATGC CC C AC TG TC CATAACTC
TACAACCTGG CATTCTGACTATAAGGTCAAAG GGCTATGTGATTC
TAAC CTCATTTC CATG GACATC AC CTTCTTCTCAGAGGAC G GAGA
GCTATCATCCCTG G GAAAG GAG GGCACAGGGTTCAGAAGTAACT
A CTTTG CTTATGAAACTG GA G G CAAG GCCTG CAAAATG CAATA CT
GCAAGCATTG GGGAGTCAGACTC C CATC AG GTGTCTGGTTC GAG
ATG G CT GATAA G GATCTCTTTGCTG CAG CCAGATTC C CTG AATG C
C CAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGA
TGTAAGTCTAATTCAG GACGTTGAGAGGATCTTGGATTATTC C CT
CTG C CAA GAAAC CT GGAGC AAAATCAGAG C GGGTCTTCCAATCT
CTC C AG TG GATCTCAGCTATCTTGCTCCTAAAAAC C C AG GAAC C G
GTC CTG CTTTCACCATAATCAATG GTACC CTAAAATACTTT GAGA
C CAGATACATCAGAGTC GATATTGCTGCTC CAATC CTCTCAAGAA
TGGTC GGAATGATCAGTGGAACTAC CA CAGAAAG GGAACTGTGG
GATGACTG G G CAC CATATG AA GAC GTG G AAATTG GACCCAATG G
AGTTCTGAGGAC CAGTTCAGGATATAAGTTTC CTTTATACATGATT
GGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAG G CT
C A G GTGTTCGAACATCCTCACATTCAAGACG CT G CTTC G CAAC TT
CCTGATGATGAGAGTTTA i 1111 i GGTGATACTGGGCTATCCAAA
AATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAG C
TCTATTGC CTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATT
CTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCAC
AC CAAGAAAAGACAGATTTATACAGACATAGAGATGAAC C GACTT
GGAAAGTAA (SEQ ID NO: 830) Table 23: XDP Versions and Component Encoding Sequences Versions & XDP
plasmid Design DNA Sequence CasX
number V1 MA-CA-NC-P1/P6-X pXDP17 (SEQ ID NO: 831) 491 MA-CA-NC-P1/P6-(-1)-POL pXDP1 (SEQ ID NO: 832) V4 MA-CA-NC-P1/P6-(-1)-X-PR pXDP88 (SEQ ID NO: 833) VS MA-CA-NC-P1/P6-X-PR pXDP22 (SEQ ID NO: 834) V7 MA-CA-NC-P1/P6-X pXDP17 (SEQ ID NO: 835) V8 MA-CA-NC-P1/P6-X pXDP17 (SEQ ID NO: 836) MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 837) j V9 MA-CA-NC-X (no p1/p6) pXDP23 (SEQ ID NO: 838) V10 MA-CA-NC-P 1-X pXDP24 (SEQ ID NO: 839) j V11 MA-CA-NC-X-(-1)-PR p3CDP25 (SEQ ID NO: 840) V12 MA-CA-X-(-1)-PR p3CDP26 (SEQ ID NO: 841) V13 MA-X-NC-(-1)-PR p3CDP27 (SEQ ID NO: 842) Versions & XDP
plasmid Design DNA Sequence CasX
number j V14 MA-X-(-1)-PR pXDP28 (SEQ ID NO: 843) j V15 MA-X-PR p3CDP29 (SEC) ID NO: 844) j V16 MA-CA-X-PR pXDP30 (SEQ ID NO: 845) j V17 MA-X pXDP31 (SEQ ID NO: 846) V18 MA-CA-X pXDP32 (SEQ ID NO: 847) (SEQ ID NO: 848) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 849) j V32 MA-CA-NC-P 1-X pXDP24 (SEQ ID NO: 850) 491 MA-CA-NC-PI/P6 pXDP3 (SEQ ID NO: 851) j V33 MA-CA-NC-X-(-1)-PR p3CDP25 (SEQ ID NO: 852) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 853) j V34 MA-CA-X-(-1)-PR p3CDP26 (SEQ ID NO: 854) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 855) j V35 MA-X-NC-(-1)-PR p3CDP27 (SEQ ID NO: 856) Versions & XDP
plasmid Design DNA Sequence CasX
number 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 857) V36 MA-X-(-1)-PR pXDP28 (SEQ ID NO: 858) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 859) V37 MA-X-PR pXDP29 (SEQ ID NO: 860) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 861) V38 MA-CA-X-PR pXDP30 (SEQ ID NO: 862) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 863) V39 MA-X pXDP31 (SEQ ID NO: 864) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 865) V40 MA-CA-X pXDP32 (SEQ ID NO: 866) 491 MA-CA-NC-P1/P6 pXDP3 (SEQ ID NO: 867) V122 MA-CA-P1/P6-X pXDP98 (SEC) ID NO: 868) V123 MA-CA-P1/P6-Pl/P6-X pXDP99 (SEQ ID NO: 869) V124 MA-CA-NC-P1/P6-P1/P6-X pXDP100 (SEQ ID NO: 870) V125 MA-CA-X-P1/P6 pXDP101 (SEQ ID NO: 871) V126 MA-CA-NC-X-P1/P6 pXDP102 (SEQ ID NO: 872) V128 MA-CA-P2-P1/P6-X pXDP104 (SEQ ID NO: 873) Example 22: Transfection and recovery of XDP constructs in the Gag-(4)-protease-CasX
configuration derived from Retroviruses.
10011941Editing 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 10011951 XDPs derived from Alpharetrovinises (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 pStx.42.174.12.7 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. MI plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 24.
10011961 XDPs derived from Betaretroviruses (Enzootic Nasal Tumor Virus (ENTV), mouse mammary tumor virus (IVINITV) and Mason-Pfizer monkey virus (MPMV)) in the Gag-(-1)-protease-CasX variation (Version 46, 47, 62 and 90; see FIG. 52B) were produced by transient transfection of LentiX HEIC293T cells using the three plasmids encoding the Gag-(-1)-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.
10011971 XDPs derived from Deltaretroviruses (bovine leukemia virus (BLV) and human T
lymphotropic virus (HTLV1)) in the Gag-(-1)-protease-CasX variation (Version 48, 49 and 63) were produced by transient transfection of LentiX HEIC293T cells using three plasmids encoding the Gag-(-1)-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
PAILPHS (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.
10011981XDPs 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.

10011991 XDPs derived from Gammaretroviruses (feline leukemia virus (FLY) 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 p3CDP49 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.
10012001 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-(-1)-protease-CasX variation (Version 53, 54, 55 and 91) were produced by transient transfection of LentiX HEK293T cells using three plasmids encoding the Gag-(-1)-protease-CasX, the g,lycoprotein, and the guide RNA, respectively, and listed in Table 24. The pXDP50, pXDP51, pXDP52, p3CDP53 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), SEEYPIMT (SEQ ID NO: 941), G-GNYPVQQ (SEQ ID NO:
942) and REVYPIVN (SEQ 1D 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, p3CDP53 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.
10012011 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 (RHSFY) and Simian foamy virus (SFV)) in the Gag-(-1)-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-(-1)-protease-CasX, the glycoprotein, and the guide RNA, respectively, and listed in Table 24. The pXDP54, pXDP55, p3CDP56, p3CDP57, p3CDP58, pXDP59 and p3CDP60 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 1D NO: 785), NTVHTVRQVES (SEQ 1D 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 pSix42.174.12.7 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 Version XDP plasmid SEQ ID NO of DNA
number Sequence pStx42.174.12. 880 pGP2 881 44 pX D P40 882 45 pXDP41 883 46 pX D P42 884 90 pX D P43 885 47 pX D P44 886 48 pX D P45 887 49 pXDP46 888 Version XDP plasmid SEO ID NO of DNA
number Sequence 50 pXDP47 889 51 pXDP48 890 52 pXDP49 891 91 pXDP50 892 53 pXDP51 893 54 pXDP52 894 55 pXDP53 895 56 pXDP54 896 57 pXDP55 897 58 pXDP56 898 59 pXDP57 899 92 pXDP58 900 60 pXDP59 901 61 pXDP60 902 62 pXDP61 903 63 pDP62 904 64 pXDP63 905 V29 pXDP88 906 Transfection 10012021The steps for creation of the XDP are depicted graphically in FIG. 24.

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 pig), pXDP44 (159 pg), pXDP45 (145 rig), pXDP46 (149 pg), pXDP47 (152 pg), pXDP48 (148 p.g), p3CDP49 (149 pg), pXDP50 (145 rig), pXDP51 (146 pg), p3CDP52 (147 pg), pXDP53 (144 p.g), pXDP54 (149 pg), pXDP55 (153 rig), pXDP56 (154 pig), pXDP57 (150 pg), pXDP58 (146 pg), pXDP59 (154 pg), pXDP60 (154 rig), pXDP61 (159 pig), pXDP62 (149 pg), pXDP63 (147 pg), pXDP88 (146 pg). Along with the structural plasmid, each transfection also received 26.3 pig of pStx42.174.12.7, and the 5 pg of pGP2 in 3800 RE of Opti-MEM media. 1 mg/m1 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 10012031 Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 LIM 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
10012041 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 10x 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 p.1 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 50W and 16.6 1 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 pl) Version Genus/order Virus XDP plasmid number Editing %
number 44 Alpharetrovirus ALV
pXDP40 91.5 45 Alpharetrovirus RSV
pXDP41 4.3 46 Betaretrovirus ENTV 0CDP42 9.1 90 Betaretrovirus MMTV
p3CDP43 7.3 47 Betaretrovirus MPMV
p3CDP44 30.5 62 Betaretrovirus MPMV Native p3CDP61 30,8 48 Deltaretrovirus BLV
p3CDP45 194 49 Deltaretrovirus HTLV1 pXDP46 20.1 63 Deltaretrovirus HTLV1 Native pXDP62 37.0 50 Epsilonretrovirus WDSV
pXDP47 10.9 51 Gammaretrovirus FLV
p3CDP48 6.7 52 Gammaretrovirus MMLV
p3CDP49 12.4 91 Non-primate CAEV 0CDP50 8.2 lentivirus 53 Non-primate EIAV p3CDP51 5.3 lentivirus 54 Non-primate SW
pXDP52 11.7 lentivirus 64 Non-primate SW Native pXDP63 13.5 lentivirus 55 Non-primate VMV
p3CDP53 8.7 lentivirus 56 Spumaretrovirinae BFV
pXDP54 3.6 Version Genus/order Virus XDP plasmid number Editing %
number 57 Spumaretrovirinae BGPFV
pXDP55 8.9 58 Spumaretrovirinae CCFV
pXDP56 5.5 59 Spumaretrovirinae EFV
pXDP57 4.4 92 Spumaretrovirinae FFV
pXDP58 7.3 60 Spumaretrovirinae RHSFV
pXDP59 4.2 61 Spumaretrovirinae SFV
pXDP60 4.5 29 Lentivirus HIV
p3CDP88 7.4 Table 26: Results of Editing Assay for the second dilution (16.6u0 Version Genus/order Virus XDP plasmid number Editing %
number 44 Al pharetrovi rus ALV
p3CDP40 85.7 45 Al pharetrovi rus RSV
p3CDP41 2_9 46 Betaretrovirus ENTV
pXDP42 2.3 90 Betaretrovirus MMTV
pXDP43 7.6 47 Betaretrovirus MPMV
pXDP44 2.6 62 Betaretrovirus MPMV Native pXDP61 8.5 48 Deltaretrovirus BLV
pXDP45 15.2 49 Deltaretrovirus HTLV1 p3CDP46 1.8 63 Deltaretrovirus HTLV1 Native pXDP62 13.0 50 Epsi1onretrovirus WDSV
pXDP47 1.1 51 Gammaretrovirus FLY
pXDP48 7.8 52 Gammaretrovirus MMLV
pXDP49 6.3 91 Non-primate CAEV
pXDP50 3.1 1entivirus 53 Non-primate EIAV
pXDP51 3.8 lentivirus 54 Non-primate SW
pXDP52 1.3 1entivirus Version Genus/order Virus XDP plasm id number Editing "4 number 64 Non-primate SIV Native pXDP63 1.0 lentivirus 55 Non-primate VMV
pXDP53 7.4 lentivirus 56 Spumaretrovirinae BFV
pXDP54 1.9 57 Spumaretrovirinae BGPFV
pXDP55 4.5 58 Spumaretrovirinae CCFV
pXDP56 3.7 59 Spumaretrovirinae EFV
pXDP57 2.7 92 Spumaretrovirinae FFV
pXDP58 1_7 60 Spumaretrovirinae RHSFV
pXDP59 3_4 61 Spumaretrovirinae SFV
pXDP60 1.8 29 Lentivirus HIV
pXDP88 53 Example 23: Transfection and recovery of XDP constructs in a MA-CA-CasX
configuration derived from Retroviruses 10012061Editing 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 10012071XDPs 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 pS1x42.174.12.7 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.
10012081 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.
10012091 XDPs derived from Deltaretroviruses (ELY and ITTLV1) in the MA-CA-CasX
variation (Version 71A, 72A and 88A, FIG. 5713) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG. 5711 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/ 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.
10012101 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.
10012111 XDPs derived from Gammaretroviruses (FLV and !ONLY) in the MA-CA-CasX

variation (Version 74A and 75A, FIG. 59B) were produced by transient transfection of LentiX
IfEK293T cells using the three plasmids portrayed in FIG. 5913 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.

10012121XDPs derived from Non-primate Lentiviruses (CAEV, ELAN, SIV and VMV) irk the MA-CA-CasX variation (Version 76A, 77A, 78A, 79A and 89A, FIG. 60B) were produced by transient transfection of LentiX BEK293T cells using the three plasmids portrayed in FIG. 6011 and listed in Table 27. The pXDP74, pXDP75, p3CDP76, 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 Version XDP plasmid SEQ
ID NO of DNASequence number N/A pStx42.I74.12.7 907 pGP2 66a pXDP64 909 67a pXDP65 910 68a pXDP66 911 69a pXDP67 912 70a p3CDP68 913 71a pXDP69 914 72a 0CDP70 915 73a pXDP7I 916 74a pXDP72 917 Version XDP plasmid SEQ
ID NO of DNASequence number 75a pXDP73 76a pXDP74 77a pXDP75 78a pXDP76 79a pXDP77 87a pXDP85 88a pXDP86 89a pXDP87 59 pXDP57 92 pXDP58 60 pXDP59 61 pXDP60 62 pXDP61 63 pXDP62 64 pXDP63 V29 pXDP88 Transfection 10012131 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 8th 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 jig), pXDP65 (143 jig), pXDP66 (142 jig), pXDP67 (143 jig), pXDP68 (144 jig), pXDP69 (136 jig), pXDP70 (137 g), pXDP71 (141 jig), pXDP72 (140 lig), pXDP73 (142 jig), pXDP74 (134 g), pXDP75 (134 jig), pXDP76 (134 jig), pXDP85 (144 lig), pXDP86 (137 jig), pXDP87 (138 jig), pXDP32 (114 g). Along with the structural plasmid, each transfection also received 26.3 fig of pStx42.174.12.7, and the 5 jig of pGP2 in 3800 pl 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 10012141Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0,45 LAM 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
10012151 tdTomato neural progenitor cells (tdT NPCs) were grown in DMEM F12 supplemented with glutamax, HEPES, non-essential amino acids, Pen/Strep, 2-mereaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using a Takara Bioscienc,es 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 10x 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.
[0012161Results: The results of the editing assay are shown in FIGS. 71A 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 I 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 I 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) i Version Genus/order Virus XDP plasmid number Editing V.
number 66a Alpharetrovirus ALV
pXDP64 47.0 67a Alpharetrovirus RSV
pXDP65 42.5 68a Betaretrovirus ENTV
pXDP66 18.3 69a Betaretrovirus MMTV pXDP67 11.7 70a Betaretrovirus MPMV pXDP68 28.3 87a Betaretrovirus MPMV Native pXDP85 30.8 71a Deltaretrovirus BLV
pXDP69 31.1 72a Deltaretrovirus HTLV1 pXDP70 22.4 88a Deltaretrovirus HTLV1 Native pXDP86 37.0 73a Epsilonretrovirus WDSV pXDP71 14.2 74a Gammaretrovirus FLY
pXDP72 77.5 75a Gammaretrovirus MMLV p3CDP73 67.3 76a Non-primate CAEV p3CDP74 18.5 lentivirus 77a Non-primate EIAV
p3CDP75 46.2 lentivirus 78a Non-primate SIV
pXDP76 17.6 tentivirus 89a Non-primate SIV Native p3CDP87 13.5 lentivirus 18 Lentivirus HIV
pXDP32 21.3 Table 29: Results of Editing Assay for the second dilution (16.6 I) Version Genusiorder Virus XDP plasmid number Editing %
number 66a Alpharetrovirus ALV
0CDP64 11.2 67a Alpharetrovirus RSV
pXDP65 14.3 68a Betaretrovirus ENTV
pXDP66 2.3 Version Genus/order Virus XDP plasmid number Editing %
number 69a Betaretrovirus MMTV pXDP67 1.7 70a Betaretrovirus MPMV pXDP68 6.0 87a Betaretrovirus MPMV Native pXDP85 8.5 71a Deltaretrovirus BLV
pXDP69 3.2 72a Deltaretrovirus HTLV1 pXDP70 2.9 88a Deltaretrovirus HTLV1 Native pXDP86 13.0 73a Epsilonretrovirus WDSV pXDP71 1.9 74a Gammaretrovirus FLV
pXDP72 32.0 75a Gammaretrovirus MMLV pXDP73 38.0 76a Non-primate CAEV pXDP74 5.6 lentivirus 77a Non-primate EIAV
pXDP75 29.1 lentivirus 78a Non-primate SIV
pXDP76 7.0 lentivirus 89a Non-primate SIV Native pXDP87 1.0 lentivirus 18 Lentivirus 1-11V
pXDP32 9.3 Example 24: Transfection and recovery of XDP constructs in the Gag-(-1)-protease-CasX
configuration derived from Retroviruses.
10012171Editing 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 Alphareiroviruse, Beiareiroviruse, Gammaretroviruse, Deltaretroviruse, Epsilonretroviruse, Non-primate lentiviruses and Spumaretroviruse Method for the generation of XDPs 10012181 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 arnpicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.
10012191 XDPs derived from Betaretroviruses (Enzootic Nasal Tumor Virus (ENTV), mouse mammary tumor virus (MMTV) and Mason-Pfizer monkey virus (MPMV)) in the Gag-(-1)-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.

10012201 XDPs derived from Deltaretroviruses (bovine leukemia virus (BLV) and human T
lymphotropic virus (HTI.V1)) in the Gag-(-1)-protease-CasX variation (Version 48, 49 and 63;
see FIG. 53A) were produced by transient transfection of LentiX 11EK293T 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 PAILHIS (SEQ ID NO: 945), PQVLPVIVITI (SEQ ID
NO: 946) and PQVLPVMEI (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.
10012211XDPs 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. 538 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. MI plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.
10012221 XDPs derived from Gammaretroviruses (feline leukemia virus (FLV) and murine leukemia virus (MA/11,V)) in the Gag-protease-CasX variation (Version 51 and 52; see FIG. 54A) were produced by transient transfection of LentiX ITEK293T 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. MI plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 30.
10012231 3CDPs 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-(-1)-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, 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, p3CDP51, pXDP52, p3CDP53 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.
10012241 XDPs derived Spumaretrovifinae 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-(-1)-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 p3CDP54, p3CDP55, p3CDP56, 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, p3CDP56, pXDP57, pXDP58, pXDP59 and pXDP60 plasmid respectively. The pSix42.174.12.7 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 Version XDP plasmid SEQ ID NO of DNA Sequence number pStx42.174.12.7 pGP2 44 pXDP40 45 pXDP41 46 pXDP42 90 pXDP43 47 pXDP44 48 pXDP45 49 pXDP46 50 pXDP47 51 pXDP48 52 pXDP49 91 pXDP50 53 pXDP51 54 pXDP52 Version XDP plasmid SEQ ID NO of DNA Sequence number 55 pXDP53 56 pXDP54 57 pXDP55 58 pXDP56 59 pXDP57 92 pXDP58 60 pXDP59 61 pXDP60 62 pXDP61 63 pXDP62 64 pXDP63 V29 pXDP88 Transfection 10012251 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 jig), pXDP42 (157 pg), pXDP43 (157 pg), pXDP44 (159 pg), pXDP45 (145 i's). pXDP46 (149 pg), pXDP47 (152 pg), p3CDP48 (148 pg), pXDP49 (149 pg), pXDP50 (145 i's) , pXDP51 (146 pg), pXDP52 (147 pg), pXDP53 (144 pg), pXDP54 (149 pg), pXDP55 (153 pg), pXDP56 (154 pg), p>CDP57 (150 pig), p>CD1P58 (146 lig), pXDP59 (154 pg), pXDP60 (154 pg), pXDP61 (159 pg), p3CDP62 (149 pg), p3CDP63 (147 lig), pXDP88 (146 pg). Along with the structural plasmid, each transfection also received 26.3 pg of pStx42.174,12.7, and the 5 jig of pGP2 in 3800 pl 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 10012261 Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 p.114 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
10012271tdTomato 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 10x 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.
10012281Results: 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 gl and 16.6 gl 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 pd) Version Genus/order Virus XDP plasmid number Editing %
number 44 Alpharetrovirus ALV
pXDP40 91.5 Version Genus/order Virus XDP plasmid number Editing %
number 45 Al pharetrovirus RSV
pXDP41 4.3 46 Betaretrovirus ENTV
pXDP42 9.1 90 Betaretrovirus MMTV
pXDP43 7.3 47 Betaretrovirus MPMV
pXDP44 30.5 62 Betaretrovirus MPMV Native pXDP61 30.8 48 Deltaretrovirus BLV
pXDP45 19.4 49 Deltaretrovirus HTLV1 pXDP46 20.1 63 Deltaretrovirus HTLV1 Native pXDP62 37.0 50 Epsi I onretrovirus WDSV
pXDP47 10.9 51 Gammaretrovirus FLV
pXDP48 6.7 52 Gammaretrovirus MMLV
pXDP49 12.4 91 Non-primate CAEV
pXDP50 8.2 lentivirus 53 Non-primate EIAV
pXDP51 5.3 lentivirus 54 Non-primate SW
pXDP52 11.7 lentivirus 64 Non-primate SW Native pXDP63 13.5 lentivirus 55 Non-primate VMV
pXDP53 8.7 lentivirus 56 Spumaretrovirinae BFV
pXDP54 3.6 57 Spumaretrovirinae BGPFV
pXDP55 8.9 58 Spumaretrovirinae CCFV
pXDP56 5.5 59 Spumaretrovirinae EFV
pXDP57 4.4 92 Spumaretrovirinae FFV
pXDP58 7.3 60 Spumaretrovirinae RHSFV
pXDP59 4.2 61 Spumaretrovirinae SFV
pXDP60 4.5 29 Lentivirus HIV
pXDP88 7.4 Table 32: Results of Editing Assay for the second dilution (16.6 I) Version Genus/order Virus XDP plasmid number Editing %
number 44 Al pharetrovirus ALV
pXDP40 85.7 45 Al pharetrovirus RSV
pXDP41 2.9 46 Betaretrovirus ENTV pXDP42 2.3 90 Betaretrovirus MMTV
p3CDP43 7.6 47 Betaretrovirus MPMV
pXDP44 2.6 62 Betaretrovirus MPMV Native pXDP61 8.5 48 Deltaretrovirus BLV
pXDP45 15.2 49 Deltaretrovirus HTLV1 pXDP46 1.8 63 Deltaretrovirus HTLV1 Native pXDP62 13.0 50 Epsi I onretrovirus WDSV
pXDP47 1.1 51 Gammaretrovirus FLY
pXDP48 7.8 52 Gammaretrovirus MMLV
p3CDP49 6.3 91 Non-primate CAEV p3CDP50 3.1 lentivirus 53 Non-primate EIAV pXDP51 3.8 lentivirus 54 Non-primate SW
pXDP52 1.3 lentivirus 64 Non-primate SW Native pXDP63 1.0 lentivirus 55 Non-primate VMV
pXDP53 7.4 lentivirus 56 Spumaretrovirinae BFV
p3CDP54 1.9 57 Spumaretrovirinae BGPFV
p3CDP55 4.5 58 Spumaretrovirinae CCFV p3CDP56 3.7 59 Spumaretrovirinae EFV
p3CDP57 2.7 92 Spumaretrovirinae FFV
pXDP58 1.7 60 Spumaretrovirinae RHSFV
pXDP59 3.4 Version Genus/order Virus XDP plasmid number Editing %
number 61 Spumaretrovirinae SFV
pXDP60 1.8 29 Lentivirus HIV
pXDP88 5.3 Example 25: Transfection and recovery of XDP constructs in the Gag-CasX
configuration derived from Retroviruses.
10012291Editing 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, Garnmaretroviruses, 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 10012301XDPs derived from Alpharetroviruses (avian leukosis virus (ALV) and rous sarcoma virus (RSV)) in the Gag-CasX variation (V102 and V114; see FIG. 62B) were produced by transient transfection of LentiX ITEIC293T 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.
MI plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33.
10012311XDPs derived from Betaretroviruses (Enzootic Nasal Tumor Virus (ENTV), mouse mammary tumor virus (MMTV) and Mason-Pfizer monkey virus (MPMV)) in the Gag-CasX
variation (V106, V111, V112 and V113, FIG. 64A) 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.
MI plasmids contained either an ampicillin or kanamycin resistance gene. The sequences incorporated into the plasmids are presented in Table 33 .
10012321XDPs derived from Deltaretroviruses (bovine leukemia virus (BLV) and human T
lymphotropic virus (HTLV1)) in the Gag-CasX variation (Version V103, V108 and V109, FIG.
63A) 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 .
10012331XDPs derived from Epsilonretroviruses (walleye dermal sarcoma virus (WDSV)) in the Gag-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 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.
1001231 XDPs derived from Gammaretroviruses (feline leukemia virus (FLV) and murine leukemia virus (MMLV)) in the Gag-CasX variation (V107 and V110, FIG. 64B) were produced by transient transfection of LentiX HEK293T cells using the three plasmids portrayed in FIG.
MB and listed in Table 33. The p3CDP132, 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.
10012351 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 (V104, V105, V115, V116 and V117, 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 p3CDP129, p3CDP130, pXDP140, p3CDP141 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 3CDP 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.
10012361 XDPs derived Spumaretrovirinae family (bovine foamy virus (BFV), equine foamy virus (EFV), feline foamy virus (FFV), Brown greater galas 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 Version XDP
number plasmid SEQ ID NO
of DNA Sequence pStx42.1 N/A

74.12.7 pGP2 102 pXDP127 103 pXDP128 104 pXDP129 105 pXDP130 106 pXDP131 107 pXDP132 108 pXDP133 109 pXDP134 110 pXDP135 111 pXDP136 112 pXDP137 113 pXDP138 114 pXDP139 115 pXDP140 116 pXDP141 117 pXDP142 118 pXDP143 80a pXDP78 Version XDP
number plasmid SEQ ID NO of DNA Sequence 81a pXDP79 994 82a pXDP80 995 83a pXDP81 996 84a pXDP82 997 85a pXDP83 998 86a pXDP84 999 V29 pXDP88 1000 Transfection 10012371 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 jig), pXDP131 (145 pg), pXDP132 (143 pg), pXDP135 (145 jig), pXDP136 (152 pg), pXDP138 (149 gg), pXDP139 (146 pg), pXDP140 (143 jig), pXDP141 (143 jig), pXDP142 (141 pg), pXDP143 (146 pg), pXDP78 (145 pg), pXDP81 (141 pg), pXDP82 (139 pg), pXDP83 (145 pg), pXDP0017 (122 jig). Along with the structural plasmid, each transfection also received 26.3 jig of pStx.42.174.12.7, and the 5 pig of pGP2 in 3800 pl of Opti-MEM media. 1 mg/m1 linear polyethylenimine (PEI, MVV=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 10012381 Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 p.114 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
10012391 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 10x 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.
[0012401Results: The results of the editing assay are shown in FIGS. 75A 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 (50u1) Version Genus/order Virus XDP plasmid number Editing %
102 Alpharetrovirus ALV
pXDP127 94.2 114 Alpharetrovirus RSV
pXDP139 43.4 106 Betaretrovirus ENTV
pXDP131 29.1 111 Betaretrovirus MIVITV
pXDP136 11.1 113 Betaretrovirus MPMV Native pXDP138 19.2 118 Epsilonretrovinis WDSV
pXDP143 2,5 107 Gammaretrovirus FLY
pXDP132 6.8 110 Gammaretrovirus MMLV
pXDP135 45.2 Version Genus/order Virus XDP plasmid number Editing %
Non-primate 104 CAEV pXDP129 14.6 lenti virus Non-primate 105 EIAV pXDP130 44.2 1enti virus Non-primate 115 SIV pXDP140 43.1 lenti virus Non-primate 116 S IV Native pXDP141 48.1 lenti virus Non-primate 117 VIv1V pXDP142 9.6 lenti virus 7 Lentivirus 1-DV
pXDP0017 84.5 80a Spumaretrovirus BFV
pXDP78 29.2 83a Spumaretrovirus EFV
pXDP81 4.7 84a Spumaretrovirus FFV
pXDP82 4.9 85a Spumaretrovirus RHSFV pXDP83 4.1 Table 35: Results of Editing Assay for the second dilution (16.6u1) Version Genus/order Virus XDP plasmid number Editing %
102 Alpharetrovirus ALV pXDP127 95.8 114 Alpharetrovirus RSV
pXDP139 20.9 106 Betaretrovirus ENTV
pXDP131 10.5 111 Betaretrovirus MMTV
pXDP136 1.0 113 Betaretrovirus MPMV Native pXDP138 2.7 118 Epsilonretrovirus WDSV pXDP143 2.6 107 Gammaretrovirus FLV
pXDP132 6.4 110 Gammaretrovirus MMLV
pXDP135 12.8 Non-primate 104 CAEV pXDP129 1.3 lenti virus Non-primate 105 EIAV pXDP130 21.7 lendvirus Non-primate 115 SIV pXDP140 2.7 lendvirus Version Genus/order Virus XDP plasmid number Editing %
Non-primate 116 SIV Native pXDP141 27.3 lentivirus Non-primate 117 VMV pXDP142 1.0 lentivirus 7 Lentivirus HIV
pXDP0017 80.0 80a Spumaretrovirus BFV
pXDP78 11.4 83a Spumaretrovirus EFV
pXDP81 0.4 84a Spumaretrovirus FFV
pXDP82 0.7 85a Spumaretrovirus RHSFV
pXDP83 0.7 Example 26: Transfection and recovery of XDP constructs derived from Spumaretrovirinae.
10012411Editing 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-(-1)-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 10012421XDPs derived from Spumaretrovirinae family (BFV, EFV, FFV, BGPFV, RHSFV and SFV) in the 90% Gag-CasX + 10% Gag-(-1)-Protease-CasX variation (V80b, V81b, V82b, V83b, V84b, V85b and V86b; see FIG. 62A) were produced by transient transfection of LentiX
HEK293T cells (Talcara 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. MI 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 Version XDP plasmid SEQ ID NO of DNA Sequence number N/A p5tx42.174.12.7 pGP2 80a pXDP78 81a pXDP79 82a pXDP80 83a pXDP81 84a pXDP82 85a pXDP83 86a pXDP84 Transfection 0012431 The steps for creation of the XDP are depicted graphically in FIG. 24.

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 inL 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 pig + 15 pig), pXDP81 + pXDP57 (150 pig +

15 jig), pXDP82 + pXDP58 (146 pig + 15 pig), pXDP83 + pXDP59 (154 pig + 15.4 pg). Along with the structural plasmid, each transfection also received 26.3 pig of pStx42.174.12.7, and the 5 itg of pGP2 in 3800 I 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 [0012441Media was changed on cells 24 hours post-transfection. XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 M 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
10012451tdTomato neural progenitor cells (MT NPCs) were grown in DMEM F12 supplemented with glutamax, I-IEPES, 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 10x 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.
[0012461Results: The results of the editing assay are shown in FIGS. 73A 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 I 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 1 and 16.6 .I 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 (50u1) Version Genus/order Virus Plasmid Editing %
668 Spumaretrovirus BFV
pXDP78 + pXDP54 33,5 698 Spumaretrovirus EFV
pXDP81 + pXDP57 3.3 Version Genus/order Virus Plasm id Editing %
70B Spumaretrovirus FFV
OCDP82 + OCDP58 3.5 87B Spumaretrovirus RHSFV
OCDP83 + OCDP59 213 Table 38: Results of Editing Assay for the second dilution (16.6u1) Version Genus/order Virus Plasm id Editing %
66B Spumaretrovirus BFV
pXDP78 + pXDP54 1.8 69B Spumaretrovirus EFV
pXDP81 + pXDP57 0.7 708 Spumaretrovirus FFV
pXDP82 + pXDP58 0.6 878 Spumaretrovirus RHSFV
pXDP83 + pXDP59 9.3

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 lD 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, 501õ 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 )CDP 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 CasI2a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 )CDP 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-pal 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 Orthoreirovirinae 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, Deharetrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus.

18. The XDP system of claim 16, wherein the Spumareirovirinae virus is selected from the group consisting of Bovispumavirus, Equispumavirus, Felispumcrvirus, 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.

2L 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

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 P28 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, 501õ 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 1D 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 I) NO: 130), KRPAATKKAGQMCKKK (SEQ ID NO: 131), PAAKRVKLD (SEQ ID NO: 132), RQRRNELKRSP (SEQ ID NO: 133), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 134), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (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 II) NO: 143), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 144), RKCLQAGMNLEARKTKK (SEQ ID
NO: 145), PRPRICIPR (SEQ ID NO: 146), PPRKKRTVV (SEQ ID NO: 147), NLSKKKKRKREK (SEQ I) NO: 148), RRPSRPFRKP (SEQ ID NO: 149), KRPRSPSS (SEQ
lD NO: 150), KRGINDRNFWRGENERKTR (SEQ ID NO: 151), PRPPKMARYDN (SEQ 11) NO: 152), KRSFSKAF (SEQ ID NO: 153), KLKIKRPVK (SEQ ID NO: 154), PKTRRRPRRSQRKRPPT (SEQ ID NO: 156), RRKKRRPRRKKRR (SEQ ID NO: 159), PKICKSRKPKKKSRK (SEQ ID NO: 160), HKKKHPDASVNFSEFSK (SEQ ID NO: 161), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 162), LSPSLSPLLSPSLSPL (SEQ ID NO: 163), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 164), PKRGRGRPKRGRGR (SEQ ID NO: 165), MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 157), PKKKRKVPPPPAAKRVKLD
(SEQ ID NO: 155), and PKKKRKVPPPPKKKRKV (SEQ 11) NO: 166), wherein the NLS are located at or near the N-terminus andlor 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 (AS0s), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, and a CRISPR guide nucleic acid.

44, The )(DP 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 )CDP 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 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 pl 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 1D 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, 501õ 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, 501õ 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 LD 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 )(DP system of claim 64, wherein the therapeutic payload is a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (AS0s), 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 pl 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 (0 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, 501õ 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, 501õ 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 haying 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 (AS0s), 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 Epsilonreirovinis 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 1-11V pl peptide;
(b) an HIV p6 peptide;
(c) a Gag-Pol polyprotein;
(d) one or more protease cleavage sites;
a non-retroviral, heterologous protease capable of cleaving the cleavage sites; and (0 a gag-transframe region-pal 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, 501õ 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, 501õ 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 )CDP system of claim 123, wherein the CR1SPR protein is a Class 1 or Class 2 CRISPR protein.

125. The XDP system of claim 124, wherein the CR1SPR protein is a Class 2 CRISPR protein selected from the group consisting of Type H, 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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: 597-78 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.

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 Gammaretrovims 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 p12 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 p12 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 pl 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-pal 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 lD 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, 501õ 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 )0P 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 (AS0s), 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 pl 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 pl 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 )0P 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 vims (SIV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

177. The )CDP system of claim 176, wherein the lentivims 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, 501õ 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 enzyrne, 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 CR1SPR 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j and CasX.

187. The XDP system of claim 186, wherein the CR1SPR 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 (ASCis), 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: 597-78 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.

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 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 pl 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 (0 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, 501õ 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, 501õ 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 H, 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 (AS0s), 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 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 P1 protease, PreScission (HRV3C protease), b virus Nla protease, B
virus RNA-2-encoded protease, aphthovims L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovims 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.

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 vims (BIDV) 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_b1, kissing loop_b2, G quadfiplex 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, BHIC 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, N11-13T3 cells, COS, WI38, MRCS, A549, HeLa cells, CHO cells, and 11T1080 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, intracisternal, 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 10s 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 loto 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 10" 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 p12 peptide (0 a PP21/24 peptide;
(g) a P12/P3/P8 peptide;
(h) a P20 peptide;
(0 a p1 peptide; and (i) 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 H, 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 Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j 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 (AS0s), 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 ancl 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 vims is selected from the group consisting of Alpharetrovings, Betaretrovirus, Dellareirovirus, Epsilonreirovirus, Gammareirovirus, and Lentivirus.

295. The XDP particle of claim 293, wherein the Spumaretrovirinae virus is selected from the group consisting of Bovispumavinis, Equispumavirus, Felispumavirus, Prosimnspumavirus, 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.