WO2023225670A2

WO2023225670A2 - Ex vivo programmable gene insertion

Info

Publication number: WO2023225670A2
Application number: PCT/US2023/067265
Authority: WO
Inventors: Chong Luo; Patrick Mendes TAVARES; Jonathan Douglas FINN
Original assignee: Tome Biosciences, Inc.
Priority date: 2022-05-20
Filing date: 2023-05-19
Publication date: 2023-11-23
Also published as: WO2023225670A3

Abstract

Described herein is a method of editing a primary cell genome (e.g., a human primary cell genome). In typical embodiments, an integration target recognition site (i.e., attB or attP site) is incorporated (i.e., beacon placement) into a human primary cell genome by delivering into the cell a gene editor, attachment site-containing guide RNA (atgRNA), and, optionally, a nicking guide RNA (ngRNA). Further, integrating a donor polynucleotide template into the human primary cell genome at the incorporated target recognition site is described herein thereby producing a genetically modified human primary cell.

Description

EX VIVO PROGRAMMABLE GENE INSERTION 1. CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/344,272, filed May 20, 2022; which is hereby incorporated in its entirety by reference. 2. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing with 605 sequences, which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on May 19, 2023 is named 52324WO-sequencelisting.xml, and is 813,441 bytes in size. 3. BACKGROUND [0003] Programmable, efficient, and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. [0004] There is a need in the art for techniques and gene editor compositions and gene editor guide RNAs which address and overcome these shortcomings and enable the programmable genomic integration of a large gene(s) in primary human cells such as T cells. 4. SUMMARY [0005] Described herein is a method of editing a primary cell genome (e.g., a human primary cell genome). In typical embodiments, an integration target recognition site (i.e., attB or attP site) is incorporated (i.e., beacon placement) into a human primary cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the integration target recognition site into the genome, (ii) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site (i.e., attachment site-containing guide RNA, atgRNA), and (iii) optionally, a nicking guide RNA (ngRNA). Further, integrating a donor polynucleotide template into the human primary cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, wherein the donor polynucleotide is integrated into the human primary cell at the incorporated genomic integration target recognition site by an integrase, thereby producing a genetically modified human primary cell. [0006] The present disclosure provides nucleic acid compositions, methods, and an overall platform for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE) (see Ionnidi et al.; doi: 10.1101/2021.11.01.466786; the entirety of which is incorporated herein by reference), transposon-mediated gene editing, or other suitable gene editing, or gene incorporation technology. Non-limiting examples of PASTE are also described in U.S. Pat.11,572,556 and PCT Publication No. WO 2023/077148A1, which is hereby incorporated by reference in its entirety. [0007] In one aspect, this disclosure features a method of generating a primary cell comprising an integration recognition site, the method comprising: (a) site-specifically incorporating at least a first integration recognition site into a target sequence in the genome of the primary cell. [0008] In some embodiments, site-specifically incorporating the at least first integration recognition site is affected by introducing into the primary cell: (i) a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at the target sequence; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor polypeptide to nick the primary cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site- specifically incorporated into the genome of the primary cell. [0009] In some embodiments, the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0010] In some embodiments, the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is an atgRNA that further includes an RT template that comprises at least a portion of the first integration recognition site, wherein the atgRNA encodes the entirety of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a nicking gRNA. [0011] In some embodiments, the method further comprises incorporating a plurality of integration recognition sites. [0012] In some embodiments, the method further comprises: (b) integrating at least a first donor polynucleotide template into the primary cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal/cognate integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the primary cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a primary cell with a site-specifically integrated donor polynucleotide template. [0013] In some embodiments, steps (a) and (b) are performed concurrently. [0014] In some embodiments, step (a) is performed prior to step (b). [0015] In some embodiments, step (a) and step (b) are performed at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks apart. [0016] In another aspect, this disclosure features a method of generating an edited primary cell, the method comprising: integrating, into the genome of the primary cell at the first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal/cognate integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the primary cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a primary cell with a site-specifically integrated donor polynucleotide template. [0017] In another aspect, this disclosure features a method of site specifically integrating a donor polynucleotide template into a primary cell genome, the method comprising: incorporating an integration recognition site into the primary cell genome by delivering into the cell: a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at a target sequence; an at least a first attachment site-containing guide RNA (atgRNA), wherein the one or more guide RNA encodes all or a portion of an at least first integration recognition site; and integrating a donor polynucleotide template into the human primary cell genome at the incorporated recognition site by delivering into the cell: the donor polynucleotide template, an integration enzyme or a polynucleotide encoding an integration enzyme, wherein the donor polynucleotide template is linked to a sequence that is an integration cognate of the integration recognition site present in the atgRNA, and wherein the donor polynucleotide template is integrated into the genome at the incorporated genomic integration recognition site by the integration enzyme; thereby producing a primary cell having the donor polynucleotide template site specifically integrated into its genome. [0018] In some embodiments, the gene editor polypeptide or the polynucleotide encoding a gene editor polypeptide, the at least first atgRNA, the donor polynucleotide template, and the integration enzyme or the polynucleotide encoding an integration enzyme are concurrently delivered. [0019] In some embodiments, the gene editor protein or the polynucleotide encoding a gene editor protein, and the at least first atgRNA, are delivered at a first time point and the donor polynucleotide template, and the integration enzyme or the polynucleotide encoding an integration enzyme are delivered at a second time point. [0020] In some embodiments, the first time point and the second time point are at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks apart. [0021] In some embodiments, the gene editor polypeptide is configured such that the nickase is linked to the reverse transcriptase. [0022] In some embodiments, the nickase is linked to the reverse transcriptase by in-frame fusion. [0023] In some embodiments, the nickase is linked to the reverse transcriptase by a linker. [0024] In some embodiments, the linker is a peptide fused in-frame between the nickase and reverse transcriptase. [0025] In some embodiments, the gene editor polynucleotide further comprises a polynucleotide sequence encoding at least an integration enzyme or the gene editor polypeptide further comprises an integration enzyme. [0026] In some embodiments, the linked nickase-reverse transcriptase are further linked to the integration enzyme. [0027] In some embodiments, an at least first atgRNA comprises: (i) a domain that is capable of guiding the prime editor system to the target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of an at least first integration recognition site. [0028] In some embodiments, the RT template comprises the entirety of the first integration recognition site. [0029] In some embodiments, site-specifically incorporating an integration recognition site into the primary cell genome further comprises delivering into the cell a second atgRNA. [0030] In some embodiments, the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence; the first atgRNA further includes a first RT template that comprises at least a portion of an at least first integration recognition site; the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0031] In some embodiments, the first RT template encodes a first single-stranded DNA sequence and the second RT template encodes a second single-stranded DNA sequence. [0032] In some embodiments, the first single-stranded DNA sequence comprises a complementary region with the first single-stranded DNA sequence. [0033] In some embodiments, the first single-stranded DNA sequence and the first single-stranded DNA sequence form a duplex. [0034] In some embodiments, the complementary region is 5 or more consecutive bases. In some embodiments, the complementary region is 10 or more consecutive bases. In some embodiments, the complementary region is 20 or more consecutive bases. In some embodiments, the complementary region is 30 or more consecutive bases. [0035] In some embodiments, at least one of the two paired guide RNAs has a chemical modification. In some embodiments, the paired guide RNAs each have a chemical modification. [0036] In some embodiments, incorporating an integration recognition site into the human primary cell genome further comprises delivering into the cell a nicking guide RNA (gRNA). [0037] In some embodiments, the first integration recognition site is an attB or attP site. [0038] In some embodiments, the integration recognition site is a modified attB or attP site. [0039] In some embodiments, the integration recognition site is specific for BxB1 or a modified BxB1. [0040] In some embodiments, the integration recognition site is comprised of 38 or 46 nucleotides. [0041] In some embodiments, the donor polynucleotide template is a minicircle. [0042] In some embodiments, the primary cell is a human primary cell. [0043] In some embodiments, the primary cell is a T cell. [0044] In some embodiments, the T cell is a CD4⁺ T cell, a CD8⁺ T cell, or a combination thereof. [0045] In some embodiments, the T cell is an autologous T cell or allogeneic T cell. [0046] In some embodiments, the target sequence is located in the TRAC locus, the CXCR4 locus, or the IL2RB locus, whereby the integration recognition site is incorporated into the TRAC locus, the CXCR4 locus, or the IL2RB locus. [0047] In some embodiments, the introducing is performed by electroporation. [0048] In some embodiments, the introducing is achieved using one or more of a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, mRNA, RNP, or lipid nanoparticle (LNP), or a combination thereof. [0049] In some embodiments, the donor polynucleotide template encodes for a chimeric antigen receptor or a T cell receptor. [0050] In some embodiments, isolating the primary cell prior to step (a). [0051] In some embodiments, the primary cell is activated ex vivo or in vitro prior to step (a). [0052] In some embodiments, the edited primary cell is expanded to generate an expanded edited primary cell composition. [0053] In some embodiments, the expanded modified human primary cell composition is isolated. [0054] In another aspect, this disclosure features a primary cell generated by any of the methods described herein. [0055] In another aspect, this disclosure features a population of primary cells generated by any of the methods described herein. [0056] In another aspect, this disclosure features a method of treating a blood disease or disorder in a subject in need thereof, the method comprising: administering to the subject the primary cell or the population of cells of any one of the preceding embodiments. [0057] In another aspect, this disclosure features a primary cell, comprising: an at least first integration recognition site site-specifically incorporated into the primary cell genome. [0058] In some embodiments, the at least first integration recognition site is incorporated into the primary cell genome at a TRAC locus, a CXCR4 locus, or a IL2RB locus. [0059] In some embodiments, the at least first integration recognition site is specific for a serine integrase. [0060] In some embodiments, the at least first integration recognition site is an attB or attP site. [0061] In some embodiments, the at least first integration recognition site is a modified attB or attP site. [0062] In some embodiments, the at least first integration recognition site is specific for BxB1 or a modified BxB1. [0063] In some embodiments, the at least first integration recognition site is comprised of 38 or 46 nucleotides. [0064] In another aspect, this disclosure features a primary cell comprising: a donor polynucleotide template integrated into the TRAC locus, CXCR4 locus, or the IL2RB locus, wherein the donor polynucleotide comprises a residual integration recognition site (e.g., an AttR and/or an AttL site). [0065] In some embodiments, the at least first integration recognition site is incorporated into the primary cell genome at a TRAC locus, a CXCR4 locus, or a IL2RB locus. [0066] In some embodiments, the donor polynucleotide template is a minicircle. [0067] In some embodiments, the donor polynucleotide template encodes a chimeric antigen receptor or a T cell receptor. [0068] In another aspect, this disclosure features a system for site-specifically integrating a donor polynucleotide template into a primary cell genome, the system comprising: a first attachment site- containing guide RNA comprising a sequence selected from Tables 12 or 14; a second attachment site-containing guide RNA comprising a sequence selected from Tables 12 or 14, wherein the second atgRNA comprises a different spacer sequence from the first atgRNA; and a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at a target sequence. [0069] In some embodiments, the system further comprises: a donor polynucleotide template; and an integration enzyme or a polynucleotide encoding an integration enzyme, wherein the donor polynucleotide template is linked to a sequence that is an integration cognate of the integration recognition site present in the atgRNA, and wherein the donor polynucleotide template is integrated into the genome at the incorporated genomic integration recognition site by the integration enzyme. [0070] In another aspect, this disclosure features an attachment site-containing guide RNA (atgRNA) comprising: a sequence selected from Tables 12 or 14. [0071] In another aspect, this disclosure features a pair of attachment site-containing guide RNA (atgRNA) comprising: a first attachment site-containing guide RNA comprising a sequence selected from Tables 12 or 14; and a second attachment site-containing guide RNA comprising a sequence selected from Tables 12 or 14, wherein the second atgRNA comprises a different spacer sequence from the first atgRNA. 5. BRIEF DESCRIPTION OF THE DRAWINGS [0072] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where: [0073] FIG.1 illustrates reference CXCR4 and IL2RB prime editing guide RNAs (pegRNAs) to be modified into attachment site-containing guide RNAs (atgRNAs) via integrase recognition site incorporation into the reverse transcriptase template segment of the guide RNA. [0074] FIG.2 illustrates modification of pegRNAs into atgRNAs capable of beacon placement into a target genome site. Exemplary 38 or 46 base attB insertion is shown. [0075] FIG. 3 illustrates beacon placement within T cells at a high dose and low dose of reagent conditions, wherein reagents are delivered via electroporation (on Day 2 of experimental workflow). [0076] FIGs.4A-4C illustrates beacon placement efficiency within T cells at a high dose and low dose of reagent conditions, wherein reagents are delivered via electroporation (on Day 2 of experimental workflow) (see Example 1). FIG.4A shows prime editing efficiency of the reference pegRNA as described in FIG. 1. FIG. 4B shows percent beacon (integration recognition site) placement using low and high dose CXCR4 atgRNA comprising either a 38bp or a 46bp AttB integration recognition site. FIG. 4C shows percent beacon (integration recognition site) placement using low and high dose IL2RB atgRNA comprising either a 38bp or a 46bp AttB integration recognition site. [0077] FIG. 5 illustrates single atgRNA (with a paired nicking guide) optimization screen conditions for T cell beacon placement at the CXCR4 and IL2RB loci, respectively. [0078] FIG.6A illustrates two-color beacon placement quantification (top). [0079] FIG. 6B-6C show percent beacon placement efficiency is shown for CXCR4 and IL2RB loci, respectively, as detected by the assay as described in FIG.6A. FIG.6B shows percent beacon placement efficiency is shown for CXCR4. FIG. 6C shows percent beacon placement efficiency is shown for IL2RB. [0080] FIGs. 7A-7B illustrate beacon placement efficiency for CXCR4 and IL2RBi loci determined by amplicon sequencing (amp-seq) or digital droplet PCR (ddPCR). FIG.7A shows percent beacon placement for CXCR4 as detected by amp-seq and ddPCR. FIG.7B shows percent beacon placement for IL2RB as detected by amp-seq and ddPCR. [0081] FIG.8 illustrates dual atgRNA method for genomic beacon placement. [0082] FIG.9 illustrates the dual atgRNA design which includes attB site (beacon) site length, and 20 base pair overlap between synthesized DNA strands (encoded within a segment of atgRNA reverse transcription template). [0083] FIG. 10 illustrates experimental workflow for a dual atgRNA mediated beacon placement. Top panel indicates the workflow timeline. Bottom panel indicates electroporation conditions. [0084] FIGs. 11A-11B illustrate dual guide mediated beacon placement efficiency at the CXCR4 (FIG. 11A) and IL2RBi (FIG. 11B) loci, respectively, as determined by amplicon sequencing (amp-seq) or digital droplet PCR (dd-PCR). [0085] FIG.12A illustrates a non-limiting exemplary chimeric antigen receptor (CAR) or T cell receptor (TCR) knock in (insertion) at the TRAC locus (FIG.12A) (see Eyquem et al, Nature, 2017; Roth et al. Nature 2018) [0086] FIG.12B shows guide RNAs (“MS”, “AM”, “TF”) and nicking guide RNAs targeting at or near TRAC exon 1 region. [0087] FIG.13 illustrates modification of single guide RNAs (sgRNAs) identified in FIG.12 into single atgRNA (with nicking guide) or dual atgRNAs capable of beacon placement into the T cell TRAC locus. Exemplary 38 or 46 base attB insertion is shown. [0088] FIGs. 14A-14B illustrate TCR protein disruption with TRAC specific guides (“MS”, “AM”, “TF”) complexed to a SpCas9 (expressed from mRNA or a protein form). FIG.14A shows results using guides “gAM” and “gTF.” FIG. 14A, left panel shows percent live CD3⁺ T cells following introduction of the guides in a complex (e.g., RNP) with a Cas9 protein. FIG.14A, right panel shows percent INDELs following introduction of the guides in a complex (e.g., RNP) with a Cas9 protein. FIG.14B, shows results of testing of various forms of Cas9 in combination with guides “gMS”, “gAM”, or “gTF”. FIG.14B top panel show flow cytometry plots of CD3⁺ T cells following transduction with a control “EP No Substrate” or a gAM quide/Cas9 RNP. FIG. 14B bottom panel shows a summary of the live CD3⁺ T cells following transduction with the indicate conditions. In particular, various forms of Cas9 were tested, including, mRNA Company-T, mRNA PL319-001, and mRNA Pl319-002. [0089] FIG. 15 illustrates single atgRNA (and associated ngRNA) and dual atgRNA experimental screen conditions for TRAC locus beacon placement. Top tables show conditions for electroporations. Bottom panel shows experimental workflow. [0090] FIGs.16A-16C illustrates single atgRNA mediated (with nicking guide RNA) beacon placement efficiency at the TRAC locus using AM, MS, and TF spacers identified in FIG.12 and tested in FIG.14. FIG. 16A shows percent beacon placement at the TRAC locus when using an atgRNA having a AM spacer. FIG.16B shows percent beacon placement at the TRAC locus when using an atgRNA having a MS spacer. FIG.16C shows percent beacon placement at the TRAC locus when using an atgRNA having a TF spacer. [0091] FIGs. 17A-17C illustrate dual atgRNA mediated beacon placement efficiency at the TRAC locus. FIG. 17A shows percent beacon placement at the TRAC locus when using a first atgRNA having a AM spacer and a second atgRNA having a spacer from the ng4 nicking guide RNA. FIG.17B shows percent beacon placement at the TRAC locus when using a first atgRNA having a MS spacer and a second atgRNA having a spacer from the ng4 nicking guide RNA. FIG. 17C shows percent beacon placement at the TRAC locus when using a first atgRNA having a TF spacer and a second atgRNA having a spacer from the ng12 nicking guide RNA. [0092] FIG. 18 shows percent beacon placement comparing the dual atgRNA-mediated beacon placement compared to single atgRNA-mediated beacon placement across 3 genomic targets (CXCR4, IL2RB, and TRAC) and the five spacer pairs (CXCR4, IL2RB, TRAC-AM, TRAC- MS, and TRAC-TF). [0093] FIG. 19A-19E shows analysis of AttP variants. FIG. 19A shows a non-limiting schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance). FIG. 19B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths. FIG. 19C shows a non-limiting schematic of multiplexed integration of different cargo sets at specific genomic loci. Three fluorescent cargos (GFP, mCherry, and YFP) are inserted orthogonally at three different loci (ACTB, LMNB1, NOLC1) for in-frame gene tagging. FIG. 19D shows orthogonality of top 4 AttB/AttP dinucleotide pairs evaluated for GFP integration with PASTE at the ACTB locus. FIG. 19E shows efficiency of multiplexed PASTE insertion of combinations of fluorophores at ACTB, LMNB1, and NOLC1 loci. Data are mean (n= 3) ± s.e.m. [0094] FIGs. 20A-20E show the experimental details used to test programmable gene insertion (PGI) in primary T cells as tested in Example 6. FIG. 20A shows a schematic of the mRNA construct encoding BxB1 used for the Experiments in Example 6, which has a sequence of SEQ ID NO: 600. 5’ UTR XBG - 5’ UTR = 5’ untranslated region of XBG - Xenopus beta globin; NLS – nuclear localization signal; 3’ UTR XBG – 3’ UTR = 3’ unstranslated region of XBG - Xenopus beta globin; and 80A’s – 80 adenosines. FIG. 20B shows a schematic of the experimental workflow used in Example 6. nCas9-RT – Cas9 nickase (“nCas9”) – reverse transcriptase (“RT”); dual atgRNA – dual attachment site-containing guide RNA (“dual atgRNA”); 1^st EP – first electroporation; 2^nd EP – second electroporation. FIG. 20C shows a schematic of the experimental workflow in terms of days. Day 0: T cells were thawed. Day 2: T cells were transfected with a gene editor polynucleotide and dual attachment site-containing guide RNAs (atgRNAs). Day 6: T cells were transfected with a polynucleotide encoding an integrase and a donor polynucleotide template. Day 9: cells were harvested for assessment of PGI. FIG. 20D shows a table providing further details of the electroporation conditions tested in Example 6. FIG. 20E shows a histogram of percent (%) viable cells following the electroporation at day 2 (“Post EP #1”) and following the second electroporation at day 6 (“Post EP #2”). [0095] FIG. 21 shows a histogram of percent (%) beacon placement (BP) for the indicated conditions tested in Example 6. [0096] FIG.22 shows a histogram of percent (%) programmable gene insertion (PGI) for the indicated conditions tested in Example 6. [0097] FIG. 23 shows a histogram of percent (%) beacon conversion for the indicated conditions tested in Example 6. [0098] FIG.24 shows a schematic of the three mRNA constructs encoding BxB1 used for the Experiments in Example 7.5’ UTR XBG - 5’ UTR = 5’ untranslated region of XBG - Xenopus beta globin; NLS – nuclear localization signal;; 3’ UTR XBG – 3’ UTR = 3’ unstranslated region of XBG - Xenopus beta globin; and 80A’s – 80 adenosines. BxB1 #1 comprises a sequence of SEQ ID NO: 600. BxB1 #2 comprises a sequence of SEQ ID NO: 601. BxB1 #3 comprises a sequence of SEQ ID NO: 602. [0099] FIGs. 25A-25B show the experimental details used to test programmable gene insertion (PGI) in primary T cells as tested in Example 7. FIG. 25A shows a schematic of the experimental workflow in terms of days. Day 0: T cells were thawed. Day 2: T cells were transfected with a gene editor polynucleotide and at least a first attachment site-containing guide RNA (atgRNA). Day 6: T cells were transfected with a polynucleotide encoding an integrase. Day 12: cells were harvested for assessment of PGI. FIG.25B shows a table providing further details of the transfection conditions tested in Example 7. [0100] FIG. 26 shows a histogram of percent (%) viability for the indicated conditions (see Example 7). Donor polynucleotide template was provided in the form of a minicircle at 3 µg or 6 µg for No BxB1, BxB1 #1, BxB1 #2, BxB1 #3 and EH-115 BxB1 #1. Conditions on x-axis: “BP only” – Beacon placement (cells were electroporated once with program EO-115 with nCas9-RT mRNA and guide RNAs); NS – No substrate in the electroporation reaction; “EP NS 1X” – Electroporation with no substrate (Cells were electroporated once with program EO-115 without substrate/editing components); “BP + EH NS” – Cells were electroporated with program EO-115 for beacon placement (BP), then electroporated a second time with program EH-115 but no substrate (DNA cargo or BxB1 mRNA); “BP + NS” - Cells were electroporated with program EO- 115 for beacon placement (BP), then electroporated a second time with program EO-115 but no substrate (DNA cargo or BxB1 mRNA); “No BxB1 group” - Cells were electroporated with program EO-115 for beacon placement (BP), then electroporated a second time with program EO- 115 with DNA cargo (but no Bxb1 mRNA); and “EH-115” – An electroporation program for the Lonza 4D Nucleofector. [0101] FIG.27 shows a histogram of percent (%) beacon placement for the indicated conditions (see Example 7). Donor polynucleotide template was provided in the form of a minicircle at 3 µg or 6 µg for No BxB1, BxB1 #1, BxB1 #2, BxB1 #3 and EH-115 BxB1 #1. Conditions on x-axis: “BP only” – Beacon placement (cells were electroporated once with program EO-115 with nCas9- RT mRNA and guide RNAs); NS – No substrate in the electroporation reaction; “EP NS 1X” – Electroporation with no substrate (Cells were electroporated once with program EO-115 without substrate/editing components); “BP + EH NS” – Cells were electroporated with program EO-115 for beacon placement (BP), then electroporated a second time with program EH-115 but no substrate (DNA cargo or BxB1 mRNA); “BP + NS” - Cells were electroporated with program EO- 115 for beacon placement (BP), then electroporated a second time with program EO-115 but no substrate (DNA cargo or BxB1 mRNA); “No BxB1 group” - Cells were electroporated with program EO-115 for beacon placement (BP), then electroporated a second time with program EO- 115 with DNA cargo (but no Bxb1 mRNA); and “EH-115” – An electroporation program for the Lonza 4D Nucleofector. [0102] FIG. 28 shows a histogram of percent (%) beacon conversion for the indicated conditions (see Example 7). Each condition on the x-axis was tested with DNA template provided as a minicircle at 3 µg or 6 µg. Donor polynucleotide template was provided in the form of a minicircle at 3 µg or 6 µg for No BxB1, BxB1 #1, BxB1 #2, BxB1 #3 and EH-115 BxB1 #1Conditions on x-axis: “BP only” – Beacon placement (cells were electroporated once with program EO-115 with nCas9-RT mRNA and guide RNAs); NS – No substrate in the electroporation reaction; “BP + NS” - Cells were electroporated with program EO-115 for beacon placement (BP), then electroporated a second time with program EO-115 but no substrate (DNA cargo or BxB1 mRNA); “No BxB1 group” - Cells were electroporated with program EO-115 for beacon placement (BP), then electroporated a second time with program EO-115 with DNA cargo (but no Bxb1 mRNA); and “EH-115” – An electroporation program for the Lonza 4D Nucleofector. [0103] FIG.29 shows a histogram of percent (%) programmable gene insertion (PGI) for the indicated conditions (see Example 7). Each condition on the x-axis was tested with DNA template provided as a minicircle at 3 µg or 6 µg. Donor polynucleotide template was provided in the form of a minicircle at 3 µg or 6 µg for No BxB1, BxB1 #1, BxB1 #2, BxB1 #3 and EH-115 BxB1 #1. Conditions on x-axis: “BP only” – Beacon placement (cells were electroporated once with program EO-115 with nCas9-RT mRNA and guide RNAs); NS – No substrate in the electroporation reaction; “BP + NS” - Cells were electroporated with program EO-115 for beacon placement (BP), then electroporated a second time with program EO-115 but no substrate (DNA cargo or BxB1 mRNA); “No BxB1 group” - Cells were electroporated with program EO-115 for beacon placement (BP), then electroporated a second time with program EO-115 with DNA cargo (but no Bxb1 mRNA); and “EH-115” – An electroporation program for the Lonza 4D Nucleofector. [0104] FIGs. 30A-30D show the experimental details used to test programmable gene insertion (PGI) in primary T cells as tested in Example 8. FIG. 30A shows a schematic of the experimental workflow used in Example 8. nCas9-RT – Cas9 nickase (“nCas9”) – reverse transcriptase (“RT”); dual atgRNA – dual attachment site-containing guide RNA (“dual atgRNA”); One-shot – one electroporation. FIG.30B shows a schematic of the mRNA construct encoding BxB1 used for the Experiments in Example 8, which has a sequence of SEQ ID NO: 600. 5’ UTR XBG - 5’ UTR = 5’ untranslated region of XBG - Xenopus beta globin; NLS – nuclear localization signal; 3’ UTR XBG – 3’ UTR = 3’ unstranslated region of XBG - Xenopus beta globin; and 80A’s – 80 adenosines. FIG. 30C shows a schematic of the experimental workflow in terms of days. Day 0: T cells were thawed. Day 2: T cells were transfected with a gene editor polynucleotide, at least a first attachment site-containing guide RNA (atgRNA), a polynucleotide encoding an integrase, and a donor polynucleotide template. Day 6: cells were harvested for assessment of PGI. FIG. 30D shows a table providing further details of the electroporation conditions tested in Example 8. [0105] FIG. 31 shows a histogram of percent (%) beacon placement (BP) for the indicated conditions tested in Example 8. [0106] FIG.32 shows a histogram of percent (%) programmable gene insertion (PGI) for the indicated conditions tested in Example 8. [0107] FIG. 33 shows a histogram of percent (%) beacon conversion for the indicated conditions tested in Example 8. 6. DETAILED DESCRIPTION [0108] This disclosure features systems, compositions, and methods for generating a primary cell comprising an integration recognition site site-specifically integrated into the genome of the primary cell. In a non-limiting example, this disclosure features a method of generating a primary cell comprising an integration recognition site, where the method includes (a) site- specifically incorporating at least a first integration recognition site into a target sequence in the genome of the primary cell. In some embodiments, site-specifically incorporating the at least first integration recognition site is effected by introducing into the primary cell: (i) a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at the target sequence; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the primary cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site-specifically incorporated into the genome of the primary cell. [0109] This disclosure features systems, compositions, and methods methods for generating a primary cell comprising an exogenous nucleic acid (e.g., a donor polynucleotide template) site- specifically integrated into the genome of the primary cell. In one non-limiting example, this disclosure features a method incorporating an integration recognition site into the human primary cell genome by delivering into the cell: a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at a target sequence; an at least a first attachment site-containing guide RNA (atgRNA), wherein the one or more guide RNA encodes all or a portion of an at least first integration recognition site; and integrating a donor polynucleotide template into the human primary cell genome at the incorporated recognition site by delivering into the cell: the donor polynucleotide template, an integration enzyme or a polynucleotide encoding an integration enzyme, wherein the donor polynucleotide template is linked to a sequence that is an integration cognate of the integration recognition site present in the atgRNA, and wherein the donor polynucleotide template is integrated into the genome at the incorporated genomic integration recognition site by the integration enzyme; thereby producing a human subject primary cell. 6.1. Gene Editors [0110] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. As used herein, the following terms have the meanings ascribed to them below. [0111] “Gene editor” as used herein, is a protein that that can be used to perform gene editing, gene modification, gene insertion, gene deletion, or gene inversion. As used herein, the terms “gene editor polynucleotide” refers to a polynucleotide sequence encoding the gene editor polypeptide. Such an enzyme or enzyme fusion may contain DNA or RNA targetable nuclease protein (i.e., Cas protein, ADAR, or ADAT), wherein target specificity is mediated by a complexed nucleic acid (i.e., guide RNA). Such an enzyme or enzyme fusion may be a DNA/RNA targetable protein, wherein target specificity is mediated by internal, conjugated, fused, or linked amino acids, such as within TALENs, ZFNs, or meganucleases. The skilled person in the art would appreciate that the gene editor can demonstrate targeted nuclease activity, targeted binding with no nuclease activity, or targeted nickase activity (or cleavase activity). A gene editor comprising a targetable protein may be fused or linked to one or more proteins or protein fragment motifs. Gene editors may be fused, linked, complexed, operate in cis or trans to one or more integrase, recombinase, polymerase, telomerase, reverse transcriptase, or invertase. A gene editor can be a prime editor fusion protein or a gene writer fusion protein. [0112] “Prime editor fusion protein” as used herein, describes a protein that is used in prime editing. “Prime editor system” as used herein describes the components used in prime editing. Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; the nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts. Such an enzyme can be a catalytically-impaired Cas9 endonuclease (a nickase). Such an enzyme can be a Casl2a/b, MAD7, or variant thereof. The nickase is fused to an engineered reverse transcriptase (RT). The nickase is programmed (directed) with an attachment site-containing guide RNA (or a prime-editing guide RNA (pegRNA)). The skilled person in the art would appreciate that the atgRNA (pegRNA) both specifies the target site and encodes the desired edit. Described herein, are attachment site-containing guide RNA (atgRNA) that both specifies the target and encodes for the desired integrase target recognition site. The nickase may be programmed (directed) with an atgRNA. Advantageously the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the atgRNA (pegRNA), whereby a nick or single stranded cut occurs. The reverse transcriptase domain then uses the atgRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Afterward, optionally, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA). Other enzymes that can be used to nick or cut only a single strand of double stranded DNA includes a cleavase (e.g., cleavase I enzyme). [0113] In some embodiments, an additional agent or agents may be added that improve the efficiency and outcome purity of the prime edit. In some embodiments, the agent may be chemical or biological and disrupt DNA mismatch repair (MMR) processes at or near the edit site (i.e., PE4 and PE5 and PEmax architecture by Chen et al. Cell, 184, 1-18, October 28, 2021; Chen et al. is incorporated herein by reference). In typical embodiments, the agent is a MMR-inhibiting protein. In certain embodiments, the MMR-inhibiting protein is dominant negative MMR protein. In certain embodiments, the dominant negative MMR protein is MLH1dn. In particular embodiments, the MMR-inhibiting agent is incorporated into the single nucleic acid construct design described herein. In some embodiments, the MMR-inhibiting agent is linked or fused to the prime editor protein fusion, which may or may not have a linked or fused integrase. In some embodiments, the MMR-inhibiting agent is linked or fused to the Gene Writer™ protein, which may or may not have a linked or fused integrase. [0114] The prime editor or gene editor system can be used to achieve DNA deletion and replacement. In some embodiments, the DNA deletion replacement is induced using a pair of atgRNAs (or pegRNAs) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair (i.e., PrimeDel by Choi et al. Nat. Biotechnology, October 14, 2021; Choi et al. is incorporated herein by reference and TwinPE by Anzalone et al.BioRxiv, November 2, 2021; Anzalone et al. is incorporated herein by reference). In some embodiments described herein, the DNA deletion is induced using a single atgRNA. In some embodiments, the DNA deletion and replacement is induced using a wild type Cas9 prime editor (PE-Cas9) system (i.e., PEDAR by Jiang et al. Nat. Biotechnology, October 14, 2021; Jiang et al. is incorporated herein by reference). In some embodiments, the DNA replacement is an integrase target recognition site or recombinase target recognition site. In certain embodiments, the constructs and methods described herein may be utilized to incorporate the pair of pegRNAs used in PrimeDel, TwinPE (WO2021226558, which is hereby incorporated by reference in its entirety), or PEDAR, the prime editor fusion protein or Gene Writer protein, optionally a nickase guide RNA (ngRNA), an integrase, a nucleic acid cargo, and optionally a recombinase into a single nucleic acid construct described herein. The integrase may be directly linked, for example by a peptide linker, to the prime editor fusion or gene writer protein. [0115] In some embodiments, the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a CRISPR enzyme nickase such as a Cas9 H840A nickase, a Cas9nickase. In some embodiments, the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a cleavase. In some embodiments the RT can be fused at, near or to the C-terminus of a Cas9nickase, e.g., Cas9 H840A. Fusing the RT to the C-terminus region, e.g., to the C-terminus, of the Cas9 nickase may result in higher editing efficiency. Such a complex is called PEI. In some embodiments, the CRISPR enzyme nickase, e.g., Cas9(H840A), i.e., a Cas9nickase, can be linked to a non-M-MLV reverse transcriptase such as an AMV-RT or XRT (Cas9(H840A)-AMV-RT or XRT). In some embodiments, instead of the CRISPR enzyme nickase being a Cas9 (H840A), i.e., instead of being a Cas9 nickase, the CRISPR enzyme nickase instead can be a CRISPR enzyme that naturally is a nickase or cuts a single strand of double stranded DNA; for instance, the CRISPR enzyme nickase can be Casl2a/b. Alternatively, the CRISPR enzyme nickase can be another mutation of Cas9, such as Cas9(Dl0A). A CRISPR enzyme, such as a CRISPR enzyme nickase, such as Cas9 (wild type), Cas9(H840A), Cas9(Dl0A) or Cas 12a/b nickase can be fused in some embodiments to a pentamutant of M-MLV RT (D200N/ L603W/ T330P/ T306K/ W313F), whereby there can be up to about 45-fold higher efficiency, and this is called PE2. In some embodiments, the M-MLV RT comprise one or more of the mutations Y8H, P51L, S56A, S67R, E69K, Vl29P, L139P, Tl97A, H204R, V223H, T246E, N249D, E286R, Q2911, E302K, E302R, F309N, M320L, P330E, L435G, L435R, N454K, D524A, D524G, D524N, E562Q, D583N, H594Q, E607K, D653N, and L671P. Specific M-MLV RT mutations are shown in Table 1.

[0116] In some embodiments, the reverse transcriptase can also be a wild-type or modified transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV RT), Feline Immunodeficiency Virus reverse transcriptase (FIV-RT), FeLV-RT (Feline leukemia virus reverse transcriptase), HIV-RT (Human Immunodeficiency Virus reverse transcriptase). In some embodiments, the reverse transcriptase can be a fusion of MMuLV to the Sto7d DNA binding domain (see Ionnidi et al.; https://doi.org/10.1101/2021.11.01.466786). The fusion of MMuLV to the Sto7d DNA binding domain sequence is given in Table 2.

[0117] PE3, PE3b, PE4, PE5, and/or PEmax, which a skilled person can incorporate into the gene editor polypeptide, involves nicking the non-edited strand, potentially causing the cell to remake that strand using the edited strand as the template to induce HR. The nicking of the non-edited strand can involve the use of a nicking guide RNA (ngRNA). [0118] The skilled person can readily incorporate into a gene editor polypeptide described herein a prime editing or CRISPR system. Examples of prime editors can be found in the following: WO2020/191153, WO2020/191171, WO2020/191233, WO2020/191234, WO2020/191239, WO2020/191241, WO2020/191242, WO2020/191243, WO2020/191245, WO2020/191246, WO2020/191248, WO2020/191249, each of which is incorporated by reference herein in its entirety. In addition, mention is made, and can be used herein, of CRISPR Patent Applications and Patents of the Zhang laboratory and/or Broad Institute, Inc. and Massachusetts Institute of Technology and/or Broad Institute, Inc., Massachusetts Institute of Technology and President and Fellows of Harvard College and/or Editas Medicine, Inc. Broad Institute, Inc., The University of Iowa Research Foundation and Massachusetts Institute of Technology, including those claiming priority to US Application 61/736,527, filed December 12, 2012, including US Patents 11,104,937, 11,091,798, 11,060,115, 11,041,173, 11,021,740, 11,008,588, 11,001,829, 10,968,257, 10,954,514, 10,946,108, 10,930,367, 10,876,100, 10,851,357, 10,781,444, 10,711,285, 10,689,691, 10,648,020, 10,640,788, 10,577,630, 10,550,372, 10,494,621, 10,377,998, 10,266,887, 10,266,886, 10,190,137, 9,840,713, 9,822,372, 9,790,490, 8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945, and 8,697,359; CRISPR Patent Applications and Patents of the Doudna laboratory and/or of Regents of the University of California, the University of Vienna and Emmanuelle Charpentier, including those claiming priority to US application 61/652,086, filed May 25, 2012, and/or 61/716,256, filed October 19, 2012, and/or 61/757,640, filed January 28, 2013, and/or 61/765,576, filed February 15, 2013 and/or 13/842,859, including US Patents 11,028,412, 11,008,590, 11,008,589, 11,001,863, 10,988,782, 10,988,780, 10,982,231, 10,982,230, 10,900,054, 10,793,878, 10,774,344, 10,752,920, 10,676,759, 10,669,560, 10,640,791, 10,626,419, 10,612,045, 10,597,680, 10,577,631, 10,570,419, 10,563,227, 10,550,407, 10,533,190, 10,526,619, 10,519,467, 10,513,712, 10,487,341, 10,443,076, 10,428,352, 10,421,980, 10,415,061, 10,407,697, 10,400,253, 10,385,360, 10,358,659, 10,358,658, 10,351,878, 10,337,029, 10,308,961, 10,301,651, 10,266,850, 10,227,611, 10,113,167, and 10,000,772; CRISPR Patent Applications and Patents of Vilnius University and/or the Siksnys laboratory, including those claiming priority to US application 62/046384 and/or 61/625,420 and/or 61/613,373 and/or PCT/IB2015/056756, including US Patent 10,385,336; CRISPR Patent Applications and Patents of the President and Fellows of Harvard College, including those of George Church’s laboratory and/or claiming priority to US application 61/738,355, filed December 17, 2012, including 11,111,521, 11,085,072, 11,064,684, 10,959,413, 10,925,263, 10,851,369, 10,787,684, 10,767,194, 10,717,990, 10,683,490, 10,640,789, 10,563,225, 10,435,708, 10,435,679, 10,375,938, 10,329,587, 10,273,501, 10,100,291, 9,970,024, 9,914,939, 9,777,262, 9,587,252, 9,267,135, 9,260,723, 9,074,199, 9,023,649; CRISPR Patent Applications and Patents of the President and Fellows of Harvard College, including those of David Liu’s laboratory, including 11,111,472, 11,104,967, 11,078,469, 11,071,790, 11,053,481, 11,046,948, 10,954,548, 10,947,530, 10,912,833, 10,858,639, 10,745,677, 10,704,062, 10,682,410, 10,612,011, 10,597,679, 10,508,298, 10,465,176, 10,323,236, 10,227,581, 10,167,457, 10,113,163, 10,077,453, 9,999,671, 9,840,699, 9,737,604, 9,526,784, 9,388,430, 9,359,599, 9,340,800, 9,340,799, 9,322,037, 9,322,006, 9,228,207, 9,163,284, and 9,068,179; and CRISPR Patent Applications and Patents of Toolgen Incorporated and/or the Kim laboratory and/or claiming priority to US application 61/717,324, filed October 23, 2012 and/or 61/803,599, filed March 20, 2013 and/or 61/837,481, filed June 20, 2013 and/or 62/033,852, filed August 6, 2014 and/or PCT/KR2013/009488 and/or PCT/KR2015/008269, including US Patent 10,851,380, and 10,519,454; and CRISPR Patent Applications and Patents of Sigma and/or Millipore and/or the Chen laboratory and/or claiming priority to US application 61/734,256, filed December 6, 2012 and/or 61/758,624, filed January 30, 2013 and/or 61/761,046, filed February 5, 2013 and/or 61/794,422, filed March 15, 2013, including US Patent 10,731,181, each of which is hereby incorporated herein by reference, and from the disclosures of the foregoing, the skilled person can readily make and use a prime editing or CRISPR system, and can especially appreciate impaired endonucleases, such as a mutated Cas9 that only nicks a single strand of DNA and is hence a nickase, or a CRISPR enzyme that only makes a single-stranded cut that can be employed in a PASTE system of the invention. Further, from the disclosures of the foregoing, the skilled person can incorporate the selected CRISPR enzyme, as part of the gene editor composition described herein. Additional gene editor polypeptides are as described in WO 2023/076898; WO 2023/015014; WO 2023/070062; WO2023288332; WO2023015318; WO2023004439; WO2023070110; WO2022256714; and WO2023283092, each of which are hereby incorporated by reference in their entireties. Additional gene editor polypeptides are as described in U.S. Patent Pub.2023/0059368, which is hereby incorporated by reference in its entirety. [0119] Prior to RT-mediated edit incorporation, the prime editor protein (1) site- specifically targets a genomic locus and (2) performs a catalytic cut or nick. These steps are typically performed by a CRISPR-Cas. However, in some embodiments the Cas protein may be substituted by other nucleic acid programmable DNA binding proteins (napDNAbp) such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or meganucleases. In addition, to the extent the “targeting rules” of other napDNAbp are known or are newly determined, it becomes possible to use new napDNAbp, beyond Cas9, to site specifically target and modify genomic sites of interest. [0120] Similar to a prime editor protein, a Gene Writer can introduce novel DNA elements, such as an integration target site, into a DNA locus. A Gene Writer protein comprises: (A) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA comprising (i) a sequence that binds the polypeptide and (ii) a heterologous insert sequence. Examples of such Gene Writer™ proteins and related systems can be found in US20200109398, which is incorporated by reference herein in its entirety. [0121] In some embodiments, the prime editor or Gene Writer protein fusion or prime editor protein linked or fused to an integrase is expressed as a split construct. In typical embodiments, the split construct in reconstituted in a cell. In some embodiments, the split construct can be fused or ligated via intein protein splicing. In some embodiments, the split construct can be reconstituted via protein-protein inter-molecular bonding and/or interactions. In some embodiments, the split construct can be reconstituted via chemical, biological, or environmental induced oligomerization. In some embodiments, the split construct can be reconstituted via nanobody binding ALFA-tagged proteins. In certain embodiments, the split construct can be adapted into one or more single nucleic acid polynucleotides. [0122] In some embodiments, an integrase or recombinase is directly linked or fused, for example by a peptide linker, which may be cleavable or non-cleavable, to the prime editor fusion protein (i.e., fused Cas9 nickase-reverse transcriptase) or Gene Writer protein. Suitable linkers, for example between the Cas9, RT, and integrase, may be selected from Table 3:

6.2. Type II CRISPR proteins [0123] The skilled person can incorporate a selected CRISPR enzyme, described below, as part of the prime editor fusion, as a component of the gene editor polypeptide described herein. Streptococcus pyogenes Cas9 (SpCas9), the most common enzyme used in genome-editing applications, is a large nuclease of 1368 amino acid residues. Advantages of SpCas9 include its short, 5′-NGG-3′ PAM and very high average editing efficiency. SpCas9 consists of two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe can be divided into three regions, a long a helix referred to as the bridge helix (residues 60–93), the REC1 (residues 94–179 and 308–713) domain, and the REC2 (residues 180–307) domain. The NUC lobe consists of the RuvC (residues 1–59, 718–769, and 909–1098), HNH (residues 775–908), and PAM-interacting (PI) (residues 1099–1368) domains. The negatively charged sgRNA:target DNA heteroduplex is accommodated in a positively charged groove at the interface between the REC and NUC lobes. In the NUC lobe, the RuvC domain is assembled from the three split RuvC motifs (RuvC I–III) and interfaces with the PI domain to form a positively charged surface that interacts with the 30 tail of the sgRNA. The HNH domain lies between the RuvC II–III motifs and forms only a few contacts with the rest of the protein. Structural aspects of SpCas9 are described by Nishimasu et al., Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA, Cell 156, 935-949, February 27, 2014. [0124] REC lobe: The REC lobe includes the REC1 and REC2 domains. The REC2 domain does not contact the bound guide:target heteroduplex, indicating that truncation of REC lobe may be tolerated by SpCas9. Further, SpCas9 mutant lacking the REC2 domain (D175–307) retained ~50% of the wild-type Cas9 activity, indicating that the REC2 domain is not critical for DNA cleavage. In striking contrast, the deletion of either the repeat-interacting region (D97–150) or the anti-repeat-interacting region (D312–409) of the REC1 domain abolished the DNA cleavage activity, indicating that the recognition of the repeat:anti-repeat duplex by the REC1 domain is critical for the Cas9 function. [0125] PAM-Interacting domain: The NUC lobe contains the PAM-interacting (PI) domain that is positioned to recognize the PAM sequence on the noncomplementary DNA strand. The PI domain of SpCas9 is required for the recognition of 5’-NGG-3’ PAM, and deletion of the PI domain (Δ1099-1368) abolished the cleavage activity, indicating that the PI domain is critical for SpCas9 function and a major determinant for the PAM specificity. [0126] RuvC domain: The RuvC nucleases of SpCas9 have an RNase H fold and four catalytic residues, Asp10 (Ala), Glu762, His983, and Asp986, that are critical for the two-metal cleavage of the noncomplementary strand of the target DNA. In addition to the conserved RNase H fold, the Cas9 RuvC domain has other structural elements involved in interactions with the guide:target heteroduplex (an end-capping loop between α42 and α43) and the PI domain/stem loop 3 (β hairpin formed by β3 and β4). [0127] HNH domain: SpCas9 HNH nucleases have three catalytic residues, Asp839, His840, and Asn863 and cleave the complementary strand of the target DNA through a single-metal mechanism. [0128] sgRNA:DNA recognition: The sgRNA guide region is primarily recognized by the REC lobe. The backbone phosphate groups of the guide region (nucleotides 2, 4–6, and 13–20) interact with the REC1 domain (Arg165, Gly166, Arg403, Asn407, Lys510, Tyr515, and Arg661) and the bridge helix (Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78). The 20-hydroxyl groups of G1, C15, U16, and G19 hydrogen bond with Val1009, Tyr450, Arg447/Ile448, and Thr404, respectively. [0129] A mutational analysis demonstrated that the R66A, R70A, and R74A mutations on the bridge helix markedly reduced the DNA cleavage activities, highlighting the functional significance of the recognition of the sgRNA ‘‘seed’’ region by the bridge helix. Although Arg78 and Arg165 also interact with the ‘‘seed’’ region, the R78A and R165A mutants showed only moderately decreased activities. These results are consistent with the fact that Arg66, Arg70, and Arg74 form multiple salt bridges with the sgRNA backbone, whereas Arg78 and Arg165 form a single salt bridge with the sgRNA backbone. Moreover, the alanine mutations of the repeat:anti- repeat duplex-interacting residues (Arg75 and Lys163) and the stemloop-1-interacting residue (Arg69) resulted in decreased DNA cleavage activity, confirming the functional importance of the recognition of the repeat:anti-repeat duplex and stem loop 1 by Cas9. [0130] RNA-guided DNA targeting: SpCas9 recognizes the guide:target heteroduplex in a sequence-independent manner. The backbone phosphate groups of the target DNA (nucleotides 1, 9–11, 13, and 20) interact with the REC1 (Asn497, Trp659, Arg661, and Gln695), RuvC (Gln926), and PI (Glu1108) domains. The C2’ atoms of the target DNA (nucleotides 5, 7, 8, 11, 19, and 20) form van der Waals interactions with the REC1 domain (Leu169, Tyr450, Met495, Met694, and His698) and the RuvC domain (Ala728). The terminal base pair of the guide:target heteroduplex (G1:C20’) is recognized by the RuvC domain via end-capping interactions; the sgRNA G1 and target DNA C20’ nucleobases interact with the Tyr1013 and Val1015 side chains, respectively, whereas the 20-hydroxyl and phosphate groups of sgRNA G1 interact with Val1009 and Gln926, respectively. [0131] Repeat:Anti-Repeat duplex recognition: The nucleobases of U23/A49 and A42/G43 hydrogen bond with the side chain of Arg1122 and the main-chain carbonyl group of Phe351, respectively. The nucleobase of the flipped U44 is sandwiched between Tyr325 and His328, with its N3 atom hydrogen bonded with Tyr325, whereas the nucleobase of the unpaired G43 stacks with Tyr359 and hydrogen bonds with Asp364. [0132] The nucleobases of G21 and U50 in the G21:U50 wobble pair stack with the terminal C20:G10 pair in the guide:target heteroduplex and Tyr72 on the bridge helix, respectively, with the U50 O4 atom hydrogen bonded with Arg75. Notably, A51 adopts the syn conformation and is oriented in the direction opposite to U50. The nucleobase of A51 is sandwiched between Phe1105 and U63, with its N1, N6, and N7 atoms hydrogen bonded with G62, Gly1103, and Phe1105, respectively. [0133] Stem-loop recognition: Stem loop 1 is primarily recognized by the REC lobe, together with the PI domain. The backbone phosphate groups of stem loop 1 (nucleotides 52, 53, and 59– 61) interact with the REC1 domain (Leu455, Ser460, Arg467, Thr472, and Ile473), the PI domain (Lys1123 and Lys1124), and the bridge helix (Arg70 and Arg74), with the 20-hydroxyl group of G58 hydrogen bonded with Leu455. A52 interacts with Phe1105 through a face-to-edge p-p stacking interaction, and the flipped U59 nucleobase hydrogen bonds with Asn77. [0134] The single-stranded linker and stem loops 2 and 3 are primarily recognized by the NUC lobe. The backbone phosphate groups of the linker (nucleotides 63–65 and 67) interact with the RuvC domain (Glu57, Lys742, and Lys1097), the PI domain (Thr1102), and the bridge helix (Arg69), with the 20-hydroxyl groups of U64 and A65 hydrogen bonded with Glu57 and His721, respectively. The C67 nucleobase forms two hydrogen bonds with Val1100. [0135] Stem loop 2 is recognized by Cas9 via the interactions between the NUC lobe and the non-Watson-Crick A68:G81 pair, which is formed by direct (between the A68 N6 and G81 O6 atoms) and water-mediated (between the A68 N1 and G81 N1 atoms) hydrogen-bonding interactions. The A68 and G81 nucleobases contact Ser1351 and Tyr1356, respectively, whereas the A68:G81 pair interacts with Thr1358 via a water-mediated hydrogen bond. The 20-hydroxyl group of A68 hydrogen bonds with His1349, whereas the G81 nucleobase hydrogen bonds with Lys33. [0136] Stem loop 3 interacts with the NUC lobe more extensively, as compared to stem loop 2. The backbone phosphate group of G92 interacts with the RuvC domain (Arg40 and Lys44), whereas the G89 and U90 nucleobases hydrogen bond with Gln1272 and Glu1225/Ala1227, respectively. The A88 and C91 nucleobases are recognized by Asn46 via multiple hydrogen- bonding interactions. [0137] Cas9 proteins smaller than SpCas9 allow more efficient packaging of nucleic acids encoding CRISPR systems, e.g., Cas9 and sgRNA into one rAAV (“all-in-one-AAV”) particle. In addition, efficient packaging of CRISPR systems can be achieved in other viral vector systems (i.e., lentiviral, hd-AAV, etc.) and non viral vector systems (i.e., lipid nanoparticle). Small Cas9 proteins can be advantageous for multidomain-Cas-nuclease-based systems for prime editing. Well characterized smaller Cas9 proteins include Staphylococcus aureus (SauCas9, 1053 amino acid residues) and Campylobacter jejuni (CjCas9, 984 amino residues). However, both recognize longer PAMs, 5′-NNGRRT-3′ for SauCas9 (R = A or G) and 5′-NNNNRYAC-3′ for CjCas9 (Y = C or T), which reduces the number of uniquely addressable target sites in the genome, in comparison to the NGG SpCas9 PAM. Among smaller Cas9s, Schmidt et al. identified Staphylococcus lugdunensis (Slu) Cas9 as having genome-editing activity and provided homology mapping to SpCas9 and SauCas9 to facilitate generation of nickases and inactive (“dead”) enzymes (Schmidt et al., 2021, Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases. Nat Commun 12, 4219. doi.org/10.1038/s41467-021- 24454-5) and engineered nucleases with higher cleavage activity by fragmenting and shuffling Cas9 DNAs. The small Cas9s and nickases are useful in the instant disclosure. [0138] Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other “Cas9 variants” having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9. [0139] In some embodiments, the disclosure also may utilize Cas9 fragments that retain their functionality and that are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. [0140] In various embodiments, the prime editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants.

[0141] In some embodiments, prime editors utilized herein comprise CRISPR-Cas system enzymes other than type II enzymes. In certain embodiments, prime editors comprise type V or type VI CRISPR-Cas system enzymes. It will be appreciated that certain CRISPR enzymes exhibit promiscuous ssDNA cleavage activity and appropriate precautions should be considered. In certain embodiments, prime editors comprise a nickase or a dead CRISPR with nuclease function comprised in a different component. [0142] In various embodiments, the nucleic acid programmable DNA binding proteins utilized herein include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12a (Cpf1), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c5, C2c8, C2c9, C2c10, Cas13a (C2c2), Cas13b (C2c6), Cas13c (C2c7), Cas13d, and Argonaute. Cas-equivalents further include those described in Makarova et al., “C2c2 is a single-component programmable RNA- guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the contents of which are incorporated herein by reference. One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e, Cas12a (Cpf1)). Similar to Cas9, Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas12a (Cpf1) mediates robust DNA interference with features distinct from Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T- rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p.949-962; the entire contents of which is hereby incorporated by reference. 6.3. Type V CRISPR proteins [0143] In some embodiments, prime editors used herein comprise the type V CRISPR family includes Francisella novicida U112 Cpf1 (FnCpf1) also known as FnCas12a. FnCpf1 adopts a bilobed architecture with the two lobes connected by the wedge (WED) domain. The N-terminal REC lobe consists of two a-helical domains (REC1 and REC2) that have been shown to coordinate the crRNA-target DNA heteroduplex. The C-terminal NUC lobe consists of the C-terminal RuvC and Nuc domains involved in target cleavage, the arginine-rich bridge helix (BH), and the PAM- interacting (PI) domain. The repeat-derived segment of the crRNA forms a pseudoknot stabilized by intra-molecular base-pairing and hydrogen-bonding interactions. The pseudoknot is coordinated by residues from the WED, RuvC, and REC2 domains, as well as by two hydrated magnesium cations. Notably, nucleotides 1–5 of the crRNA are ordered in the central cavity of FnCas12a and adopt an A-form-like helical conformation. Conformational ordering of the seed sequence is facilitated by multiple interactions between the ribose and phosphate moieties of the crRNA backbone and FnCpf1 residues in the WED and REC1 domains. These include residues Thr16, Lys595, His804, and His881 from the WED domain and residues Tyr47, Lys51, Phe182, and Arg186 from the REC1 domain. The structure of the FnCas12a-crRNA complex further reveals that the bases of the seed sequence are solvent exposed and poised for hybridization with target DNA. Structural aspects of FnCpf1 are described by Swarts et al., Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a, Molecular Cell 66, 221-233, April 20, 2017. [0144] Pre-crRNA processing: Essential residues for crRNA processing include His843, Lys852, and Lys869. Structural observations are consistent with an acid-base catalytic mechanism in which Lys869 acts as the general base catalyst to deprotonate the attacking 2’-hydroxyl group of U(-19), while His843 acts as a general acid to protonate the 5’-oxygen leaving group of A(-18). In turn, the side chain of Lys852 is involved in charge stabilization of the transition state. Collectively, these interactions facilitate the intra-molecular attack of the 20-hydroxyl group of U(-19) on the scissile phosphate and promote the formation of the 2’,3’-cyclic phosphate product. [0145] R-loop formation: The crRNA-target DNA strand heteroduplex is enclosed in the central cavity formed by the REC and NUC lobes and interacts extensively with the REC1 and REC2 domains. The PAM-containing DNA duplex comprises target strand nucleotides dT0–dT8 and non-target strand nucleotides dA(8)*–dA0* and is contacted by the PI, WED, and REC1 domains. The 5’-TTN-3’ PAM is recognized in FnCas12a by a mechanism combining the shape- specific recognition of a narrowed minor groove, with base-specific recognition of the PAM bases by two invariant residues, Lys671 and Lys613. Directly downstream of the PAM, the duplex of the target DNA is disrupted by the side chain of residue Lys667, which is inserted between the DNA strands and forms a cation-π stacking interaction with the dA0–dT0* base pair. The phosphate group linking target strand residues dT(-1) and dT0 is coordinated by hydrogen-bonding interactions with the side chain of Lys823 and the backbone amide of Gly826. Target strand residue dT(-1) bends away from residue T0, allowing the target strand to interact with the seed sequence of the crRNA. The non-target strand nucleotides dT1*–dT5* interact with the Arg692- Ser702 loop in FnCas12a through hydrogen-bonding and ionic interactions between backbone phosphate groups and side chains of Arg692, Asn700, Ser702, and Gln704, as well as main-chain amide groups of Lys699, Asn700, and Ser702. Alanine substitution of Q704 or replacement of residues Thr698–Ser702 in FnCas12a with the sequence Ala-Gly3 (SEQ ID NO: 115) substantially reduced DNA cleavage activity, suggesting that these residues contribute to R-loop formation by stabilizing the displaced conformation of the nontarget DNA strand. [0146] In the FnCas12a R-loop complex, the crRNA-target strand heteroduplex is terminated by a stacking interaction with a conserved aromatic residue (Tyr410). This prevents base pairing between the crRNA and the target strand beyond nucleotides U20 and dA(-20), respectively. Beyond this point, the target DNA strand nucleotides re-engage the non-target DNA strand, forming a PAM-distal DNA duplex comprising nucleotides dC(-21)–dA(-27) and dG21*–dT27*, respectively. The duplex is confined between the REC2 and Nuc domains at the end of the central channel formed by the REC and NUC lobes. [0147] Target DNA cleavage: FnCpf1 can independently accommodate both the target and non-target DNA strands in the catalytic pocket of the RuvC domain. The RuvC active site contains three catalytic residues (D917, E1006, and D1255). Structural observations suggest that both the target and non-target DNA strands are cleaved by the same catalytic mechanism in a single active site in Cpf1/Cas12a enzymes. [0148] Another type V CRISPR is AsCpf1 from Acidaminococcus sp BV3L6 (Yamano et al., Crystal structure of Cpf1 in complex with guide RNA and target DNA, Cell 165, 949-962, May 5, 2016) [0149] In certain embodiments, the nuclease comprises a Cas12f effector. Small CRISPR- associated effector proteins belonging to the type V-F subtype have been identified through the mining of sequence databases and members classified into Cas12f1 (Cas14a and type V-U3), Cas12f2 (Cas14b) and Cas12f3 (Cas14c, type V-U2 and U4). (See, e.g., Karvelis et al., PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Research, 21 May 2020, 48(9), 5016–23 doi.org/10.1093/nar/gkaa208). Xu et al. described development of a 529 amino acid Cas12f-based system for mammalian genome engineering through multiple rounds of iterative protein engineering and screening. (Xu, X. et al., Engineered Miniature CRISPR-Cas System for Mammalian Genome Regulation and Editing. Molecular Cell, October 21, 2021, 81(20): 4333-45, doi.org/10.1016/j.molcel.2021.08.008). [0150] Exemplary CRISPR-Cas proteins and enzymes used in the Prime Editors herein include the following without limitation.

6.4. Protospacer Adjacent Motif [0151] As used herein, the term “protospacer adjacent sequence” or “protospacer adjacent motif” or “PAM” refers to an approximately 2-6 base pair DNA sequence (or a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-long nucleotide sequence) that is an important targeting component of a Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5' to 3' direction of Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5'-NGG-3' wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence. [0152] For example, with reference to the canonical SpCas9 amino acid sequence, the PAM specificity can be modified by introducing one or more mutations, including (a) D1135V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) D1135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG. In addition, the D1135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein. [0153] It will also be appreciated that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities and in some embodiments are therefore chosen based on the desired PAM recognition. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In still another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These examples are not meant to be limiting. It will be further appreciated that non- SpCas9s bind a variety of PAM sequences, which makes them useful to expand the range of sequences that can be targeted according to the invention. Furthermore, non-SpCas9s may have other characteristics that make them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV). Further reference may be made to Shah et al., “Protospacer recognition motifs: mixed identities and functional diversity,” RNA Biology, 10(5): 891-899 (which is incorporated herein by reference). Gasiunas used cell-free biochemical screens to identify protospacer adjacent motif (PAM) and guide RNA requirements of 79 Cas9 proteins. (Gasiunas et al., A catalogue of biochemically diverse CRISPR-Cas9 orthologs, Nature Communications 11:5512 doi.org/10.1038/s41467-020-19344-1) The authors described 7 classes of gRNA and 50 different PAM requirement. [0154] Oh, Y. et al. describe linking reverse transcriptase to a Francisella novicida Cas9 [FnCas9(H969A)] nickase module. (Oh, Y. et al., Expansion of the prime editing modality with Cas9 from Francisella novicida, bioRxiv 2021.05.25.445577; doi.org/10.1101/2021.05.25.445577). By increasing the distance to the PAM, the FnCas9(H969A) nickase module expands the region of a reverse transcription template (RTT) following the primer binding site. 6.5. Prime Editors [0155] “Prime editor fusion protein” describes a protein that is used in prime editing. Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; and a nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts. Such an enzyme can be a catalytically-impaired Cas9 endonuclease (a nickase). Such an enzyme can be a Casl2a/b, MAD7, or variant thereof. The nickase is fused to an engineered reverse transcriptase (RT). The nickase is programmed (directed) with an attachment site-containing gRNA (atgRNA) or a prime-editing guide RNA (pegRNA). The skilled person in the art would appreciate that the atgRNA (or pegRNA) both specifies the target site and encodes the desired edit. Advantageously the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA, whereby a nick or single stranded cut occurs. The reverse transcriptase domain then uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Afterward, optionally, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA). [0156] As used herein, “PE1” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(wt)] + a desired atgRNA. In various embodiments, the prime editors disclosed herein is comprised of PE1. [0157] As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired PEgRNA. In various embodiments, the prime editors disclosed herein is comprised of PE2. [0158] In various embodiments, the prime editors disclosed herein is comprised of PE2 and co-expression of MMR protein MLH1dn, that is PE4. [0159] As used herein, “PE3” refers to PE2 plus a second-strand nicking guide RNA that complexes with the PE2 and introduces a nick in the non-edited DNA strand. The induction of the second nick increases the chances of the unedited strand, rather than the edited strand, to be repaired. In various embodiments, the prime editors disclosed herein is comprised of PE3. [0160] In various embodiments, the prime editors disclosed herein is comprised of PE3 and co-expression of MMR protein MLH1dn, that is PE5. [0161] As used herein, “PE3b” refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence with mismatches to the unedited original allele that matches only the edited strand. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. 6.6. Guides for Prime Editing [0162] Anzalone et al., 2019 (Nature 576:149) describes prime editing and a prime editing complex using a type II CRISPR and can be used herein. A prime editing complex consists of a type II CRISPR PE protein containing an RNA-guided DNA-nicking domain fused to a reverse transcriptase (RT) domain and complexed with a pegRNA. The pegRNA comprises (5’ to 3’) a spacer that is complementary to the target sequence of a genomic DNA, a nickase (e.g. Cas9) binding site, a reverse transcriptase template including editing positions, and primer binding site (PBS). The PE–pegRNA complex binds the target DNA and the CRISPR protein nicks the PAM- containing strand. The resulting 3′ end of the nicked target hybridizes to the primer-binding site (PBS) of the pegRNA, then primes reverse transcription of new DNA containing the desired edit using the RT template of the pegRNA. The overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end. The structure leaves the PBS at the 3’ end of the pegRNA free to bind to the nicked strand complementary to the target which forms the primer for reverse transcription. [0163] Guide RNAs of CRISPRs differ in overall structure. For example, while the spacer of a type II gRNA is located at the 5’ end, the spacer of a type V gRNA is located towards the 3’ end, with the CRISPR protein (e.g. Cas12a) binding region located toward the 5’ end. Accordingly, the regions of a type V pegRNA are rearranged compared to a type II pegRNA. The overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end. The pegRNA comprises (5’ to 3’) a CRISPR protein-binding region, a spacer which is complementary to the target sequence of a genomic DNA, a reverse transcriptase template including editing positions, and primer binding site (PBS). 6.7. Attachment Site-Containing Guide RNA (atgRNA) [0164] As used herein, the term “attachment site-containing guide RNA” (atgRNA) and the like refer to an extended single guide RNA (sgRNA) comprising a primer binding site (PBS), a reverse transcriptase (RT) template sequence, and wherein the RT template encodes for an integration recognition site or a recombinase recognition site that can be recognized by a recombinase, integrase, or transposase. In some embodiments, the RT template comprises a clamp sequence and an integration recognition site. As referred to herein an atgRNA may be referred to as a guide RNA. An integration target recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA). [0165] As used herein, the term “cognate integration recognition site” or “integration cognate” or “cognate pair” refers to a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined. Recombination between a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second recognition site (e.g., any of the integration recognition sites described herein) is mediated by functional symmetry between the two integration recognition sites and the central dinucleotide of each of the two integration recognition sites. In some cases, a first integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined with a second integration recognition site (e.g., any of the integration recognition sites described herein) are referred to as a “cognate pair.” A non-limiting example of a cognate pair include an attB site and an attP site, whereby a serine integrase mediates recombination between the attB site and the attP site. FIGs.1A-1E show optimization of the integration recognition site. [0166] In typical embodiments, an atgRNA comprises a reverse transcriptase template that encodes, partially or in its entirety, an integration recognition site (also referred to as an integration target recognition site) or a recombinase recognition site (also referred to as a recombinase target recognition site). The integration target recognition site, which is to be placed at a desired location in the genome or intracellular nucleic acid, is referred to as a “beacon,” a “beacon” site or an “attachment site” or a “landing pad” or “landing site.” An integration target recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA). [0167] In typical embodiments, an atgRNA comprises a reverse transcriptase template that encodes, partially or in entirety, for an integration target recognition site or recombinase target recognition site. The integration target recognition site, which is to be place at a desired location in the genome, is referred to as a “beacon site” or an “attachment site” or a “landing pad” or “landing site.” [0168] During genome editing, the primer binding site allows the 3' end of the nicked DNA strand to hybridize to the atgRNA, while the RT template serves as a template for the synthesis of edited genetic information. The atgRNA is capable for instance, without limitation, of (i) identifying the target nucleotide sequence to be edited and (ii) encoding new genetic information that replaces (or in some cases adds) the targeted sequence. In some embodiments, the atgRNA is capable of (i) identifying the target nucleotide sequence to be edited and (ii) encoding an integration site that replaces (or inserts/deletes within) the targeted sequences. [0169] In some embodiments, where the system contains a first atgRNA and a second atgRNA (see FIG. 2 or FIG. 8), the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, where the at least first pair of atgRNAs have domains that are capable of guiding the gene editor polypeptide or prime editor fusion protein to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the first integration recognition site; and the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0170] In some embodiments, the first atgRNA’s reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second atgRNA comprising a second reverse transcriptase template. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site. Use of two guide RNAs that are (or encode DNA that is) partially complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs). In certain embodiments, use of two guide RNAs that are (or encode DNA that is) full complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site- containing guide RNAs (atgRNAs). [0171] In some embodiments, the atgRNA are selected from Tables 12 or 14. In some embodiments, one or more of the atgRNA are selected from SEQ ID NO: 559, 560, 563, 564, 567- 569, 570, 573, 574, 577, 578, or 579-598. In some embodiments, one or more of the atgRNA are selected from 603 and 604. [0172] As shown in FIG. 19A-19E, AttP variants (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance) can be optimized. [0173] Table 9 includes atgRNAs, sgRNAs and nicking guides that can be used herein. Spacers are labeled in capital font (SPACER), RT regions in bold capital (RT REGION), AttB sites in bold lower case (attb site), and PBS in capital italics (PBS). Unless otherwise denoted, the AttB is for Bxb1.

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Table 9

Table 9

6.8. Integrases/Recombinases and Integration/Recombination Sites [0174] In typical embodiments, a gene editor polypeptide described herein contains an integrase or recombinase. In some embodiments, the integrase is delivered as a protein or the integrase is encoded in a delivered polynucleotide. In some embodiments, the integration enzyme is selected from the group consisting of Dre, Vika, Bxb1, ϕC31, RDF, ϕBTl, R1, R2, R3, R4, R5, TP901-1, A118, ϕFCl, ϕC1, MR11, TG1, ϕ370.l, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, ϕRV, retrotransposases encoded by a Tc1/mariner family member including but not limited to retrotransposases encoded by LI, Tol2, Tel, Tc3, Himar 1 (isolated from the horn fly, Haematobia irritans), Mos1 (Mosaic element of Drosophila mauritiana), and Minos, and any mutants thereof. As can be used herein, Xu et al describes methods for evaluating integrase activity in E. coli and mammalian cells and confirmed at least R4, φC31, φBT1, Bxb1, SPBc, TP901-1 and Wβ integrases to be active on substrates integrated into the genome of HT1080 cells (Xu et al., 2013, Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol.2013 Oct 20;13:87. doi: 10.1186/1472-6750-13-87). Durrant describes new large serine recombinases (LSRs) divided into three classes distinguished from one another by efficiency and specificity, including landing pad LSRs which outperform wild-type Bxb1 in episomal and chromosomal integration efficiency, LSRs that achieve both efficient and site- specific integration without a landing pad, and multi-targeting LSRs with minimal site-specificity. Additionally, embodiments can include any serine recombinase such as BceINT, SSCINT, SACINT, and INT10 (see Ionnidi et al., 2021; Drag-and-drop genome insertion without DNA cleavage with CRISPR directed integrases. bioRxiv 2021.11.01.466786, doi.org/10.1101/2021.11.01.466786). In some embodiments, the integration site can be selected from an attB site, an attP site, an attL site, an attR site, a lox71 site a Vox site, or a FRT site. [0175] It will be appreciated that desired activity of integrases, transposases and the like can depend on nuclear localization. In certain embodiments, prokaryotic enzymes are adapted to modulate nuclear localization. In certain embodiments, eukaryotic or vertebrate enzymes are adapted to modulate nuclear localization. In certain embodiments, the disclosure provides fusion or hybrid proteins. Such modulation can comprise addition or removal of one or more nuclear localization signal (NLS) and/or addition or removal of one or more nuclear export signal (NES). Xu et al compared derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal (NLS) to conclude that certain integrases benefit from addition of an NLS whereas others are transported efficiently without addition, and a major determinant of activity in yeast and vertebrate cells is avoidance of toxicity. (Xu et al., 2016, Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae. BMC Biotechnol. 2016 Feb 9; 16:13. doi: 10.1186/s12896-016-0241-5). Ramakrishnan et al. systematically studied the effect of different NES mutants developed from mariner-like elements (MLEs) on transposase localization and activity and concluded that nuclear export provides a means of controlling transposition activity and maintaining genome integrity. (Ramakrishnan et al. Nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmar1 and Ppmar2 of moso bamboo. Mob DNA. 2019 Aug 19;10:35. doi:10.1186/s13100-019-0179-y). The methods and constructs are used to modulate nuclear localization of system components of the disclosure. [0176] In typical embodiments, the integrase used herein is selected from below.

[0177] Sequences of insertion sites (i.e., recognition target sites) suitable for use in embodiments of the disclosure are presented below.

6.9. Programmable Gene Insertion in Primary Cells [0178] This disclosure also features systems, compositions, and methods for generating a primary cell comprising an integration recognition site site-specifically integrated into the genome of the primary cell. [0179] In one embodiment, a method of generating a primary cell comprising an integration recognition site, where the method includes (a) site-specifically incorporating at least a first integration recognition site into a target sequence in the genome of the primary cell. In some embodiments, site-specifically incorporating the at least first integration recognition site is effected by introducing into the primary cell: (i) a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at the target sequence; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the primary cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site- specifically incorporated into the genome of the primary cell. [0180] In some embodiments of the method of site-specifically incorporating the at least first integration recognition site the genome of the primary cell, the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0181] In some embodiments of the method of site-specifically incorporating the at least first integration recognition site the genome of the primary cell, the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is an atgRNA that further includes an RT template that comprises at least a portion of the first integration recognition site, wherein the atgRNA encodes the entirety of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a nicking gRNA. [0182] This disclosure also features systems, compositions, and methods for generating a primary cell comprising an exogenous nucleic acid (e.g., a donor polynucleotide template) site-specifically integrated into the genome of the primary cell. [0183] In some embodiments, the method of site-specifically integrating an exogenous nucleic acid (e.g., a donor polynucleotide template) into a primary cell genome includes: (b) integrating at least a first donor polynucleotide template into the primary cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal/cognate integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the primary cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a primary cell with a site-specifically integrated donor polynucleotide template. [0184] In some embodiments, the method of site-specifically integrating an exogenous nucleic acid (e.g., a donor polynucleotide template) into a primary cell genome includes performing steps (a) and (b) concurrently. In some embodiments of the method of site-specifically incorporating the at least first integration recognition site the genome of the primary cell, the method includes performing step (a) prior to performing step (b). In some embodiments of the method of site- specifically incorporating the at least first integration recognition site the genome of the primary cell, the method includes performing step (a) and step (b) at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks apart. [0185] In one embodiment, the method of site specifically integrating an exogenous nucleic acid (e.g., a donor polynucleotide template) into a primary cell genome includes integrating, into the genome of any of the primary cells described herein at the first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal/cognate integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the primary cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a primary cell with a site- specifically integrated donor polynucleotide template. [0186] In one embodiment, the method of site specifically integrating an exogenous nucleic acid (e.g., a donor polynucleotide template) into a primary cell genome, the method comprising: incorporating an integration recognition site into the human primary cell genome by delivering into the cell: a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at a target sequence; an at least a first attachment site-containing guide RNA (atgRNA), wherein the one or more guide RNA encodes all or a portion of an at least first integration recognition site; and integrating a donor polynucleotide template into the human primary cell genome at the incorporated recognition site by delivering into the cell: the donor polynucleotide template, an integration enzyme or a polynucleotide encoding an integration enzyme, wherein the donor polynucleotide template is linked to a sequence that is an integration cognate of the integration recognition site present in the atgRNA, and wherein the donor polynucleotide template is integrated into the genome at the incorporated genomic integration recognition site by the integration enzyme; thereby producing a human subject primary cell. [0187] In some embodiments, the gene editor polypeptide or the polynucleotide encoding a gene editor polypeptide, the at least first atgRNA, the donor polynucleotide template, and the integration enzyme or the polynucleotide encoding an integration enzyme are concurrently delivered into the primary cell. [0188] In some embodiments, the gene editor protein or the polynucleotide encoding a gene editor protein, and the at least first atgRNA, are delivered at a first time point and the donor polynucleotide template, and the integration enzyme or the polynucleotide encoding an integration enzyme are delivered at a second time point. In such embodiments, the first time point and the second time point are at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks apart. [0189] In some embodiments, the gene editor polypeptide is configured such that the nickase is linked to the reverse transcriptase. In some embodiments, the nickase is linked to the reverse transcriptase by in-frame fusion. In some embodiments, the nickase is linked to the reverse transcriptase by a linker. In some embodiments, the linker is a peptide fused in-frame between the nickase and reverse transcriptase. [0190] In some embodiments, the gene editor polynucleotide further comprises a polynucleotide sequence encoding at least an integration enzyme or the gene editor polypeptide further comprises an integration enzyme. In some embodiments, the linked nickase-reverse transcriptase are further linked to the integration enzyme. [0191] In some embodiments, an at least first atgRNA comprises: (i) a domain that is capable of guiding the prime editor system to the target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of an at least first integration recognition site. [0192] In some embodiments, the RT template comprises the entirety of the first integration recognition site. [0193] In some embodiments, site-specifically incorporating an integration recognition site into the primary cell genome further comprises delivering into the cell a second atgRNA. [0194] In some embodiments, the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein: the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence; the first atgRNA further includes a first RT template that comprises at least a portion of an at least first integration recognition site; the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0195] In some embodiments, the first RT template encodes a first single-stranded DNA sequence and the second RT template encodes a second single-stranded DNA sequence. [0196] In some embodiments, the first single-stranded DNA sequence comprises a complementary region with the first single-stranded DNA sequence. In some embodiments, the first single- stranded DNA sequence and the first single-stranded DNA sequence form a duplex. In some embodiments, the complementary region is 5 or more consecutive bases. In some embodiments, the complementary region is 10 or more consecutive bases. In some embodiments, the complementary region is 20 or more consecutive bases. In some embodiments, the complementary region is 30 or more consecutive bases. [0197] In some embodiments, incorporating an integration recognition site into the human primary cell genome further comprises delivering into the cell a nicking guide RNA (gRNA). [0198] In some embodiments, the first integration recognition site is an attB or attP site. In some embodiments, the integration recognition site is a modified attB or attP site. In some embodiments, the integration recognition site is specific for BxB1 or a modified BxB1. [0199] In some embodiments, the integration recognition site is comprised of 38 or 46 nucleotides. [0200] In certain embodiments, this disclosure provides a method of editing a human subject primary cell genome. In typical embodiments, an integration target recognition site (i.e., integration recognition site or beacon) is incorporated into a primary cell genome (e.g., a human primary cell) by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the integration target recognition site into the genome, (ii) one or more guide RNA (e.g., one or more atgRNA), wherein the one or more guide RNA encodes an integrase target recognition site (i.e., atgRNA), and (iii) optionally, a nicking guide RNA (ngRNA). Further, integrating a donor polynucleotide template into the human primary cell (e.g., the human primary cell) genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, wherein the donor polynucleotide is integrated into the primary cell (e.g., the human primary cell) at the incorporated genomic integration target recognition site by an integrase, thereby producing a genetically modified human primary cell. [0201] In some embodiments, an integration target recognition site (i.e., integration recognition site or beacon) is incorporated into a primary cell genome (e.g., a human primary cell genome) by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the integration target recognition site into the genome, (ii) one or more guide RNA complex, wherein the one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site (i.e., atgRNA), and (iii) optionally, a nicking guide RNA (ngRNA). Further, integrating a donor polynucleotide template into the primary cell genome (e.g., the human primary cell genome) at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, wherein the donor polynucleotide is integrated into the primary cell (e.g., the human primary cell) at the incorporated genomic integration target recognition site by an integrase, thereby producing a genetically modified primary cell (e.g., a modified human primary cell). [0202] In typical embodiments, the one or more guide RNA complex is a split guide RNA complex or split guide RNA system. In typical embodiments, the one or more guide RNA complex is a split atgRNA complex or split atgRNA system. [0203] In some embodiments, the guide RNA or the guide RNA complex reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second guide RNA comprised of a reverse transcriptase template. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single- stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site. Use of two guide RNAs that are (or encode DNA that is) partially complementary to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs). [0204] In some embodiments, an integration target recognition site is incorporated (referred to herein as beacon placement) into a primary cell (e.g., a human primary cell) genome using a single atgRNA and a single nicking guide RNA (ngRNA). In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) using two atgRNAs (dual atgRNAs). Beacon placement efficiency can be determined, for example, by a two-color digital droplet PCR assay that compares signal from no-insertion amplicons (i.e., wild type) with signal from beacon inserted/placed amplicons. Beacon placement efficiency can be determined, for example, by amplicon-sequencing (amp-seq). [0205] In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at an efficiency of at least 60%. In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at an efficiency of at least 70%. In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at an efficiency of at least 80%. In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at an efficiency of at least 90%. [0206] In some embodiments, the integration target site is incorporated at one or more of the TRAC locus, CXCR4 locus, TRBC1 locus, TRBC2 locus, PDCD1 locus, B2M locus, CIITA locus, or IL2RB locus. In some embodiments, a donor polynucleotide is integrated at one or more incorporated integration target site. [0207] In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at the CXCR4 locus at an of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using a single atgRNA and a single ngRNA. In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at the CXCR4 locus at an efficiency of at least 0.5% using a single atgRNA and a single ngRNA at a 1.5:1 or 3:1 molar ratio of atgRNA to ngRNA. In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at the CXCR4 locus at an efficiency of at least 0.5% using a single atgRNA and a single ngRNA at a 1.5:1 or 3:1 molar ratio of atgRNA to ngRNA at 900 picomoles total of atgRNA plus ngRNA. In some embodiments, an integration target recognition site is incorporated into a primary cell genome (e.g., a human primary cell genome) at the CXCR4 locus at an efficiency of at least 0.5% using a single atgRNA and a single ngRNA at a 1.5:1 or 3:1 molar ratio of atgRNA to ngRNA at 900 picomoles total of atgRNA plus ngRNA and 3 or 6 micrograms of mRNA encoding a prime editor protein. [0208] In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using a single atgRNA and a single ngRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 12.5% using a single atgRNA and a single ngRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 12.5% using a single atgRNA and a single ngRNA at a 3:1 molar ratio of atgRNA to ngRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 12.5% using a single atgRNA and a single ngRNA at a 3:1 molar ratio of atgRNA to ngRNA at 900 picomoles total of atgRNA plus ngRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 12.5% using a single atgRNA and a single ngRNA at a 3:1 molar ratio of atgRNA to ngRNA at 900 picomoles total of atgRNA plus ngRNA and 3 or 6 micrograms of mRNA encoding a prime editor protein. [0209] In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the CXCR4 locus at an efficiency of at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using dual atgRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the CXCR4 locus at an efficiency of at least 57.2% using dual atgRNAs. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the CXCR4 locus at an efficiency of at least 57.2% using dual atgRNAs at a 1:1 molar ratio of each atgRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the CXCR4 locus at an efficiency of at least 57.2% using dual atgRNAs at a 1:1 molar ratio of each atgRNA at 400 picomoles total of atgRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the CXCR4 locus at an efficiency of at least 57.2% using dual atgRNAs at a 1:1 molar ratio of each atgRNA at 400 picomoles total of atgRNA and 3 or 6 micrograms of mRNA encoding a prime editor protein. [0210] In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using dual atgRNAs. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 25% using dual atgRNAs at a 1:1 molar ratio of each atgRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 25% using dual atgRNAs at a 1:1 molar ratio of each atgRNA at 400 or 200 picomoles total of atgRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the IL2RB locus at an efficiency of at least 25% using dual atgRNAs at a 1:1 molar ratio of each atgRNA at 400 or 200 picomoles total of atgRNA and 3 or 6 micrograms of mRNA encoding a prime editor protein. [0211] In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the TRAC locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using a single atgRNA and a single ngRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the TRAC locus at an efficiency of at least 10% using a single atgRNA and a single ngRNA at a 1.5:1 or 3:1 molar ratio of atgRNA to ngRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the TRAC locus at an efficiency of at least 10% using a single atgRNA and a single ngRNA at a 1.5:1 or 3:1 molar ratio of atgRNA to ngRNA at 900 picomoles total of atgRNA plus ngRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the TRAC locus at an efficiency of at least 10% using a single atgRNA and a single ngRNA at a 1.5:1 or 3:1 molar ratio of atgRNA to ngRNA at 900 picomoles total of atgRNA plus ngRNA and 3 or 6 micrograms of mRNA encoding a prime editor protein. [0212] In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the TRAC locus at an efficiency of at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using dual atgRNAs. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the TRAC locus at an efficiency of at least 20% using dual atgRNAs at a 1:1 molar ratio of each dual atgRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the TRAC locus at an efficiency of at least 20% using dual atgRNAs at a 1:1 molar ratio of each dual atgRNA at total of 800 picomoles total of dual atgRNA. In some embodiments, an integration target recognition site is incorporated into a human primary cell genome at the TRAC locus at an efficiency of at least 20% using dual atgRNAs at a 1:1 molar ratio of each dual atgRNA at total of 800 picomoles total of dual atgRNA and 3 or 6 micrograms of mRNA encoding a prime editor protein. [0213] In certain embodiments, the integration target recognition site is specific for an integrase. In some embodiments, the integration target recognition site is specific for a recombinase or transposase. In typical embodiments, the integration target recognition site is an attB or attP site. In some embodiments, the integration target recognition site is a modified or orthogonal attB or attP site. In certain embodiments, the integrase target recognition site is a Bxb1 attB or attP sequence. In some embodiments, the att-site central dinucleotide is not GT or CA. [0214] In some embodiments, the integration target recognition site is comprised of 38 or 46 nucleotides. In some embodiments, the integration target recognition site is less than 46 nucleotides. In some embodiments, the integration target recognition site is less than 40 nucleotides. In some embodiments, the integration target recognition site is less than 35 nucleotides. In some embodiments, the integration target recognition site is less than 30 nucleotides. In some embodiments, the integration target recognition site is less than 25 nucleotides. [0215] In some embodiments, the one or more guide RNA or the one or more guide RNA complex comprises a chemical modification. In certain embodiments, the chemical modification is selected from one or more of a 2’ O-methyl and phosphorothioate. [0216] Sequences of guide RNAs (i.e., attachment site-containing guide RNAs) suitable for partial replacement use in embodiments of the disclosure are presented below. In the below table: [0217] Non-modified RNA are entered as ‘r_’. For example, as rA or rU. [0218] 2’ O-methyl RNA are entered as ‘m_’. [0219] Phosphorothioated RNA are entered as ‘r_*’ [0220] Phosphorothioated 2’ O-methyl RNA are entered as ‘m_*’

[0221] In typical embodiments, the guide RNA for beacon placement comprises, in 5’ to 3’ order, (i) a spacer complementary to a first target genomic site, (ii) a scaffold capable of binding to a DNA binding nuclease or nuclease with nickase activity, (iii) a reverse transcriptase template comprised of at least an integrase target recognition site, and (iv) a primer binding site. In typical embodiments, the reverse transcriptase template encodes for a first single-stranded DNA sequence or first DNA flap sequence.

[0222] In certain embodiments, the cellular integrated polynucleotide (i.e., donor polynucleotide template) encodes for one or more of a chimeric antigen receptor, a T cell receptor, a cytokine receptor, a chemokine receptor, or a modified receptor. In some embodiments, the cellular integrated polynucleotide encodes for an exogenous T cell receptor. In some embodiments, the cellular integrated polynucleotide encodes for a secreted cytokine. In some embodiments, the cellular integrated polynucleotide encodes for a recombinant protein. In some embodiments, the cellular integrated polynucleotide further comprises one or more of a co-stimulatory receptor such suitable co-stimulatory signaling regions are also well known in the art, and include members of the B7/CD28 family such as B7-1, B7-2, E7-H1 , B7-H2, E7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-l, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LII .RA3. MI .R.A4. MLRB L L1LRB2, I..ILRB3 or LILRB4; or tumor necrosis factor (TNF) superfamily members such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT. Lymphotoxin-alpha, 0X40, RELT, TACT TL1 A, TN F-alpha or TNF RII, or members of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CDS, CD84, CD229, CRACC, NTB-.A or SLAM: or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other costimulatory molecules such as CD7, CD96, CD160, CD200, CD30()a, CRTAM, DAP12, Dectin- 1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG-3 or TSLP R.

[0223] In some embodiments, the cellular integrated polynucleotide (i.e., donor polynucleotide template) is comprised of a suicide switch. In certain embodiments, the suicide switch is comprised of an inducible caspase 9 or HSV thymidine kinase.

[0224] The selection of the co-stimulatory signaling regions may be selected depending upon the particular use intended for the transformed cells. In particular, the co-stimulatory signaling regions selected for those which may work co-operatively or synergistically together. For example, the co-stimulatory signaling regions may be selected from CD28, CD27, ICOS, 4-1BB, 0X40, CD30, GI TR, HVEM, DIM or CD40.

[0225] In some embodiments, the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor, further comprises one or more integrase target recognition site. In some embodiments, the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor, further comprises at least two integrase target recognition sites. In certain embodiments, the one or more integrase target recognition sites flank the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor. In certain embodiments, the flanking integrase target recognition sites allow for excision of any inserted genetic sequence. [0226] In certain embodiments, the human primary cell is isolated ex vivo or differentiated from isolated human primary cells. In some embodiments, the human primary cell is isolated from a human subject prior to incorporating an integration target recognition site into the human primary cell genome. In some embodiments the human primary cell is autologous. In some embodiments, the human primary cell is allogeneic. “Allogeneic” as used herein, means cells from different individuals of the same species. In some embodiments, the human primary cell is activated ex vivo or in vitro prior to incorporating an integration target recognition site into the human primary cell genome. In some embodiments, the human cell is non-activated (i.e., non-stimulated) prior to incorporating an integration target recognition site into the human primary cell genome. In some embodiments, the genetically modified human primary cell is expanded in vitro or ex vivo after integrating a donor polynucleotide template. In some embodiments, the expanded modified human primary cell composition is isolated. In certain embodiments, the isolated human primary cell composition is administered in an effective amount to a human subject. In certain embodiments, the human subject is concurrently or subsequently administered a suitable dose of an anti-IL-6R antibody, such as tocilizumab, alone or in combination with a suitable corticosteroid to decrease or prevent cytokine release syndrome (CRS) and/or neurotoxicity in the subject, also referred to as immune effector cell-associated neurotoxicity syndrome (ICANS) (See, e.g., Front. Immunol., 28 August 2020; doi.org/10.3389/fimmu.2020.01973). 6.10. Split Gene Editor Guide RNA Compositions [0227] The present disclosure contemplates a split guide RNA comprised of two or more polynucleotides that are capable of forming a guide RNA complex (see Liu et al; doi: 10.1038/s41587-022-01255-9 the entirety of which is incorporated by reference herein). In some embodiments, the guide RNA may be a guide RNA complex, wherein the guide RNA complex is comprised of at least two polynucleotide components. In certain embodiments, the guide RNA complex is comprised of a first polynucleotide component and a second polynucleotide component. In certain embodiments, the first polynucleotide component comprises a spacer complementary to a target first genomic site and a scaffold capable of binding to a DNA binding nickase, and wherein the second polynucleotide component comprises a reverse transcriptase template comprised of at least an integrase target recognition site, a primer binding site and a RNA- protein recruitment domain. In some embodiments, the RNA-protein recruitment domain is a MS2 hairpin. [0228] In some embodiments, at least one of the first polynucleotide component and the second polynucleotide component are circularized. In some embodiments, circularization is mediated by a ribozyme or ligase. In some embodiments, circularization is mediated by covalently or non-covalently linking the 5’ and 3’ termini. [0229] In certain embodiments, the guide RNA or the guide RNA complex further comprises one or more of an RNA-protein recruitment domain, RNA-RNA recruitment domain, a transcriptional termination signal, a reverse transcription termination signal, an RNA ribozyme, or a chemical linker. [0230] In typical embodiments, one or more guide RNA complex is comprised in one or more RNA polynucleotides or DNA polynucleotides. 6.11. Guide RNA Compositions for Dual Guide RNA Systems [0231] In some embodiments, the guide RNA or the guide RNA complex reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second guide RNA comprised of a reverse transcriptase template. [0232] In some embodiments, the complementary region between the first and second single- stranded DNA sequences is comprises of 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 11, at least 12, at least 13, at least 14, at least 15 , at least 16 , at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive bases. [0233] In certain embodiments, the complementary region between the first and second single- stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site. [0234] In some embodiments, the integrase target recognition site is an Bxb1 attB or attP sequence. In some embodiments, the att-site central dinucleotide is not GT or CA. [0235] In typical embodiments, one or more guide RNA or the guide RNA complex is comprised in one or more RNA polynucleotides or DNA polynucleotides. 6.12. Guide RNA-Gene Editor Complex [0236] In typical embodiments, the guide RNA or guide RNA complex is capable of binding a DNA binding nickase selected from the group consisting of: Cas9-D10A, Cas9-H840A, Cas12a/b/c/d/e nickase, CasX nickase, SaCas9 nickase, and CasY nickase. In certain embodiments, the nickase is linked or fused to one or more of a reverse transcriptase. In certain embodiments, the nickase is linked or fused to one or more of a reverse transcriptase and integrase. In certain embodiments, the nickase is linked or fused to one or more of an integrase. 6.13. Genes and Targets [0237] This disclosure provides compositions, systems and methods for correcting or replacing genes or gene fragments (including introns or exons) or inserting genes in new locations. In certain embodiments, such a method comprises recombination or integration into a safe harbor site (SHS). A frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion. Another locus comprises the human homolog of the murine Rosa26 locus. Yet another SHS comprises the human H11 locus on chromosome 22. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. In certain embodiments, a method of the disclosure comprises recombining corrective gene fragments into a defective locus. [0238] The methods and compositions can be used to target, without limitation, stem cells for example induced pluripotent stem cells (iPSCs), HSCs, HSPCs, mesenchymal stem cells, or neuronal stem cells and cells at various stages of differentiation. In certain embodiments, methods and compositions of the disclosure are adapted to target organoids, including patient derived organoids. [0239] In certain embodiments, methods and compositions of the disclosure are adapted to treat muscle cells, not limited to cardiomyocytes for Duchene Muscular Dystrophy (DMD). The dystrophin gene is the largest gene in the human genome, spanning ∼2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14-kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon. An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs). In some embodiments, the methods and systems described herein are used to treat DMD by site- specifically integrating in the genome a polynucleotide template that repairs or replaces all or a portion of the defective DMD gene. [0240] The following are non-limiting diseases that may be treated utilizing the methods and compositions of the present disclosure: Inherited Retinal Diseases: • Stargardt Disease (ABCA4) • Leber congenital amaurosis 10 (CEP290) • X linked Retinitis Pigmentosa (RPGR) • Autosomal Dominant Retinitis Pigmentosa (RHO) Liver Diseases: • Wilson’s disease (ATP7B) • Alpha-1 antitrypsin (SERPINA1) Intellectual Disabilities: • Rett Syndrome (MECP2) • SYNGAP1-ID (SYNGAP1) • CDKL5 deficiency disorder (CDKL5) Peripheral Neuropathies: • Charcot-Marie-Tooth 2A (MFN2) Lung Diseases: • Cystic Fibrosis (CFTR) • Alpha-1 Antitrypsin (SERPINA1) Autoimmune diseases: • IgA Nephropathy (Berger’s disease) • Anti-Neutrophil Cytoplasmic Antibody (ANCA) Vasculitis • Systemic Lupus Erythematosus (SLE) / Lupus Nephritis (LN) • Membranous Nephropathy (MN) • C3 glomerulonephritis (C3GN) Blood disorders: • Sickle Cell • Hemophilia • Factor VIII or • Factor IX • Ornithine transcarbamylase deficiency (OTCD) • Homocystinuria (HCU) • Phenylketonuria (PKU) Cancer • Prostate cancer • Renal cell cancer • Thyroid cancer [0241] CFTR (cystic fibrosis transmembrane conductance regulator). The most common cystic fibrosis (CF) mutation F508del removes a single amino acid. In some embodiments, recombining human CFTR into an SHS of a cell that expresses CFTR F508del is a corrective treatment path. In some embodiments, the methods and systems described herein are used to CF by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing CF. Proposed validation is detection of persistent CFTR mRNA and protein expression in transduced cells. [0242] Sickle cell disease (SCD) is caused by mutation of a specific amino acid — valine to glutamic acid at amino acid position 6. In some embodiments, SCD is corrected by recombination of the HBB gene into a safe harbor site (SHS) and by demonstrating correction in a proportion of target cells that is high enough to produce a substantial benefit. In some embodiments, the methods and systems described herein are used to sickle cell disease by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the disease. In some embodiments, validation is detection of persistent HBB mRNA and protein expression in transduced cells. [0243] DMD — Duchenne Muscular Dystrophy. The dystrophin gene is the largest gene in the human genome, spanning ∼2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14- kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon. An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs). [0244] In some embodiments, recombination will be into safe harbor sites (SHS). A frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion. In some embodiments, the site is the human homolog of the e murine Rosa26 locus (pubmed.ncbi.nlm.nih.gov/18037879). In some embodiments, the site is the human H11 locus on chromosome 22. Proposed target cells for recombination include stem cells for example induced pluripotent stem cells (iPSCs) and cells at various stages of differentiation. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. In such instances, rescuing mutants by recombining in corrected gene fragments with the methods and systems described herein is a corrective option. [0245] In some embodiments, correcting mutations in exon 44 (or 51) by recombining in a corrective coding sequence downstream of exon 43 (or 50), using the methods and systems described herein is a corrective option. Proposed validation is detection of persistent DMD mRNA and protein expression in transduced cells. [0246] F8 (Factor VIII). A large proportion of severe hemophilia A patients harbor one of two types of chromosomal inversions in the FVIII gene. The recombinase technology and methods described herein are well suited to correcting such inversions (and other mutations) by recombining of the FVIII gene into a SHS. [0247] In some embodiments, correcting factor VIII deficiency by recombining the FVIII gene into an SHS is a corrective path. In some embodiments, the methods and systems described herein are used to correct factor VIII deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency. Proposed validation is detection of persistent FVIII mRNA and protein expression in transduced cells. [0248] Factor 9 (Factor IX) Hemophilia B, also called factor IX (FIX) deficiency is a genetic disorder caused by missing or defective factor IX, a clotting protein. [0249] In some embodiments, the methods and systems described herein are used to correct factor IX deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency. Proposed validation is detection of persistent FiX mRNA and protein expression in transduced cells. [0250] Ornithine transcarbamylase deficiency (OTCD). Ornithine transcarbamylase deficiency is a rare genetic condition that causes ammonia to build up in the blood. The condition – more commonly called OTC deficiency – is more common in boys than girls and tends to be more severe when symptoms emerge shortly after birth. [0251] In some embodiments, the methods and systems described herein are used to correct OTC deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the OTC deficiency or integrates a polynucleotide encoding a functional ornithine transcarbamylase enzyme. Proposed validation is detection of persistent OTC mRNA and protein expression in transduced cells. [0252] Phenylketonuria, also called PKU, is a rare inherited disorder that causes an amino acid called phenylalanine to build up in the body. PKU is caused by a change in the phenylalanine hydroxylase (PAH) gene. This gene helps create the enzyme needed to break down phenylalanine. [0253] In some embodiments, the methods and systems described herein are used to correct PKU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the PKU deficiency or integrates a polynucleotide encoding a functional phenylalanine hydroxylase (PAH) gene. Proposed validation is detection of persistent PAH mRNA and protein expression in transduced cells. [0254] Homocystinuria (HCU). Homocystinuria is elevation of the amino acid, homocysteine (protein building block coming from our diet) in the urine or blood. Common causes of HCU include: problems with the enzyme cystathionine beta synthase (CBS), which converts homocysteine to the amino acid cystathionine (which then becomes cysteine) and needs the vitamin B6 (pyridoxine); and roblems with converting homocysteine to the amino acid methionine. [0255] In some embodiments, the methods and systems described herein are used to correct HCU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the HCU or integrates a polynucleotide encoding a functional copy of a gene (e.g., CBS) able to reduce or prevent buildup of homocysteine in the urine. Proposed validation is detection of persistent CBS mRNA and protein expression in transduced cells. [0256] IgA Nephropathy (Berger’s disease). IgA nephropathy, also known as Berger's disease, is a kidney/autoimmune disease that occurs when an antibody called immunoglobulin A (IgA) builds up in the kidneys. [0257] In some embodiments, the methods and systems described herein are used to treat Berger’s disease by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of Berger’s disease. [0258] Anti-Neutrophil Cytoplasmic Antibody (ANCA) Vasculitis. ANCA vasculitis is an autoimmune disease affecting small blood vessels in the body. It is caused by autoantibodies called ANCAs, or Anti-Neutrophilic Cytoplasmic Autoantibodies. ANCAs target and attack a certain kind of white blood cells called neutrophils. [0259] In some embodiments, the methods and systems described herein are used to treat ANCA vasculitis by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of ANCA vasculitis. [0260] Systemic Lupus Erythematosus (SLE) / Lupus Nephritis (LN). Lupus is an autoimmune—a disorder in which the body’s immune system attacks the body’s own cells and organs. [0261] In some embodiments, the methods and systems described herein are used to treat SLE/LN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of SLE/LN. [0262] Membranous Nephropathy (MN). MN is a kidney disease that affects the filters (glomeruli) of the kidney and can cause protein in the urine, as well as decreased kidney function and swelling. It can sometimes be called membranous glomerulopathy as well (these terms can be used interchangeably and mean the same thing). [0263] In some embodiments, the methods and systems described herein are used to treat MN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of MN. [0264] C3 glomerulonephritis (C3GN). C3 glomerulopathy is a group of related conditions that cause the kidneys to malfunction. The major features of C3 glomerulopathy include high levels of protein in the urine (proteinuria), blood in the urine (hematuria), reduced amounts of urine, low levels of protein in the blood, and swelling in many areas of the body. Affected individuals may have particularly low levels of a protein called complement component 3 (or C3) in the blood. [0265] In some embodiments, the methods and systems described herein are used to treat C3 glomerulopathy by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of C3 glomerulopathy. 6.14. Methods of treatment [0266] In another aspect, methods of treatment are presented. The method comprises administering an effective amount of the pharmaceutical composition comprising the nucleic acid construct or vectorized nucleic acid construct described above to a patient in need thereof. [0267] In some embodiments, the method of cell delivery used here occurs using electroporation. [0268] In some embodiments, the method of cell delivery used here occurs using a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, mRNA, RNP, or lipid nanoparticle. Delivery of the nucleic acid construct can also be by fusosome or exosome, (See, e.g., WO2019222403 which is incorporated by reference herein). Delivery of nucleic acid construct can also be by VesiCas (See, e.g., US20210261957A1 which is incorporated by reference herein). [0269] DNA or RNA viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems to be used herein could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. [0270] Methods of non-viral delivery include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). 6.14.1. Lipid Nanoparticle Delivery [0271] In some embodiments, the gene editor, guide RNA (i.e. atgRNA or atgRNAs), nicking guide RNA, and/or donor polynucleotide can be packaged in a LNP and administered intravenously. In some embodiments, the gene editor, guide RNA (i.e. atgRNA or atgRNAs), nicking guide RNA, and/or donor polynucleotide can be packaged in a LNP and administered intrathecally. In some embodiments, In some embodiments, the gene editor, guide RNA (i.e. atgRNA or atgRNAs), nicking guide RNA, and/or donor polynucleotide can be packaged in a LNP and administered by intracerebral ventricular injection. In some embodiments, In some embodiments, the gene editor, guide RNA (i.e. atgRNA or atgRNAs), nicking guide RNA, and/or donor polynucleotide can be packaged in a LNP and administered by intracisternal magna administration. In some embodiments, t In some embodiments, the gene editor, guide RNA (i.e. atgRNA or atgRNAs), nicking guide RNA, and/or donor polynucleotide are packaged in a LNP and administered by intravitreal injection. [0272] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther.2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787). [0273] In another embodiment, LNP doses of about 0.01 to about 1 mg per kg of body weight administered intravenously are contemplated. Medications to reduce the risk of infusion-related reactions are contemplated, such as dexamethasone, acetampinophen, diphenhydramine or cetirizine, and ranitidine are contemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeks for five doses are also contemplated. [0274] The charge of the LNP must be taken into consideration. As cationic lipids combined with negatively charged lipids to induce nonbilayer structures that facilitate intracellular delivery. Because charged LNPs are rapidly cleared from circulation following intravenous injection, ionizable cationic lipids with pKa values below 7 were developed (see, e.g., Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, December 2011). Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Four species of ionizable cationic lipids have been focused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl- 3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). It has been shown that LNP siRNA systems containing these lipids exhibit remarkably different gene silencing properties in hepatocytes in vivo, with potencies varying according to the series DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII gene silencing model (see, e.g., Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, December 2011). A dosage of 1 µg/ml of LNP in or associated with the LNP may be contemplated, especially for a formulation containing DLinKC2-DMA. [0275] In some embodiments, the LNP composition comprises one or more one or more ionizable lipids. As used herein, the term "ionizable lipid" has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged or negatively charged. In principle, there are no specific limitations concerning the ionizable lipids of the LNP compositions disclosed herein. In some embodiments, the one or more ionizable lipids are selected from the group consisting of 3-(didodecylamino)- N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4- tridodecyl-1,4-piperazinediethanami- ne (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza- octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), 2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octad- eca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-- octadeca-9,12-dien-1- yloxy]propan-1-amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-- octadeca-9,12-dien-1-y loxy]propan-1-amine (Octyl-CLinDMA (2S)). In one embodiment, the ionizable lipid may be selected from, but not limited to, an ionizable lipid described in International Publication Nos. WO2013086354 and WO2013116126. [0276] In some embodiments, the lipid nanoparticle may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids. Such cationic and/or ionizable lipids include, but are not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2- (didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanami- ne (KL22), 14,25- ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin- MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2-({8- [(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- -octadeca-9,12-dien-1- yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3.beta.)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z- ,12Z)-octadeca-9,12-dien-1-yl oxy]propan-1-amine (Octyl-CLinDMA (2R)), (2S)-2-({8-[(3βcholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z- ,12Z)-octadeca-9,12-dien-1-yl oxy]propan-1-amine (Octyl-CLinDMA (2S)).N,N-dioleyl-N,N- dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N,N--N-triethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); 1,2-Dioleyloxy-3- trimethylaminopropane chloride salt ("DOTAP.Cl"); 3-.beta.-(N--(N',N'-dimethylaminoethane)- carbamoyl)cholesterol ("DC-Chol"), N-(1-(2,3-dioleyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethyl- -ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl carboxyspermine ("DOGS"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), N,N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and N-(1,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"). Additionally, a number of commercial preparations of cationic and/or ionizable lipids can be used, such as, e.g., LIPOFECTIN.RTM. (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE.RTM. (including DOSPA and DOPE, available from GIBCO/BRL). KL10, KL22, and KL25 are described, for example, in U.S. Pat. No.8,691,750. [0277] In some embodiments, the LNP composition comprises one or more amino lipids. The terms "amino lipid" and "cationic lipid" are used interchangeably herein to include those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group). In principle, there are no specific limitations concerning the amino lipids of the LNP compositions disclosed herein. The cationic lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and is substantially neutral at a pH above the pKa. The cationic lipids can also be termed titratable cationic lipids. In some embodiments, the one or more cationic lipids include: a protonatable tertiary amine (e.g., pH-titratable) head group; alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DOTMA, DLinDMA, DLenDMA, .gamma.-DLenDMA, DLin-K-DMA, DLin-K-C2- DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2-DMA, C12-200, cKK-E12, cKK-A12, cKK-O12, DLin-MC2- DMA (also known as MC2), and DLin-MC3-DMA (also known as MC3). [0278] Anionic lipids suitable for use in lipid nanoparticles include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids. [0279] Neutral lipids (including both uncharged and zwitterionic lipids) suitable for use in lipid nanoparticles include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, sterols (e.g., cholesterol) and cerebrosides. In some embodiments, the lipid nanoparticle comprises cholesterol. Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains and cyclic regions can be used. In some embodiments, the neutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine. In some embodiments, the neutral lipid may be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol. [0280] In some embodiments, amphipathic lipids are included in nanoparticles. Exemplary amphipathic lipids suitable for use in nanoparticles include, but are not limited to, sphingolipids, phospholipids, fatty acids, and amino lipids. [0281] The lipid composition of the pharmaceutical composition may comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. [0282] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. [0283] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. [0284] Particular amphipathic lipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. [0285] Non-natural amphipathic lipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). [0286] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. [0287] In some embodiments, the LNP composition comprises one or more phospholipids. In some embodiments, the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn- glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3- phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (ME 16:0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine1,2- didocosahexaenoyl-- sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, and any mixtures thereof. [0288] Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and beta.-acyloxyacids, may also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols. [0289] In some embodiments, the LNP composition comprises one or more helper lipids. The term "helper lipid" as used herein refers to lipids that enhance transfection (e.g., transfection of an LNP comprising an mRNA that encodes a site-directed endonuclease, such as a SpCas9 polypeptide). In principle, there are no specific limitations concerning the helper lipids of the LNP compositions disclosed herein. Without being bound to any particular theory, it is believed that the mechanism by which the helper lipid enhances transfection includes enhancing particle stability. In some embodiments, the helper lipid enhances membrane fusogenicity. Generally, the helper lipid of the LNP compositions disclosure herein can be any helper lipid known in the art. Non-limiting examples of helper lipids suitable for the compositions and methods include steroids, sterols, and alkyl resorcinols. Particularly helper lipids suitable for use in the present disclosure include, but are not limited to, saturated phosphatidylcholine (PC) such as distearoyl-PC (DSPC) and dipalymitoyl-PC (DPPC), dioleoylphosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn- glycero-3-phosphocholine (DLPC), cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In some embodiments, the helper lipid of the LNP composition includes cholesterol. [0290] In some embodiments, the LNP composition comprises one or more structural lipids. As used herein, the term "structural lipid" refers to sterols and also to lipids containing sterol moieties. Without being bound to any particular theory, it is believed that the incorporation of structural lipids into the LNPs mitigates aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. [0291] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. In some embodiments, the LNP composition disclosed herein comprise one or more polyethylene glycol (PEG) lipid. The term "PEG-lipid" refers to polyethylene glycol (PEG)-modified lipids. Such lipids are also referred to as PEGylated lipids. Non-limiting examples of PEG-lipids include PEG- modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan- 3-amines For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C.sub.14 to about C.sub.22, preferably from about C.sub.14 to about C.sub.16. In some embodiments, a PEG moiety, for example a mPEG-NH.sub.2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiment, the PEG-lipid is PEG2k-DMG. In some embodiments, the one or more PEG lipids of the LNP composition comprises PEG-DMPE. In some embodiments, the one or more PEG lipids of the LNP composition comprises PEG-DMG. [0292] In some embodiments, the ratio between the lipid components and the nucleic acid molecules of the LNP composition, e.g., the weight ratio, is sufficient for (i) formation of LNPs with desired characteristics, e.g., size, charge, and (ii) delivery of a sufficient dose of nucleic acid at a dose of the lipid component(s) that is tolerable for in vivo administration as readily ascertained by one of skill in the art. [0293] In certain embodiments, it is desirable to target a nanoparticle, e.g., a lipid nanoparticle, using a targeting moiety that is specific to a cell type and/or tissue type. In some embodiments, a nanoparticle may be targeted to a particular cell, tissue, and/or organ using a targeting moiety. In particular embodiments, a nanoparticle comprises a targeting moiety. Exemplary non-limiting targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, or F(ab')2 fragments), single domain antibodies, camelid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies)). In some embodiments, the targeting moiety may be a polypeptide. The targeting moiety may include the entire polypeptide (e.g., peptide or protein) or fragments thereof. A targeting moiety is typically positioned on the outer surface of the nanoparticle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor. A variety of different targeting moieties and methods are known and available in the art, including those described, e.g., in Sapra et al., Prog. Lipid Res.42(5):439-62, 2003 and Abra et al., J. Liposome Res.12:1-3, 2002. [0294] In some embodiments, a lipid nanoparticle (e.g., a liposome) may include a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) chains (see, e.g., Allen et al., Biochimica et Biophysica Acta 1237: 99-108, 1995; DeFrees et al., Journal of the American Chemistry Society 118: 6101-6104, 1996; Blume et al., Biochimica et Biophysica Acta 1149: 180- 184,1993; Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; U.S. Pat. No. 5,013,556; Zalipsky, Bioconjugate Chemistry 4: 296-299, 1993; Zalipsky, FEBS Letters 353: 71- 74, 1994; Zalipsky, in Stealth Liposomes Chapter 9 (Lasic and Martin, Eds) CRC Press, Boca Raton Fla., 1995). In one approach, a targeting moiety for targeting the lipid nanoparticle is linked to the polar head group of lipids forming the nanoparticle. In another approach, the targeting moiety is attached to the distal ends of the PEG chains forming the hydrophilic polymer coating (see, e.g., Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; Kirpotin et al., FEBS Letters 388: 115-118, 1996). [0295] Standard methods for coupling the targeting moiety or moieties may be used. For example, phosphatidylethanolamine, which can be activated for attachment of targeting moieties, or derivatized lipophilic compounds, such as lipid-derivatized bleomycin, can be used. Antibody- targeted liposomes can be constructed using, for instance, liposomes that incorporate protein A (see, e.g., Renneisen et al., J. Bio. Chem., 265:16337-16342, 1990 and Leonetti et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451, 1990). Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726. Examples of targeting moieties can also include other polypeptides that are specific to cellular components, including antigens associated with neoplasms or tumors. Polypeptides used as targeting moieties can be attached to the liposomes via covalent bonds (see, for example Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc.1987)). Other targeting methods include the biotin-avidin system. [0296] In some embodiments, a lipid nanoparticle includes a targeting moiety that targets the lipid nanoparticle to a cell including, but not limited to, hepatocytes, colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells (including primary tumor cells and metastatic tumor cells). In particular embodiments, the targeting moiety targets the lipid nanoparticle to a hepatocyte. [0297] The lipid nanoparticles described herein may be lipidoid-based. The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem. 201021:1448-1454; Schroeder et al., J Intern Med. 2010267:9-21; Akinc et al., Nat. Biotechnol. 200826:561-569; Love et al., Proc Natl Acad Sci USA. 2010107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA.2011108:12996-3001). [0298] The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see e.g., Akinc et al., Mol Ther.200917:872-879), use of lipidoid oligonucleotides to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited. [0299] In one aspect, effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including, but not limited to, a neutral lipid (e.g., diacylphosphatidylcholine), cholesterol, a PEGylated lipid (e.g., PEG-DMPE), and a fatty acid (e.g., an omega-3 fatty acid) may be used to optimize the formulation of the mRNA or system for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc. Exemplary lipidoids include, but are not limited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 (including variants and derivatives), DLin-MC3-DMA and analogs thereof. The use of lipidoid formulations for the localized delivery of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may also not require all of the formulation components which may be required for systemic delivery, and as such may comprise the lipidoid and the mRNA or system. [0300] According to the present disclosure, a system described herein may be formulated by mixing the mRNA or system, or individual components of the system, with the lipidoid at a set ratio prior to addition to cells. In vivo formulations may require the addition of extra ingredients to facilitate circulation throughout the body. After formation of the particle, a system or individual components of a system is added and allowed to integrate with the complex. The encapsulation efficiency is determined using a standard dye exclusion assays. [0301] In vivo delivery of systems may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy. Formulations with the different lipidoids, including, but not limited to penta[3-(1- laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010)), C12-200 (including derivatives and variants), MD1, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA and DLin-MC3-DMA can be tested for in vivo activity. The lipidoid referred to herein as "98N12-5" is disclosed by Akinc et al., Mol Ther.200917:872-879). The lipidoid referred to herein as "C12-200" is disclosed by Love et al., Proc Natl Acad Sci USA. 2010107:1864-1869 and Liu and Huang, Molecular Therapy. 2010669-670. [0302] LNPs in which a nucleic acid is entrapped within the lipid portion of the particle and is protected from degradation, can be formed by any method known in the art including, but not limited to, a continuous mixing method, a direct dilution process, and an in-line dilution process. Additional techniques and methods suitable for the preparation of the LNPs described herein include coacervation, microemulsions, supercritical fluid technologies, phase-inversion temperature (PIT) techniques. [0303] In some embodiments, the LNPs used herein are produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution a nucleic acid described herein in a first reservoir, providing an organic lipid solution in a second reservoir (wherein the lipids present in the organic lipid solution are solubilized in an organic solvent, e.g., a lower alkanol such as ethanol), and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution so as to substantially instantaneously produce a lipid vesicle (e.g., liposome) encapsulating the nucleic acid molecule within the lipid vesicle. This process and the apparatus for carrying out this process are known in the art. More information in this regard can be found in, for example, U.S. Patent Publication No.20040142025. The action of continuously introducing lipid and buffer solutions into a mixing environment, such as in a mixing chamber, causes a continuous dilution of the lipid solution with the buffer solution, thereby producing a lipid vesicle substantially instantaneously upon mixing. By mixing the aqueous solution comprising a nucleic acid molecule with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (e.g., aqueous solution) to produce a nucleic acid-lipid particle. [0304] In some embodiments, the LNPs used herein are produced via a direct dilution process that includes forming a lipid vesicle (e.g., liposome) solution and immediately and directly introducing the lipid vesicle solution into a collection vessel containing a controlled amount of dilution buffer. In some embodiments, the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution. In some embodiments, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid vesicle solution introduced thereto. [0305] In some embodiments, the LNPs are produced via an in-line dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region. In these embodiments, the lipid vesicle (e.g., liposome) solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region. These processes and the apparatuses for carrying out direct dilution and in-line dilution processes are known in the art. More information in this regard can be found in, for example, U.S. Patent Publication No. 20070042031. 6.14.2. Viral Vector Delivery [0306] In certain aspects, the disclosure includes vectors, e.g. for delivering or introducing in a cell, but also for propagating these components (e.g. in prokaryotic cells). A used herein, a “vector“ is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. In general, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double- stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors." Vectors for and that result in expression in a eukaryotic cell can be referred to herein as "eukaryotic expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. [0307] Recombinant expression vectors can comprise a nucleic acid of the disclosure in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). With regards to recombination and cloning methods, mention is made of U.S. patent application Ser. No.10/815,730, published Sep.2, 2004 as US 2004-0171156 A1, the contents of which are herein incorporated by reference in their entirety. [0308] Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, for instance a Type V protein such as C2c1 or C2c3, and/or any of the present RNAs, for instance a guide RNA, can be delivered using any suitable vector, e.g., plasmid or viral vectors, such as adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof. Effector proteins and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors. In some embodiments, the vector, e.g., plasmid or viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc. [0309] Such a dosage may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceutically-acceptable excipient, and/or other compounds known in the art. The dosage may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form. Suitable exemplary ingredients include microcrystalline cellulose, carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J.1991) which is incorporated by reference herein. [0310] In an embodiment herein the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 x 10⁵ particles (also referred to as particle units, pu) of adenoviral vector. In an embodiment herein, the dose preferably is at least about 1 x 10⁶ particles (for example, about 1 x 10⁶-1 x 10¹¹ particles), more preferably at least about 1 x 10⁷ particles, more preferably at least about 1 x 10⁸ particles (e.g., about 1 x 10⁸-1 x 10¹¹ particles or about 1 x 10⁹-1 x 10¹² particles), and most preferably at least about 1 x 10¹⁰ particles (e.g., about 1 x 10⁹-1 x 10¹⁰ particles or about 1 x 10⁹-1 x 10¹² particles), or even at least about 1 x 10¹⁰ particles (e.g., about 1 x 10¹⁰-1 x 10¹² particles) of the adenoviral vector. Alternatively, the dose comprises no more than about 1 x 10¹⁴ particles, preferably no more than about 1 x 10¹³ particles, even more preferably no more than about 1 x 10¹² particles, even more preferably no more than about 1 x 10¹¹ particles, and most preferably no more than about 1 x 10¹⁰ particles (e.g., no more than about 1 x 10⁹ particles). Thus, the dose may contain a single dose of adenoviral vector with, for example, about 1 x 10⁶ particle units (pu), about 2 x 10⁶ pu, about 4 x 10⁶ pu, about 1 x 10⁷ pu, about 2 x 10⁷ pu, about 4 x 10⁷ pu, about 1 x 10⁸ pu, about 2 x 10⁸ pu, about 4 x 10⁸ pu, about 1 x 10⁹ pu, about 2 x 10⁹ pu, about 4 x 10⁹ pu, about 1 x 10¹⁰ pu, about 2 x 10¹⁰ pu, about 4 x 10¹⁰ pu, about 1 x 10¹¹ pu, about 2 x 10¹¹ pu, about 4 x 10¹¹ pu, about 1 x 10¹² pu, about 2 x 10¹² pu, or about 4 x 10¹² pu of adenoviral vector. See, for example, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel, et. al., granted on Jun.4, 2013; incorporated by reference herein, and the dosages at col 29, lines 36-58 thereof. In an embodiment herein, the adenovirus is delivered via multiple doses. [0311] In an embodiment herein, the delivery is via an AAV. A therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 x 10¹⁰ to about 1 x 10¹⁰ functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects. In an embodiment herein, the AAV dose is generally in the range of concentrations of from about 1 x 10⁵ to 1 x 10⁵⁰ genomes AAV, from about 1 x 10⁸ to 1 x 10²⁰ genomes AAV, from about 1 x 10¹⁰ to about 1 x 10¹⁶ genomes, or about 1 x 10¹¹ to about 1 x 10¹⁶ genomes AAV. A human dosage may be about 1 x 10¹³ genomes AAV. Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col.27, lines 45-60. [0312] The promoter used to drive nucleic acid-targeting effector protein coding nucleic acid molecule expression can include: AAV ITR can serve as a promoter: this is advantageous for eliminating the need for an additional promoter element (which can take up space in the vector). The additional space freed up can be used to drive the expression of additional elements (gRNA, etc.). Also, ITR activity is relatively weaker, so can be used to reduce potential toxicity due to over expression of nucleic acid-targeting effector protein. For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc. For brain or other CNS expression, can use promoters: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For liver expression, can use Albumin promoter. For lung expression, can use SP-B. For endothelial cells, can use ICAM. For hematopoietic cells can use IFNbeta or CD45. For Osteoblasts can use OG-2. [0313] The promoter used to drive guide RNA can include: Pol III promoters such as U6 or H1 Use of Pol II promoter and intronic cassettes to express guide RNA Adeno Associated Virus (AAV) [0314] Nucleic acid-targeting effector protein and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No.8,454,972 (formulations, doses for adenovirus), U.S. Pat. No.8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For examples, for AAV, the route of administration, formulation and dose can be as in U.S. Pat. No.8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids. Doses may be based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. The viral vectors can be injected into the tissue of interest. For cell- type specific genome modification, the expression of nucleic acid-targeting effector can be driven by a cell-type specific promoter. For example, liver-specific expression might use the Albumin promoter and neuron-specific expression (e.g., for targeting CNS disorders) might use the Synapsin I promoter. [0315] In terms of in vivo delivery, AAV is advantageous over other viral vectors for a couple of reasons: Low toxicity (this may be due to the purification method not requiring ultra centrifugation of cell particles that can activate the immune response) and Low probability of causing insertional mutagenesis because it does not integrate into the host genome. [0316] AAV has a packaging limit of 4.5 or 4.75 Kb. This means that nucleic acid-targeting effector protein (such as a Type V protein such as C2c1 or C2c3) as well as a promoter and transcription terminator have to be all fit into the same viral vector. Therefore embodiments of the disclosure include utilizing homologs of nucleic acid-targeting effector protein (such as a Type V protein such as C2c1 or C2c3) that are shorter. [0317] As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. The herein promoters and vectors are preferred individually. [0318] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and psi2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference. [0319] Millington-Ward et al. (Molecular Therapy, vol.19 no.4, 642-649 April 2011) describes adeno-associated virus (AAV) vectors to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. An injection of either 6.0 x 10⁸ vp or 1.8 x 10¹⁰ vp AAV were subretinally injected into the eyes by Millington-Ward et al. The AAV vectors of Millington-Ward et al. may be applied to the system of the present dislcosure, contemplating a dose of about 2 x 10¹¹ to about 6 x 10¹¹ vp administered to a human. [0320] Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also relates to in vivo directed evolution to fashion an AAV vector that delivers wild-type versions of defective genes throughout the retina after noninjurious injection into the eyes' vitreous humor. Dalkara describes a 7 mer peptide display library and an AAV library constructed by DNA shuffling of cap genes from AAV1, 2, 4, 5, 6, 8, and 9. The rcAAV libraries and rAAV vectors expressing GFP under a CAG or Rho promoter were packaged and deoxyribonuclease-resistant genomic titers were obtained through quantitative PCR. The libraries were pooled, and two rounds of evolution were performed, each consisting of initial library diversification followed by three in vivo selection steps. In each such step, P30 rho-GFP mice were intravitreally injected with 2 ml of iodixanol-purified, phosphate- buffered saline (PBS)-dialyzed library with a genomic titer of about 1.times.10.sup.12 vg/ml. The AAV vectors of Dalkara et al. may be applied to the nucleic acid-targeting system of the present disclosure, contemplating a dose of about 1 x 10¹⁵ to about 1 x 10¹⁶ vg/ml administered to a human. [0321] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SW), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol.66:1635-1640 (1992); Sommnerfelt et al., Virol.176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol.5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989). [0322] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and yr2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference. [0323] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. Cells taken from a subject include, but are not limited to, hepatocytes or cells isolated from muscle, the CNS, eye or lung. Immunological cells are also contemplated, such as but not limited to T cells, HSCs, B-cells and NK cells. [0324] Another useful method to deliver proteins, enzymes, and guides comprises transfection of messenger RNA (mRNA). Examples of mRNA delivery methods and compositions that may be utilized in the present disclosure including, for example, PCT/US2014/028330, US8822663B2, NZ700688A, ES2740248T3, EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BR112016030852A2, and EP3362461A1. Expression of CRISPR systems in particular is described by WO2020014577. Each of these publications are incorporated herein by reference in their entireties. Additional disclosure hereby incorporated by reference can be found in Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4): 710-728. [0325] In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO- IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr-/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR- L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB- 435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI- H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. [0326] In some embodiments, one or more vectors described herein are used to produce a non- human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. In certain embodiments, the organism or subject is a plant. In certain embodiments, the organism or subject or plant is algae. Methods for producing transgenic plants and animals are known in the art, and generally begin with a method of cell transfection, such as described herein. [0327] In one aspect, the disclosure provides for methods of modifying a target polynucleotide in a prokaryotic or eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including micro-algae) and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including micro-algae). [0328] In plants, pathogens are often host-specific. For example, Fusariumn oxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato, and F. oxysporum f. dianthii Puccinia graminis f. sp. tritici attacks only wheat. Plants have existing and induced defenses to resist most pathogens. Mutations and recombination events across plant generations lead to genetic variability that gives rise to susceptibility, especially as pathogens reproduce with more frequency than plants. In plants there can be non-host resistance, e.g., the host and pathogen are incompatible. There can also be Horizontal Resistance, e.g., partial resistance against all races of a pathogen, typically controlled by many genes and Vertical Resistance, e.g., complete resistance to some races of a pathogen but not to other races, typically controlled by a few genes. In a Gene-for-Gene level, plants and pathogens evolve together, and the genetic changes in one balance changes in other. Accordingly, using Natural Variability, breeders combine most useful genes for Yield. Quality, Uniformity, Hardiness, Resistance. The sources of resistance genes include native or foreign Varieties, Heirloom Varieties, Wild Plant Relatives, and Induced Mutations, e.g., treating plant material with mutagenic agents. Using the present disclosure, plant breeders are provided with a new tool to induce mutations. Accordingly, one skilled in the art can analyze the genome of sources of resistance genes, and in Varieties having desired characteristics or traits employ the present disclosure to induce the rise of resistance genes, with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs. [0329] Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown and may be at a normal or abnormal level. 7. ADDITIONAL EMBODIMENTS [0330] Embodiment 1. A method of editing a human subject primary cell genome, the method comprising: a) incorporating an integration target recognition site into the human primary cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the integration target recognition site into the genome; (ii) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA; and b) integrating a donor polynucleotide template into the human primary cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target recognition site, wherein the donor polynucleotide is integrated into the human primary cell at the incorporated genomic integration target recognition site by an integrase; thereby producing a genetically modified human subject primary cell. [0331] Embodiment 2. A method of editing a human subject primary cell genome, the method comprising: a) incorporating an integration target recognition site into the human primary cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; (ii) one or more guide RNA complex, wherein the one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA; and b) integrating a donor polynucleotide template into the human primary cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target recognition site, wherein the donor polynucleotide is integrated into the human primary cell at the incorporated genomic integration target recognition site by an integrase; thereby producing a genetically modified human subject primary cell. [0332] Embodiment 3. The method of any one of embodiments 1-2, wherein the human subject primary cell is an autologous T cell or allogeneic T cell. [0333] Embodiment 4. The method of any one of embodiments 1-3, wherein the integration target recognition site is incorporated at one or more of the TRAC locus, CXCR4 locus, or IL2RB locus. [0334] Embodiment 5. The method of any one of embodiments 1-4, wherein delivery is performed by electroporation. [0335] Embodiment 6. The method of any one of embodiments 1-5, wherein delivery into the human primary cell occurs using one or more of a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, mRNA, RNP, or lipid nanoparticle. [0336] Embodiment 7. The method of any one of embodiments 1-6, wherein the integration target recognition site is an attB or attP site. [0337] Embodiment 8. The method of any one of embodiments 1-7, wherein the integration target recognition site is a modified attB or attP site. [0338] Embodiment 9. The method of any one of embodiments 1-8, the integration target recognition site is specific for BxB1 or a modified BxB1. [0339] Embodiment 10. The method of any one of embodiments 1-9, the integration target recognition site is comprised of 38 or 46 nucleotides. [0340] Embodiment 11. The method of any one of embodiments 1-10, wherein the one or more guide RNA or the one or more guide RNA complex comprises a chemical modification. [0341] Embodiment 12. The method of any one of embodiments 1 or 2, wherein the cellular genome integrated polynucleotide encodes for a chimeric antigen receptor or a T cell receptor. [0342] Embodiment 13. The method of embodiment 12, wherein the chimeric antigen receptor further comprises an integrase target recognition site. [0343] Embodiment 14. The method of embodiment 12, wherein the T cell receptor further comprises an integrase target recognition site. [0344] Embodiment 15. The method of any one of embodiments 1 or 2, isolating the human primary cell prior to step (a). [0345] Embodiment 16. The method of any one of embodiments 1 or 2, wherein the human primary cell is activated ex vivo or in vitro prior to step (a). [0346] Embodiment 17. The method of any one of embodiments 1 or 2, wherein the modified human primary cell is expanded ex vivo after step (b)(i) to generate an expanded modified human primary cell composition. [0347] Embodiment 18. The method of embodiment 17, wherein the expanded modified human primary cell composition is isolated. [0348] Embodiment 19. The method of embodiment 18, further comprising a subsequent step administering in an effective amount to a human subject. 8. EXAMPLES 8.1. Example 1: Primary T cell single atgRNA-mediated beacon placement within the CXCR4 and IL2RB loci [0349] To demonstrate and optimize attB beacon placement (i.e., integration target recognition site placement or incorporation) at the CXCR4 and IL2RB loci within T cells, two reference pegRNAs paired with the associated reference ngRNAs were initially selected from literature (Chen et al., 2021, Cell 184, 5635-5652, the entirety which is incorporated by reference herein) (FIG. 1). The reference CXCR4 pegRNA and the reference IL2RB pegRNA, respectively, were modified to atgRNAs by attB insertion (38 or 46 nucleotide attB site insertion) within a segment of the reverse transcriptase template (FIG.2) (Table 12). [0350] Primary human T cells were harvested from peripheral blood mononuclear cells of healthy donors. CD3⁺ T cells were purified by negative selection using immunomagnetic cell isolation procedures (Charles River Laboratories). Cells were activated with TranAct (Miltenyi) in CTS OpTmizer media (Gibco) supplemented with Immune Cell Serum Replacement (Gibco) and GlutaMax (Gibco). [0351] T cell beacon placement was verified using high and low dose of a gene editor polynucleotide (2 or 6 micrograms of PE2 encoding mRNA), atgRNA (180 or 540 picomoles), and ngRNA (120 or 360 picomoles). Human PBMC-derived T cells on Day 0 were thawed and activated followed by electroporating with low dose and high dose composition, respectively, on Day 2 of the experimental workflow (FIG.3). FIGs.4B-4C demonstrates CXCR438-base beacon (integration recognition site) insertion at the high dose condition provided ~0.3% efficiency and the IL2RB 38-base beacon (integration recognition site) insertion at the high dose condition provided ~6% efficiency (efficiency determined by total sequence read analysis). As shown in FIG. 4A, the pegRNA produced about 30% efficiency when used to create 1-2 base pair (bp) substitutions when using the high dose. Quantification of beacon placement was conducted on Day 5 (FIG.3). 8.2. Example 2: Primary T cell improved single atgRNA-mediated beacon placement within the CXCR4 and IL2RB loci [0352] To further enhance beacon placement in primary T cells, a range of single atgRNA (with associated nicking guide RNA) were investigated (FIG.5). [0353] Primary human T cells were harvested from peripheral blood mononuclear cells of healthy donors. CD3⁺ T cells were purified by negative selection using immunomagnetic cell isolation procedures (Charles River Laboratories). Cells were activated with TranAct (Miltenyi) in CTS OpTmizer media (Gibco) supplemented with Immune Cell Serum Replacement (Gibco) and GlutaMax (Gibco). [0354] Following T cell thaw and activation on Day 0 and reagent electroporation on Day 2, improved beacon placement was seen for both the CXCR4 and IL2RB loci, respectively (as quantified on Day 5). FIG.6A demonstrates the two-color droplet digital PCR (ddPCR) assay for quantifying beacon placement. FIG. 6B illustrates at least ~0.5% beacon placement within the CXCR4 locus across several conditions and a single condition (condition E in FIG. 5) demonstrated >1.2% beacon placement. FIG.6C illustrates at least ~5% beacon placement within the IL2RB locus across several conditions and two conditions (condition B and F in FIG. 5) demonstrated >10% beacon placement. This data showed increased beacon placement compared with the results in Example 1. Additionally, this data showed that the higher ratio of atgRNA to ngRNA (atgRNA:ngRNA) did not have a significant impact on percent beacon placement. [0355] FIGs. 7A-7B illustrates beacon placement across tested conditions as determined by ddPCR and amp-seq. As shown in FIG.7A, beacon placement in the CXCR4 locus validated the results of the 2C-ddPC. Similarly, beacon placement in the IL2RB locus validated the pattern of results shown in the 2C-ddPCR data although the amp-seq showed lower percent beacon placement (see FIG.7B). 8.3. Example 3: Primary T cell dual atgRNA-mediated beacon placement within the CXCR4 and IL2RB loci [0356] A dual atgRNA approach was assessed for percent beacon placement efficiency in the the CXCR4 and IL2RB loci. FIG.8 illustrates a non-limiting example of a dual atgRNA approach to beacon placement within a genome. Here, the tested paired (forward and reverse atgRNA pairs, Table 12) atgRNAs mediate either a 38 base pair or 46 base pair between Bxb1 attB site beacon placement (Table 12). The paired atgRNAs tested herein allow for a 20 base anneal overlap between the two synthesized DNA strands, wherein the annealed DNA strands are templated within the atgRNAs reverse transcription template (FIG.9). [0357] Primary human T cells were harvested from peripheral blood mononuclear cells of healthy donors. CD3⁺ T cells were purified by negative selection using immunomagnetic cell isolation procedures (Charles River Laboratories). Cells were activated with TranAct (Miltenyi) in CTS OpTmizer media (Gibco) supplemented with Immune Cell Serum Replacement (Gibco) and GlutaMax (Gibco). [0358] Following T cell thaw and activation on Day 0 and reagent electroporation on Day 2 (FIG. 10) cells were harvested, and beacon placement was quantified on Day 5. High beacon placement was seen for both the CXCR4 and IL2RB loci, respectively, using the dual atgRNA approach. Using dual atgRNAs, a maximum of 57.2% beacon placement was measured at the CXCR4 locus (FIG. 11A). Using dual atgRNAs, a maximum of 29.7% beacon placement was measured at the IL2RB locus (FIG. 11B). Assessment of percent beacon placement using Amp- seq validated the dd-PCR data (see FIGs.11A-11B). 8.4. Example 4: Primary T cell single atgRNA-mediated beacon placement within the TRAC locus [0359] To demonstrate and optimize attB beacon placement (i.e., integration target recognition site placement or incorporation) at the TRAC locus within T cells, three TRAC (“MS”, “AM”, and “TF”) targeting guide RNAs were initially selected from literature (FIGs.12A-12B) (See, Eyquem et al., Nature 543, 113-117 (2017), the entirety of which is incorporated by reference herein. See, Roth et al., Nature 559, 405-409 (20180, the entirety of which is incorporated by reference herein). In some cases, the targeting the TRAC1 using the strategy in FIG.12A enables more uniform CAR or TCR expression, it disrupts endogenous TCR signaling and can delay effector T cell differentiation and exhaustion. In addition, 4 nicking guide RNAs were designed (FIG. 12B). FIG.13 shows the workflow for optimizing the sgRNA identified above (see FIG.12). [0360] The sgRNA identified above were assessed for indel generation in the TRAC locus in CD3⁺ T cells. As shown in FIGs.14A-14B illustrates all 3 TRAC targeting guide RNAs resulted in T cell TCR protein disruption via indel generation when electroporated with a mRNA expressing SpCas9. [0361] Having identified spacer sequences that can target the TRAC locus, these spacer sequences were used in atgRNA to introduce beacons (integration recognition sites) into the gnome of the T cells at the TRAC locus (i.e., the target sequence). Beacon placement (attB; 38bp) was tested across a range of single atgRNA (wherein the spacer sequence is the same as “MS”, “AM”, and “TF” guide RNA) and nicking guide reagent conditions (FIG.15). [0362] Primary human T cells were harvested from peripheral blood mononuclear cells of healthy donors. CD3⁺ T cells were purified by negative selection using immunomagnetic cell isolation procedures (Charles River Laboratories). Cells were activated with TranAct (Miltenyi) in CTS OpTmizer media (Gibco) supplemented with Immune Cell Serum Replacement (Gibco) and GlutaMax (Gibco). [0363] Following T cell thaw and activation on Day 0 and reagent electroporation on Day 2 (FIG.15), cells were harvested, and beacon placement was quantified on Day 5. FIGs.16A-16C demonstrates AM and MS spacer use resulted in more efficient beacon placement than TF spacer use. Moreover, nicking guide 9 is more efficient than nicking guide 4 when paired with AM atgRNA or MS atgRNA (FIGs. 16A-16B). Overall, this data showed that tuning experimental conditions (as shown in FIG.15) such as PE encoding mRNA amount, atgRNA amount, ratio of atgRNA to ngRNA, can impact beacon placement nearly 2-fold. Additionally, this data showed that spacer sequence can play a role in beacon placement and that the specific combination of atgRNA and ngRNA can also play a role in beacon placement. 8.5. Example 5: Primary T cell dual atgRNA-mediated beacon placement within the TRAC locus [0364] Beacon placement (attB) at the T cell TRAC locus was tested in a dual atgRNA format. Nicking guide spacers (FIG. 13) were modified into a second atgRNA by retaining the exact spacer sequence and further including RT-template elements (attB site, PBS) of an atgRNA (forward and reverse atgRNA pairs, Table 12). [0365] Primary human T cells were harvested from peripheral blood mononuclear cells of healthy donors. CD3⁺ T cells were purified by negative selection using immunomagnetic cell isolation procedures (Charles River Laboratories). Cells were activated with TranAct (Miltenyi) in CTS OpTmizer media (Gibco) supplemented with Immune Cell Serum Replacement (Gibco) and GlutaMax (Gibco). [0366] Following T cell thaw and activation on Day 0 and reagent electroporation on Day 2 (FIG.15), cells were harvested, and beacon placement was quantified on Day 5. Beacon placement was significantly higher within the dual atgRNA context for both conditions tested (FIG.15 “E” and “F”) and across all spacers tested as compared to single atRNA use. FIG.17A shows percent beacon placement at the TRAC locus when using a first atgRNA having a AM spacer and a second atgRNA having a spacer from the ng4 nicking guide RNA. FIG. 17B shows percent beacon placement at the TRAC locus when using a first atgRNA having a MS spacer and a second atgRNA having a spacer from the ng4 nicking guide RNA. FIG.17C shows percent beacon placement at the TRAC locus when using a first atgRNA having a TF spacer and a second atgRNA having a spacer from the ng12 nicking guide RNA.As shown in FIGs. 17A-17C, across the 3 TRAC spacers/dual atgRNA tested, the corresponding atgRNAs mediated at least ~20% beacon insertion (for the 38 nucleotide attB site). As shown in FIG.18, for each of the 3 gene targets and 5 spacer pairs, the dual atgRNA format resulted in higher beacon placement efficiency compared to the single atgRNA method. 8.6. Example 6: Ex vivo PGI in primary human T cells [0367] Programmable gene insertion (PGI) was assessed in T cells at the TRAC locus. In these experiments electroporation protocols: EO-115 and EH-115 were compared. Additionally, different BxB1 formats were used at different concentrations (see, e.g., FIG.20A). [0368] Primary T cells were electroporated with a first set of components: an mRNA encoding a gene editor protein (e.g., a Cas9 nickase (nCas9) and a reverse transcriptase (RT)) and a first and second atgRNA followed by another electroporation with a second set of components: a donor polynucleotide template (in the form of a minicircle) and a mRNA encoding the BxB1 (see, e.g., FIG. 20B). In particular, as described in FIG. 20C, primary T cells were thawed at day 0, electroporated with the first set of components at day 2, electroporated a second time at day 6 with the second components, and harvested at day 9. Electroporation conditions included those as described in FIG. 20D. Conditions I and J compared EO-115/EO-115 to EO-115/HE-115. Conditions N and O were included as no BxB1 controls but were otherwise similar to conditions I and J, respectively. [0369] Cell viability was assessed after each electroporation (see FIG. 20B). As shown in FIG. 20E, cell viability was greater than 60% except in condition I following the second electroporation. [0370] As noted above, at day 9, T cells were collected and genomic DNA was harvested. As shown in FIG. 21, beacon placement was nearly 100% for condition I and around 50% for condition J. As shown in FIG.22, PGI was 12.6% for condition I and round 5% for condition J. Assessment of beacon conversion was consistent with the percent PGI for condition I but was slightly than the percent PGI for condition J (See FIG.23). [0371] Overall, this data is the first successful demonstration of programmable gene insertion in a primary T cell using a dual electroporation strategy to introduce the PGI components. 8.7. Example 7: Optimization of ex vivo PGI in primary human T cells [0372] Programmable gene insertion (PGI) was assessed in T cells at the TRAC locus to see if the results described in Example 6 could be recapitulated using different versions of BxB1 (i.e., tagged or untagged BxB1). Variants of BxB1 tested are as shown in FIG.24 and Table 13.

[0373] Similar to Example 6, primary T cells were electroporated with a first set of components: an mRNA encoding a gene editor protein (e.g., a Cas9 nickase (nCas9) and a reverse transcriptase (RT)) and a first and second atgRNA (Table 14) followed by another electroporation with a second set of components: a donor polynucleotide template (in the form of a minicircle (see Table 15)) and a mRNA encoding the various BxB1 constructs. In particular, as described in FIG. 25A, primary T cells were thawed at day 0, electroporated with the first set of components at day 2, electroporated a second time at day 6 with the second components and harvested at day 9. Electroporation conditions included those as described in FIG.25B. The first electroporation was performed with EO-115. The second electroporation included the components as described in FIG.25B.

[0374] Cell viability was assessed at day 9 prior to harvesting the cells. As shown in FIG.26, controls conditions, including EP NS 1x, BP only, BP + EH NS, and BP + NS had viability greater than 80%. In contrast, cells electroporated according to the conditions in FIG.25B as well as no BxB1 control has significantly reduced cell viability compared to the controls (EP NS 1x, BP only, BP + EH NS, and BP + NS). [0375] At day 9, T cells were harvested and genomic was collected. Assessment of beacon placement revealed around 60% beacon placement for each of the conditions tested (FIG. 27). Assessment of beacon conversion rate revealed that conversion rate was highest for cells electroporated with BxB1 #3 (see FIG.28). [0376] As shown in FIG.28, consistent with the beacon conversion data in FIG.29, PGI was highest when BxB1 #3 was used along with 3 µg of donor template polynucleotide (minicircle). PGI in the BP + NS condition was likely a technical error. [0377] Overall, this data showed ex vivo PGI in human primary T cells. 8.8. Example 8: Ex vivo PGI in primary human T cells [0378] Programmable gene insertion (PGI) was assessed in T cells at the TRAC locus using a single electroporation to introduce the nCas9-RT, a BxB1, dual atgRNAs, and template. (see, e.g., FIG.30A). [0379] As shown in FIG. 30A, primary T cells were electroporated using a single electroporation to introduce: an mRNA encoding a gene editor protein (e.g., a Cas9 nickase (nCas9) and a reverse transcriptase (RT)), a first and second atgRNA, a donor polynucleotide template (in the form of a minicircle) and a mRNA encoding the BxB1 (see, e.g., FIG.30B). In particular, as described in FIG.30C, primary T cells were thawed at day 0, electroporated with all of the PGI components at day 2, and harvested at day 6. Electroporation conditions included those as described in FIG.30D. [0380] As noted above, at day 6, T cells were collected and genomic DNA was harvested. As shown in FIG.31, beacon placement was about 50% for condition E and around 30% for condition G. As shown in FIG.32, PGI was 2% for condition E and round 1% for condition G. Assessment of beacon conversion showed about 4% for condition E and about 3.5% for condition G (See FIG. 33). [0381] Overall, this data is the first successful demonstration of programmable gene insertion in a primary T cell using a single electroporation to introduce the PGI components. [0382] EQUIVALENTS AND INCORPORATION BY REFERENCE [0383] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. [0384] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it is understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS: 1. A method of generating a primary cell comprising an integration recognition site, the method comprising: (a) site-specifically incorporating at least a first integration recognition site into a target sequence in the genome of the primary cell.

2. The method of claim 1, wherein site-specifically incorporating the at least first integration recognition site is affected by introducing into the primary cell: (i) a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at the target sequence; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor polypeptide to nick the primary cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site-specifically incorporated into the genome of the primary cell.

3. The method of claim 2, wherein the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.

4. The method of claim 2, wherein the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is an atgRNA that further includes an RT template that comprises at least a portion of the first integration recognition site, wherein the atgRNA encodes the entirety of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a nicking gRNA.

5. The method of any one of claims 1-4, further comprising incorporating a plurality of integration recognition sites.

6. The method of any one of claims 1-5, further comprising: (b) integrating at least a first donor polynucleotide template into the primary cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal/cognate integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the primary cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a primary cell with a site-specifically integrated donor polynucleotide template.

7. The method of any one of claims 1-6, wherein steps (a) and (b) are performed concurrently.

8. The method of any one of claims 1-6, wherein step (a) is performed prior to step (b).

9. The method of any one of claims 1-8, wherein step (a) and step (b) are performed at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks apart.

10. A method of generating an edited primary cell, the method comprising: (a) integrating, into the genome of the primary cell of any one of claims 1-9 at the first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal/cognate integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the primary cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a primary cell with a site-specifically integrated donor polynucleotide template.

11. A method of site specifically integrating a donor polynucleotide template into a primary cell genome, the method comprising: incorporating an integration recognition site into the primary cell genome by delivering into the cell: a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at a target sequence; an at least a first attachment site-containing guide RNA (atgRNA), wherein the one or more guide RNA encodes all or a portion of an at least first integration recognition site; and integrating a donor polynucleotide template into the human primary cell genome at the incorporated recognition site by delivering into the cell: the donor polynucleotide template, an integration enzyme or a polynucleotide encoding an integration enzyme, wherein the donor polynucleotide template is linked to a sequence that is an integration cognate of the integration recognition site present in the atgRNA, and wherein the donor polynucleotide template is integrated into the genome at the incorporated genomic integration recognition site by the integration enzyme; thereby producing a primary cell having the donor polynucleotide template site specifically integrated into its genome.

12. The method of claim 11, wherein the gene editor polypeptide or the polynucleotide encoding a gene editor polypeptide, the at least first atgRNA, the donor polynucleotide template, and the integration enzyme or the polynucleotide encoding an integration enzyme are concurrently delivered.

13. The method of claim 11, wherein the gene editor protein or the polynucleotide encoding a gene editor protein, and the at least first atgRNA, are delivered at a first time point and the donor polynucleotide template, and the integration enzyme or the polynucleotide encoding an integration enzyme are delivered at a second time point.

14. The method of claim 13, wherein the first time point and the second time point are at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks apart.

15. The method of any one of claims 2-14, wherein the gene editor polypeptide is configured such that the nickase is linked to the reverse transcriptase.

16. The method of claim 15, wherein the nickase is linked to the reverse transcriptase by in- frame fusion.

17. The method of claim 16, wherein the nickase is linked to the reverse transcriptase by a linker.

18. The method of claim 17, wherein the linker is a peptide fused in-frame between the nickase and reverse transcriptase.

19. The method of any one of claims 2-18, wherein the gene editor polynucleotide further comprises a polynucleotide sequence encoding at least an integration enzyme or the gene editor polypeptide further comprises an integration enzyme.

20. The method of claim 19, wherein the linked nickase-reverse transcriptase are further linked to the integration enzyme.

21. The method of any one of claims 1-20, wherein an at least first atgRNA comprises: (i) a domain that is capable of guiding the prime editor system to the target sequence; and (ii) a reverse transcriptase (RT) template that comprises at least a portion of an at least first integration recognition site.

22. The method of claim 21, wherein the RT template comprises the entirety of the first integration recognition site.

23. The method of any one of claims 1-22, wherein site-specifically incorporating an integration recognition site into the primary cell genome further comprises delivering into the cell a second atgRNA.

24. The method of any one of claims 21-23, wherein the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, wherein the at least first pair of atgRNAs have domains that are capable of guiding the prime editor system to a target sequence; the first atgRNA further includes a first RT template that comprises at least a portion of an at least first integration recognition site; the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.

25. The method of claim 24, wherein the first RT template encodes a first single-stranded DNA sequence and the second RT template encodes a second single-stranded DNA sequence.

26. The method of claim 24 or 25, wherein the first single-stranded DNA sequence comprises a complementary region with the first single-stranded DNA sequence.

27. The method of any one of claims 24-26, wherein the first single-stranded DNA sequence and the first single-stranded DNA sequence form a duplex.

28. The method of any one of claims 26 or 27, wherein the complementary region is 5 or more consecutive bases.

29. The method of any one of claims 26 or 27, wherein the complementary region is 10 or more consecutive bases.

30. The method of any one of claims 26 or 27, wherein the complementary region is 20 or more consecutive bases.

31. The method of any one of claims 26 or 27, wherein the complementary region is 30 or more consecutive bases.

32. The method of any one of claims 2-31, wherein at least one of the paired guide RNAs has a chemical modification.

33. The method of any one of claims 2-32, wherein the paired guide RNAs each have a chemical modification.

34. The method of any one of claims 1-33, wherein incorporating an integration recognition site into the human primary cell genome further comprises delivering into the cell a nicking guide RNA (gRNA).

35. The method of any one of claims 1-34, wherein the first integration recognition site is an attB or attP site.

36. The method of any one of claims 1-34, wherein the integration recognition site is a modified attB or attP site.

37. The method of any one of claims 1-36, wherein the integration recognition site is specific for BxB1 or a modified BxB1.

38. The method of any one of claims 1-37, wherein the integration recognition site is comprised of 38 or 46 nucleotides.

39. The method of any one of claims 6-38, wherein the donor polynucleotide template is a minicircle.

40. The method of any one of claims 1-39, wherein the primary cell is a human primary cell.

41. The method of any one of claim 1-40, wherein the primary cell is a T cell.

42. The method of claim 41, wherein the T cell is a CD4⁺ T cell, a CD8⁺ T cell, or a combination thereof.

43. The method of any one of claims 41-42, wherein the T cell is an autologous T cell or allogeneic T cell.

44. The method of any one of claims 1-43, wherein the target sequence is located in the TRAC locus, the CXCR4 locus, or the IL2RB locus, whereby the integration recognition site is incorporated into the TRAC locus, the CXCR4 locus, or the IL2RB locus.

45. The method of any one of claims 1-44, wherein the introducing is performed by electroporation.

46. The method of any one of claims 1-44, wherein the introducing is achieved using one or more of a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, mRNA, RNP, or lipid nanoparticle (LNP), or a combination thereof.

47. The method of any one of claims 6-46, wherein the donor polynucleotide template encodes for a chimeric antigen receptor or a T cell receptor.

48. The method of any one of claims 1-47, further comprising isolating the primary cell prior to step (a).

49. The method of any one of claims 1-48, wherein the primary cell is activated ex vivo or in vitro prior to step (a).

50. The method of any one of claims 1-49, wherein the edited primary cell is expanded to generate an expanded edited primary cell composition.

51. The method of claim 50, wherein the expanded primary cell composition is isolated.

52. A primary cell generated by the method of any one of claims 1-51.

53. A population of primary cells generated by the method of any one of claims 1-51.

54. A method of treating a blood disease or disorder in a subject in need thereof, the method comprising: administering to the subject the cell of claim 52 or the population of cells of claim 53.

55. A primary cell, comprising: an at least first integration recognition site site-specifically incorporated into the primary cell genome.

56. The primary cell of claim 55, wherein the at least first integration recognition site is incorporated into the primary cell genome at the TRAC locus, the CXCR4 locus, or the IL2RB locus.

57. The primary cell of claim 55 or 56, wherein the at least first integration recognition site is specific for a serine integrase.

58. The primary cell of any one of claims 55-57, wherein the at least first integration recognition site is an attB or attP site.

59. The primary cell of any one of claims 55-58, wherein the at least first integration recognition site is a modified attB or attP site.

60. The primary cell of any one of claims 55-59, wherein the at least first integration recognition site is specific for BxB1 or a modified BxB1.

61. The primary cell of any one of claims 55-60, wherein the at least first integration recognition site is comprised of 38 or 46 nucleotides.

62. A primary cell comprising: a donor polynucleotide template integrated into the TRAC locus, the CXCR4 locus, or the IL2RB locus, wherein the donor polynucleotide comprises a residual integration recognition site.

63. The primary cell of claim 62, wherein the at least first integration recognition site is incorporated into the primary cell genome at the TRAC locus, the CXCR4 locus, or the IL2RB locus.

64. The primary cell of claim 62 or 63, wherein the donor polynucleotide template is a minicircle.

65. The primary cell of any one of claims 62-64, wherein the donor polynucleotide template encodes a chimeric antigen receptor or a T cell receptor.

66. A system for site-specifically integrating a donor polynucleotide template into a primary cell genome, the system comprising: a first attachment site-containing guide RNA comprising a sequence selected from Tables 12 or 14; a second attachment site-containing guide RNA comprising a sequence selected from Tables 12 or 14, wherein the second atgRNA comprises a different spacer sequence from the first atgRNA; and a gene editor polypeptide or a polynucleotide encoding a gene editor polypeptide, wherein the gene editor polypeptide comprises a DNA binding nickase domain linked to a reverse transcriptase domain and is capable of incorporating the integration recognition site into the genome of the primary cell at a target sequence.

67. The system of claim 66, further comprising: a donor polynucleotide template; and an integration enzyme or a polynucleotide encoding an integration enzyme, wherein the donor polynucleotide template is linked to a sequence that is an integration cognate of the integration recognition site present in the atgRNA, and wherein the donor polynucleotide template is integrated into the genome at the incorporated genomic integration recognition site by the integration enzyme.

68. An attachment site-containing guide RNA (atgRNA) comprising: a sequence selected from Tables 12 or 14.

69. A pair of attachment site-containing guide RNA (atgRNA) comprising: a first attachment site-containing guide RNA comprising a sequence selected from Tables 12 or 14; and a second attachment site-containing guide RNA comprising a sequence selected from Tables 12 or 14, wherein the second atgRNA comprises a different spacer sequence from the first atgRNA.