Compositions and methods for genomic knock-in and epigenetic editing

Abstract

Provided herein are, inter alia, methods, compositions, and systems for engineering a immune cell (i.e., the generating an engineered antigen-specific T cell). The methods, compositions, and systems for engineering an immune cell provided herein are, inter alia, useful for treating cancer in a subject.

Description

PATENT Attorney Docket No. 048536-774001WOCOMPOSITIONS AND METHODS FOR GENOMIC KNOCK-IN AND EPIGENETIC EDITING RELATED APPLICATION DATA

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of the U.S. Patent Application Nos. 63 / 746,871, filed on January 17, 2025, and 63 / 785,544, filed on April 8, 2025, each of which is hereby incorporated by reference in its entirety and for all purposes.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under HR0011-19-2-0007 awarded by the Defense Advanced Research Projects Agency, and CA239597, and K08 CA273529 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The material in the accompanying Sequence Listing is hereby incorporated by reference in its entirety. The accompanying file, named “048536-774001WO_SL_ST26.xml” was created on January 16, 2026, and is 267,783 bytes in size.BACKGROUND

[0004] Engineered cellular therapies are providing revolutionary treatments for an increasing range of human diseases. Epigenetic reprogramming of target gene expression is an emerging approach to engineer therapeutic cellular phenotypes while sidestepping potential toxicity risks of genetic editing. Despite the opportunity, we lack efficient and readily programmable tools that can introduce targeted, durable, and multiplexed epigenetic changes into therapeutically -relevant primary human cell types. The methods and compositions provided herein, inter alia, address these and other problems in the art.BRIEF SUMMARY

[0005] In an aspect is provided a polynucleotide including the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 2.

[0006] In an aspect is provided a polynucleotide including the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136.PATENT Attorney Docket No. 048536-774001WO

[0007] In another aspect is provided a vector including the polynucleotide provided herein including embodiments thereof.

[0008] In another aspect is provided a cell including the polynucleotide provided herein including embodiments thereof or the vector provided herein including embodiments thereof.

[0009] In another aspect is provided a T cell gene editing system, the system including: (i) a first polynucleotide encoding a fusion protein, wherein the fusion protein includes a nuclease-deficient RNA-guided DNA endonuclease domain, a Kruppel associated box domain, and a DNA methyltransferase domain, wherein the first polynucleotide includes the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2; (ii) a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guided DNA endonuclease domain; (iii) a ribonucleoprotein including an RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; and (iv) a third polynucleotide encoding a transgene.

[0010] In another aspect is provided a T cell gene editing system, the system including: (i) a first polynucleotide encoding a fusion protein, wherein the fusion protein includes a nuclease-deficient RNA-guided DNA endonuclease domain, a Kruppel associated box domain, a DNA methyltransferase domain, a first XTEN linker, and a second XTEN linker, wherein the first polynucleotide includes the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2; (ii) a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guided DNA endonuclease domain; (iii) a ribonucleoprotein including an RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; and (iv) a third polynucleotide encoding a transgene.

[0011] In another aspect is provided a T cell gene editing system, the system including: (i) a first polynucleotide encoding a fusion protein, wherein the fusion protein includes a nuclease-deficient RNA-guided DNA endonuclease domain, a Kruppel associated box domain, and a DNA methyltransferase domain; (ii) a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guided DNA endonuclease domain; (iii) a ribonucleoprotein including an orthogonal RNA-guided DNAPATENT Attorney Docket No. 048536-774001WOendonuclease enzyme domain bound to a second sgRNA; and (iv) a third polynucleotide encoding a transgene.

[0012] In another aspect is provided a method of generating an engineered antigen-specific T cell, the method including: (i) delivering a first polynucleotide to a T cell, wherein the first polynucleotide encodes a fusion protein including a nuclease-deficient RNA-guided DNA endonuclease domain, a Kruppel associated box domain, and a DNA methyltransferase domain; (ii) delivering a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guided DNA endonuclease domain; (iii) delivering a ribonucleoprotein to the T cell, wherein the ribonucleoprotein includes an orthogonal RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; (iv) delivering a third polynucleotide to the T cell, wherein the third polynucleotide encodes a transgene; and (v) allowing the T cell to express the first polynucleotide, the ribonucleoprotein, and the third polynucleotide, thereby generating an engineered antigen-specific T cell.

[0013] In another aspect is provided a method of treating a cancer in a subject in need thereof, the method including administering a therapeutically effective amount of an engineered antigenspecific T cell generated according to the method provided herein including embodiments thereof to the subject in need thereof, thereby treating the cancer.

[0014] In another aspect is provided a cell including a first polynucleotide, a first single guide RNA (sgRNA), a ribonucleoprotein, and a second polynucleotide, wherein the first polynucleotide encodes a fusion protein including a nuclease-deficient RNA-guided DNA endonuclease domain, a Kruppel associated box domain, and a DNA methyltransferase domain; wherein the first sgRNA binds the nuclease deficient RNA-guided DNA endonuclease domain; wherein the ribonucleoprotein includes an orthogonal RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; and wherein the second polynucleotide encodes a transgene.

[0015] In another aspect is provided a T cell gene editing system, the system including: (i) a first polynucleotide encoding a fusion protein, wherein the fusion protein includes from N-terminus to C-terminus a DNA demethylation domain, an XTEN linker, and a nuclease-deficient RNA-guided DNA endonuclease domain; and (ii) a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guidedPATENT Attorney Docket No. 048536-774001WODNA endonuclease domain, wherein the first sgRNA hybridizes to a first target nucleic acid sequence in a forkhead box P3 (FOXP3) gene.

[0016] In another aspect is provided a method of increasing expression of a forkhead box P3 (FOXP3) gene in a T cell, the method includes transfecting the T cell with the T cell gene editing system provided herein including embodiments thereof, thereby demethylating a portion of the FOXP3 gene within the T cell and increasing expression of the FOXP3 gene relative to the absence of the T cell gene editing system.

[0017] In another aspect is provided a cell including the T cell gene editing system provided herein including embodiments thereofBRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGS. 1A-1L show specific and durable transcriptional silencing by CRISPRoff in primary human T cells. FIG. 1A: Comparison of knockdown (KD) efficiency of CD151 between seven CRISPRoff mRNA designs over a series of mRNA doses. Fold change in CD151 KD is shown relative to the unoptimized mRNA design (CRISPRoff 1) at 4 doses of mRNA. CD 151 positivity was modeled as a function of dose and mRNA variant and then computed a P value for the difference between CRISPRoff? and the rest of the mRNA variants using the standard error. P values were then adjusted using the Benjamini -Hochberg procedure. CRISPRoff? was the most potent CRISPRoff mRNA variant as assessed by the degree of CD151 silencing across CRISPRoff doses («=2 donors; CRISPRoffl, *** =0.00022; CRISPRoff2. **F=0.011;CRISPRoff3, **^=0.0096; CRISPRofM, **P=0.001; CRISPRoff5, ** =0.0044; CRISPRoff6, *P=0.04). FIG. IB: Comparison of Cas9, CRISPRi, or CRISPRoff mRNA knockout (KO) or silencing (knockdown; KD) activity on CD151, CD55, CD81 loci over a time course of 28 days post-electroporation. Black arrows along the x-axis represent restimulations with anti-CD2 / CD3 / CD28 soluble antibodies (day 9, day 18, and day 27 after electroporation)(n=4 donors except on day 10, where n=2 donors; mean ±s.d.). FIG. 1C: Representative flow cytometry histogram plots of CD151 KD (or KO) by CRISPRoff, CRISPRi, or Cas9 at day 5 and day 28 post-electroporation. FIGS. 1D-1E: Transcriptomic assessment by RNA-seq of CRISPRoff activity and specificity upon silencing of CD55 (FIG. ID) or CD81 (FIG. IE) relative to anon-targeting sgRNA (NTC). Cells were electroporated with CRISPRoff mRNA and an sgRNAPATENT Attorney Docket No. 048536-774001WOtargeting CD55 or CD81 or NTC. Cells were harvested 28 days post-electroporation for RNA extraction. Data are representative of two healthy donors. FIG. IF: Comparison of CpG methylation analyzed by WGBS within a 20 kb window centered on the CD55 TSS. CpG islands and the sgRNA targeting site are annotated. Tracks represent samples electroporated with CRISPRoff mRNA and an sgRNA targeting the CD55 TSS or NTC for two independent donors (DI, D2) in replicate. Cells were collected 30 days post-electroporation. FIG. 1G: A Manhattan plot displaying differentially methylated regions (DMRs) between cells treated with CRISPRoff and an sgRNA targeting CD55 or NTC and analyzed by WGBS (cells were collected 30 days post-electroporation). The arrow denotes the genomic position of CD55 (n = 2 donors). FIG. 1H: A graph showing the number of live T cells following cell editing by CRISPRoff and Cas9 when targeting either three, four, or five genes simultaneously compared to an empty electroporation control (EE). Live cell counts were measured five days after electroporation. Data are representative of two healthy donors. FIG. II: A plot comparing CRISPRoff versus Cas9 multiplexed gene silencing efficiency targeting three genes (CD151, CD81, CD44). four genes (CD151. CD81, CD44, CD55) or five genes (CD151. CD81, (41)44. CD55. CD46) at day 5 or day 30 post-electroporation. Cells were analyzed using flow cytometry and the fraction of cells with all genes silenced was calculated. FIG. 1J: A representative flow plot of cells targeted for triple gene silencing (CD151, CD81, CD44) silencing (top) or NTC (bottom). Cells were analyzed 30 days post-electroporation. In the top plot, cells were first gated on CD44-silenced cells, and the represented population shows CD81 and CD151 silencing. FIG. lK:Day 27 transcript levels of FAS, PTPN2, RC3H1 (Roquin 1), and SUV39H1 relative to NTC as measured by RT-qPCR (n=3 donors). FIG. IL: Transcriptomic assessment by RNA-seq of CRISPRoff activity upon silencing of FAS or an NTC and then harvested at 7 days after electroporation for RNA extraction. The labeled dot indicates the target gene (e.g., FAS), which is significantly downregulated, and other gray dots have no significance (empirical Bayes moderated statistics with Benjamini-Hochberg FDR control, adjusted P<0.05 FAS adjusted P=3.35 x IO’20; n=4 donors).

[0019] FIGS. 2A-2F show an integrated approach for simultaneous epigenetic and genetic engineering of CAR-T cells. FIG. 2A: Schematic of a method for simultaneously generating epigenetically and genetically engineered CAR-T cells with either Casl2a or Cas9 RNPs forPATENT Attorney Docket No. 048536-774001WOCAR knock-in (KI). FIG. 2B: A graph comparing CD19-specific CAR KI efficiency with no mRNA present or CRISPRoff mRNA used in combination with a NTC or sgRNA targeting RASA2. CRISPRoff was electroporated with either one sgRNA or a pool of three sgRNAs targeting the RASA2 TSS. Conditions noted as 0 sgRNA indicates a NTC control. FIG.2C: Western blot comparison of RASA2 silencing with CRISPRoff with or without a CD19-specific TRAC-CAR. CRISPRoff was co-electroporated with either a NTC, a single sgRNa targeting RASA2, or a pool of three sgRNAs targeting RA A 2. FIG. 2D: Graphs show CAR-T cell cytotoxicity by Incucyte analysis after five repetitive stimulations with target cancer cells. Gray lines indicate / .4,S42-epi-edited CAR-T cells, and black lines are control-edited CAR-T cells. The shaded areas depict the 95% confidence interval for replicates (N=3). Each row represents an E:T ratio (top: 1:1, bottom: 2: 1). FIG. 2E: Western blot images show RASA2 expression in CAR T cells that were treated with either one sgRNA or a pool of three sgRNAs targeting RASA2, which were isolated after the fifth repetitive stimulation. FIG. 2F: CD19+A375 melanoma cells were engrafted into NSG mice via flank injection. Casl2a compatible TRAC CD19-CAR T cells were generated in combination with CRISPRoff mRNA and a pool of three sgRNAs targeting CD151 or a NTC and then transferred into mice via the tail vein. 14 days after CAR T cell injection, the tumor and spleen were harvested from mice, and CD151 expression among CD45+human T cells was assessed via flow cytometry' (tumor: red; spleen: black).

[0020] FIGS. 3A-3I show optimization of CRISPRoff mRNA for durable silencing in primary human T cells. FIG. 3A: Schematic of seven CRISPRoff mRNA variants tested in primary human T cells. FIG. 3B: Comparison of CD151 KD efficiency four days post-electroporation between CRISPRoff mRNAs that incorporated base modifications (1-Me ps-UTP) versus standard UTP at four doses of mRNA. FIG.3C: Comparison of CD151 KD efficiency four days post-electroporation between codon optimization strategies for dCas9 (Design 1 versus Design 2) at four doses of mRNA. Quantification of CD151 KD is shown as fold change relative to the unoptimized mRNA design (CRISPRoff 1). FIG. 3D: Comparison of CD151 KD efficiency four days post-electroporation between mRNA cap structures (ARCA, m7G, Capl). Quantification of CD151 KD is shown as fold change relative to the unoptimized mRNA design (CRISPRoff 1).FIG. 3E: Quantification of CD151 silencing four days post-electroporation with a dose titration of each CRISPRoff mRNA and a pool of three sgRNAs targeting the CD 151 TSS. Plots showPATENT Attorney Docket No. 048536-774001WOthe mean and individual values from two healthy donors. FIG. 3F: Live cell counts collected at four days post-electroporation for each of the CRISPRoff mRNA variants. FIG. 3G: Four individual Lonza pulse codes (DS 137, EH100, EH115, and EO115) were tested for mRNA electroporation of either Cas9, CRISPRi, or CRISPRoff mRNA at four separate time points, either before or after initial activation with anti-CD2 / CD3 / CD28 Dynabeads (Day 0 indicates pre-activated cells, 2 days post-activation, 5 days post-activation, or 12 days post-activation). CD1 1 protein expression was quantified at 5 days after each electroporation. FIG. 3H:Comparison of CD1 1 KD efficiency at 4 days post-electroporation between codon optimization strategies for dCas9 (Design 1 versus Design 2) at four doses of mRNA. Each dot represents an mRNA variant across two donors (n=2 donors, mean±s.e.m; two-sided Welch’s t-test). FIG. 31: Comparison of CD151 KD efficiency at 4 days post-electroporation between mRNA cap structures (ARCA, m7G, Capl). Quantification of CD151 KD is depicted as fold change relative to the unoptimized mRNA design (CRISPRoffl). Each dot represents an mRNA variant across two donors (n=2 donors, mean±s.e.m.;*L><0.05 and **P<0.01, two-sided Welch’s / -test).

[0021] FIGS. 4A-4B show CRISPRoff silencing at genes without promoter CpG island annotations. FIG. 4A: The percentage of CD8+(top) or CD4+(bottom) T cells expressing PD1 as measured by flow-cytometry over a 30-day time course. For CRISPRoff conditions, cells were electroporated with CRISPRoff and either one sgRNA or pool of three sgRNAs targeting PD1. For Cas9 conditions, cells were electroporated with Cas9 mRNA and an sgRNA targeting PD1 as a comparison (black). Cells were restimulated with anti-CD2 / CD3 / CD28 soluble antibodies before each timepoint. NTC are shown as dotted lines with triangles, and targeting sgRNAs are shown as solid lines with circles. FIG. 4B: A graph showing the percentage of T cells expressing CD45 as measured by flow cytometry over a time course of 24 days. Cells were treated with CRISPRoff, CRISPRi, or Cas9 (black) mRNA. NTC are shown as dotted lines with triangles, and targeting sgRNAs are shown as solid lines with circles.

[0022] FIGS. 5A-5G show' durable multiplexed gene silencing with CRISPRoff. FIG.5A: Viability of cells, related to panel C, electroporated with CRISPRoff at tw o doses of mRNA (low7= 1.5 pg, high = 3 pg). Cells were co-electroporated with either one sgRNA targeting a single gene (sgRNA dose = 1.6 pg), three genes simultaneously (4.8 pg sgRNA), four genesPATENT Attorney Docket No. 048536-774001WOsimultaneously (6.4 pg of sgRNA) or five genes simultaneously (8 pg sgRNA). For multiplexed conditions, each gene target received one sgRNA to the TSS. Live cell counts were collected at day 5 post-electroporation, and cell counts were normalized and displayed as fold-change relative to an empty electroporation condition. FIG. 5B: Graphs showing CRISPRoff silencing of individual genes (CD151, CD44. CD46. CD55, or CD81) over a course of 30 days postelectroporation of CRISPRoff mRNA and the indicated sgRNA across 10 healthy donors. Cells were re-stimulated every nine days after initial activation with anti-CD2 / CD3 / CD28 soluble antibodies. Black arrows indicate restimulation timepoints. NTC are shown as dotted lines, and targeting sgRNAs are show n as solid lines. FIG. 5C: Comparison of three, four, or five multiplex gene silencing with CRISPRoff at either a high or low dose of mRNA. Flow cvtometrv data was collected at day 5 post-electroporation and day 30 post-electroporation and the fraction of cells with all genes silenced for a given multiplex combination w as calculated (see methods).FIG. 5D: Multiplex analysis of triple gene CRISPRoff silencing when targeting three distinct combinations of genes (CD151, CD81, CD44), (CD151, CD81, CD46). CD151, CD81, CD55) over a time course of 30 days post-electroporation with CRISPRoff mRNA and the indicated sgRNAs. NTC are shown as dotted lines, and targeting sgRNAs are shown as solid lines. Cells were restimulated every7nine days post initial activation, indicated by black arrows. FIGS.5E-5F: Multiplex analysis of either four-gene (FIG. 5E) or five-gene (FIG. 5F) CRISPRoff gene silencing over a time course of 30 days post-electroporation with CRISPRoff mRNA and the indicated sgRNAs. NTC are shown as dotted lines, and targeting sgRNAs are shown as solid lines. Cells were restimulated every 9 days after initial activation, indicated by black arrows. FIG. 5G: Viability of cells that were either not electroporated (No E) or empty electroporated (EE). Empty electroporated cells were treated with P3 buffer only. (n=4 donors per condition, mean ±s.d.; two-sided Welch’s / -test).

[0023] FIGS. 6A-6G show limitations of S. pyogenes Cas9 for simultaneous KD and CAR KI.FIG. 6A: A graph depicting translocation frequencies between R SA 2 and TRAC loci, measured by ddPCR, following transfection of cells with Cas9 RNPs targeting TRAC alone or along with CRISPRoff mRNA and the indicated full length sgRNAs targeting RA A 2. truncated sgRNAs targeting RASA2. or NTC. FIG. 6B: Western blot images show RASA2 KO with Cas9 mRNA, or CRISPRoff RASA2 KD with either full length sgRNAs (as one sgRNA or pool of three) orPATENT Attorney Docket No. 048536-774001WOtruncated sgRNAs (as one sgRNA or a pool of three). Cells were harvested for protein lysates on day 7 post-electroporation. FIG.6C: Schematic of the experiment to examine the functional efficacy of / 4,S'42-epi -edited CAR-T cells. FIG. 6D: Cytotoxicity as measured by Incucyte analysis after five repetitive stimulations with target cells. Each row represents an E:T ratio (top: 1:1. bottom: 2: 1). FIG. 6E: A graph comparing BCMA-specific CAR KI efficiency with Cas9 mRNA or CRISPRoff mRNA used in combination with a NTC or sgRNA targeting PAS 2. CRISPRoff was electroporated with either one sgRNA or a pool of three sgRNAs targeting the R4SA2 TSS. sgRNAs were either delivered as full length or truncated (Mean ± s.d, n = 6 donors). FIGS. 6F-6G: T cell immunophenotypes on day 7 based on CD45RA and CD62L expression, measured by flow cytometry. Data is representative of 1 donor.

[0024] FIGS. 7A-7L show an integrated approach for simultaneous epigenetic and genetic engineering of CAR-T cells. FIG. 7A: Schematic of a method for simultaneously generating epigenetically and genetically engineered CAR-T cells with either Casl2a or Cas9 RNPs for CAR knock-in (KI). FIG. 7B: A graph comparing CD19-specific CAR KI efficiency with no mRNA present or CRISPRoff mRNA used in combination with a NTC or sgRNA targeting RASA2. CRISPRoff was electroporated with either one sgRNA or a pool of three sgRNAs targeting the 4SA2 TSS. Conditions noted as 0 sgRNA indicates a NTC control. FIG. 7C: Western blot comparison of J 4SA2 silencing with CRISPRoff with or without a CD19-specific 7'AW-CAR. CRISPRoff was co-electroporated with either a NTC, a single sgRNa targeting PA A 2. or a pool of three sgRNAs targeting PASA2. FIG. 7D: Day 7 transcript levels of RASA2 normalized to GAPDH relative to NTC as measured by RT-qPCR for 3 independent donors. FIGS. 7E-7F: T cell immunophenotypes on day 7 based on CD45RA and CD62L expression, measured by flow cytometry. FIG. 7G: Graphs show CAR-T cell cytotoxicity by Incucyte analysis after five repetitive stimulations with target cancer cells. Dark gray lines indicate / S'42-epi-edited CAR-T cells, and light gray lines are control-edited CAR-T cells. The shaded areas depict the 95% confidence interval for technical replicates (N=3) across three independent donors. Each row represents an E:T ratio (top: 1:2, middle 1:1, bottom: 2:1). FIG.7H: Western blot images show RASA2 expression in CAR T cells that were treated with either one sgRNA or a pool of three sgRNAs targeting RASA2, which were isolated after the fifth repetitive stimulation. FIG. 71: NSG mice were injected with 0.5x106 Nalm6 cells, followed byPATENT Attorney Docket No. 048536-774001WO1x105 RASA2 epi-silenced CD19-specific CAR T cells or CD19-specific CAR T cells treated with aNTC. Tumor growth was monitored by bioluminescence imaging (n = 23 mice per group; Mann-Whitney U test, p-value = 0.0004). FIG. 7J: Survival of RASA2 epi-silenced CD19 CAR T cells show n in FIG. 71 Survival curves were compared with log-rank test. FIG. 7K:Representative images for the mice shown in FIG. 71. FIGS. 7L-7M: T cell immunophenotypes on day 7 based on CD45RA and CD62L expression, measured by flow cytometry. Data is representative of 1 donor. FIG. 7N: Schematic of the repetitive-stimulation assay to examine the functional efficacy of t4N42-epi-silenced CAR-T cells.

[0025] FIGS. 8A-8B show specific and durable transcriptional silencing by CRISPRoff in primary T cells. FIG. 8A: Day 27 transcript levels of FAS, PTPN2, RC3FU (Roquin-1), and SUV39H1 relative to NTC as measured by RT-qPCR (n = 3 donors). FIG. 8B: Transcriptomic assessment by RNA-seq of CRISPRoff activity upon silencing of FAS relative to NTC. Cells were electroporated with CRISPRoff mRNA and an sgRNA targeting FAS or aNTC and then harvested 27 days post-electroporation for RNA extraction. Data are representative of two independent donors.

[0026] FIGS. 9A-9K show CRISPRoff silencing at genes that lack CGI annotations. FIG. 9A:Comparison of Cas9 KO or CRISPRoff KD activity with either a single guide or a pool of three guides at CD5 over a time course of 30 days post-electroporation. Black arrows along the x-axis represent restimulations with anti-CD2 / CD3 / CD28 soluble antibodies (n = 2 healthy donors). NTC are shown as dotted lines with triangles, and targeting sgRNAs are shown as solid lines with circles. FIG. 9B: Representative flow cytometry histogram plots of CD5 KD or KO compared to NTC at day 30 post-electroporation. FIG. 9C: Comparison of Cas9 KO or CRISPRoff KD activity' with either a single guide or a pool of three guides at LAG3 over a time course of 31 days post-electroporation. Black arrows along the x-axis represent restimulations with anti-CD2 / CD3 / CD28 soluble antibodies, and cells were collected for flow cytometry 24 hours after each restimulation (n = 2 healthy donors). FIG. 9D: Representative flow cytometry histogram plots of LAG3 KD or KO compared to NTC at day 31 post-electroporation. FIG. 9E:Comparison of Cas9 KO or CRISPRoff KD activity with either a single guide or a pool of three guides at PDCD1 over a time course of 30 days post-electroporation. Black arrows along the a-axis represent restimulations with anti-CD2 / CD3 / CD28 soluble antibodies, and cells werePATENT Attorney Docket No. 048536-774001WOcollected for flow cytometry 24 hours after each restimulation (n = 2 healthy donors). FIG. 9F:Representative flow cytometry histogram plots of PD1 KDor KO compared to NTCat day 31 post-electroporation. FIG.9G: Comparison of Cas9 KO or CRISPRoff KD activity with either a single guide or a pool of three guides at CD39 over a time course of 35 days postelectroporation. Black arrows along the x-axis represent restimulations with anti-CD2 / CD3 / CD28 soluble antibodies, and cells were collected for flow cytometry 24 hours after each restimulation (n = 2 healthy donors). FIG. 9H: Representative flow cytometry histogram plots of CD39 KD or KO compared to NTC at day 31 post-electroporation. FIG.91: Comparison of Cas9 KO, CRISPRi or CRISPRoff activity with either a single guide targeting CD45 over a time course of 25 days post-electroporation. Black arrows along the x-axis represent restimulations with anti-CD2 / CD3 / CD28 soluble antibodies (n = 2 healthy donors). FIG.9J: Representative flow cytometry7histogram plots of KO, CRISPRi KD, or CRISPRoff KD compared to NTC at day 25 post-electroporation. FIG.9K: Fold change in surface protein expression (% positive cells) from the 1sttimepoint to the last timepoint for cells treated with CRISPRoff mRNA and the most potent guide targeting the TSS of each respective gene. The amount of CpG dinucleotides within a 500bp + / - the TSS of each gene is shown on the x-axis. Gray dots indicate genes with CGI annotations in UCSC genome browser and black dots indicate genes that do not have a CGI annotation.

[0027] FIGS. 10A-10E show durable multiplexed gene silencing. FIG. 10A: A graph showing the number of live T cells following cell editing by CRISPRoff and Cas9 when targeting either three, four, or five genes simultaneously compared to an empty electroporation control (EE). Live cell counts were measured five days after electroporation (n = 2 donors). FIG. 10B: A plot comparing CRISPRoff versus Cas9 multiplexed gene silencing efficiency targeting three genes (CD151, CD81, CD44), four genes (CD15 CD81, CD44, CD55) or five genes (CD151. CD81, (4)44. CD55, CD46) at day 5 or day 30 post-electroporation. Cells were analyzed using flow' cytometry' and the fraction of cells w ith all genes silenced w as calculated (n = 2 donors). FIG.10C: A representative flow' plot of cells targeted for triple gene silencing (CD151, CD81, CD44) silencing (top) or NTC (bottom). Cells were analyzed 30 days post-electroporation. In the top plot, cells were first gated on CD44-silenced cells, and the represented population shows CD81 and CD151 silencing. FIG. 10D: Pie charts depicting the breakdown of 3 gene, 4 gene, or 5 genePATENT Attorney Docket No. 048536-774001WOsilencing shown in (FIG. 10B). Data are representative of 2 healthy donors. FIG. 10E: An RNA-seq log2 CPM (normalized counts per million) plot showing triple-target-gene KD in cells electroporated with CRISPRoff mRNA and sgRNAs targeting FAS, RC3H1, and SUV39H1 or an NTC sgRNA. Cells were collected for RNA-seq 7 days after electroporation (n=4 donors; mean ±s.e.m.).

[0028] FIGS. 11A-11G show CRISPRon can target enhancer regions in primary human T cells. FIG. 11A: Schematic of the TET1 catalytic domain fused to dCas9 (CRISPRon-TETv3) and work flow of targeted demethylation via mRNA electroporation. FIG. 11B: The FOXP3 gene body with the TSDR and TSS highlighted in grey. The percent methylation of individual CpGs measured by PBAT-seq across the TSDR or TSS between activated Tregs and Tconvs is shown as a heat map. Each bar in the heat map represents an individual CpG. Data is representative of one donor. FIG. 11C: Percent FOXP3 positive Tconv cells receiving CRISPRon mRNA and an sgRNA targeting the TSDR or AAVS1 control region as measured by flow cytometry 9 days post-electroporation (n = 4 donors). FIG. 11D: Representative flow plots from day 9 post-electroporation depicting FOXP3 expression upon treatment with CRISPRon targeting the TSDR or an AAVS1 control. FIG. HE: Percent FOXP3 positive Tconv cells receiving CRISPRon targeting the TSDR or an AAVS1 control as measured by flow cytometry 28 days post initial activation (n = 2 donors). FIG. HF: Representative flow cytometry histogram plots showing FOXP3 MFI for CRISPRon treated samples with Guide 3 or an AAVS1 control at day 28 post-electroporation. FIG. 11G: Mean percent methylation across all CpGs assayed per targeted region (TSDR or TSS). Each triangle (donor 1) or circle (donor 2) represents an individual CpG.

[0029] FIGS. 12A-12E show durable at specific CRISPRoff silencing at therapeutically relevant genes in T cells. FIG. 12A: Transcript levels at day 27 post-electroporation as measured by RT-qPCR iorMED12 and RASA2 normalized to GAPDH. Gene expression is plotted as Fold Change to an NTC. Cells were electroporated with CRISPRoff mRNA and the top predicted guide, or a pool of the top 3 guides, or an NTC. (B-E) Transcriptomic assessment by RNA-seq of CRISPRoff activity upon silencing of (FIG. 12B) SUV39H1 (FIG. 12C) RC3H1 (FIG. 12D), PTPN2 (FIG. 12E) or MED 12 relative to NTC. Cells that were electroporated with the most potent guide identified from RT-qPCR for each gene in FIG. 12A or FIG. 8A were harvested forPATENT Attorney Docket No. 048536-774001WORNA extraction and bulk RNA sequencing. Bulk RNA sequencing data are representative of two healthy donors.

[0030] FIGS. 13A-13G show targeting the FOXP3 TSDR in primary human T cells with CRISPRon. FIG. 13A: Percent FOXP3 positive Tconv cells receiving CRISPRon mRNA and sgRNAs targeting the TSDR, the TSS, or AAVS1 control region as measured by flow cytometry 9 days post-electroporation (AAVS1 controls and Guides 1 & 3 targeting the TSDR: n = 4 donors. For the remaining conditions: n = 2 donors). FIG. 13B: Flow cytometry plots showing FOXP3 expression between cells treated with CRISPRon mRNA and Guide 3 targeting the FOXP3 TSDR or AAVS1 controls 48 hours after restimulation with anti -human CD2 / CD3 / CD28 soluble antibodies. FIG. 13C: Targeted bisulfite sequencing analysis showing the percent methylation of individual CpGs assayed within either the TSDR, the FOXP3 TSS, IKZF2 TSS or IL2RA TSS when cells were treated with CRISPRon mRNA and Guide 3 targeting the FOXP3 TSDR or AAVS1 controls. Each triangle (donor 1) or circle (donor 2) representants an individual CpG. FIG. 13D: The mean percent methylation across the IKZF2 or IL2RA regions shown in FIG. 13C when cells were treated with CRISPRon mRNA and Guide 3 targeting the FOXP3 TSDR or AAVS1 controls. Each triangle (donor 1) or circle (donor 2) represents an individual CpG. FIGS. 13E-13F: Percentage of FOXP3+Tconv cells after epiediting with CRISPRon mRNA variants (TETv4, TETv5) and 1-3 sgRNAs targeting the TSDR or AAVS1 control region as measured by flow cytometry at 9 days post-electroporation (FIG.13E) or 28 days post-electroporation (FIG. 13F). Cells treated with a single sgRNA received Guide 3, cells treated with a pool of two sgRNAs received Guide 1 and Guide 3, and cells treated with a pool of three sgRNAs received Guide 1, Guide 3, and Guide 4. (n=4 donors per condition, mean±s.e.m.; two-sided Welch’s / -test: For cells collected on day 9 treated with TETv4: 1 guide targeting the TSDR *F>=0.044, 2 guides *F=0.018, 3 guides *P=0.033. For cells collected on day 28 treated with TETv4: 2 guides targeting the TSDR *P=0.037, 3 guides *7)=0.026. For cells collected on day 28 treated with TETv5: 2 guides targeting the TSDR *P=0.027, 3 guides * / '=().046). FIG. 13G: Schematic of three CRISPRon variants (TETv3, TETv4, and TETv5), each of which consists of TET1 catalytic domain fused to dCas9 with varying XTEN linker lengths (e.g., XTEN16, XTEN80, and XTEN100, respectively).PATENT Attorney Docket No. 048536-774001WO

[0031] FIGS. 14A-14D show durability of CRISPRoff silencing in CAR T cells transferred in vivo. FIG. 14A: CD19+A375 melanoma cells were engrafted into NSG mice via flank injection. Casl2a compatible TRAC CD19-CAR T cells were generated in combination with CRISPRoff mRNA and a pool of three sgRNAs targeting CD151 or a NTC (left graph shows the CD 19+ CAR KI frequency prior to injection) (right graph shows the CD 151 KD prior to injection). CD151-epi-silenced CAR T cells or control-edited CAR T cells were then transferred into mice via the tail vein 7 days later. FIG. 14B: Representative gating strategy of tumors harvested from mice 14 days after CAR T cell injection. FIG. 14C: 14 days after CAR T cell injection, the tumor and spleen were harvested from mice, and CD151 expression among CD45+human T cells was assessed via flow cytometry (tumor: red; spleen: black). FIG. 14D: Representative flow cytometry graphs for T cells from the tumor or spleen that were treated with a NTC or sgRNAs targeting CD151.

[0032] FIGS. 15A-15D show RASA2 epi-silenced CAR T cells improve tumor control in vivo. FIG. 15A: Flow cytometry data showing levels of CD 19+ CAR positivity' and abTCR KO in T cells immediately prior to injection. CAR T cells were normalized based on their CAR expression levels to inject equal numbers of CAR T cells per mouse. FIG. 15B: Experimental timeline. FIG. 15C: BLI data showing Individual tumor growth over time in NSG mice engrafted with 0.5xl06Nalm6 cells and O.lxlO6RASA2-epi-silenced CD19+ CAR T cells. FIG.15D: Live cell counts collected nine days after electroporation between cells that have a TRAC CAR KI or no CAR KI. EE: empty electroporation. (n=4 donors).

[0033] FIGS. 16A-16N show durable at specific CRISPRoff silencing at therapeutically relevant genes in T cells. FIG. 16A: An RNA-sequencing log2 CPM (normalized counts per million) plot showing cells electroporated with CRISPRoff and sgRNAs targeting CD55, CD81, FAS, PTPN2, RASA2, RC3H1, MED12, or SUV39H1 as compared to NTC treated cells. The percent KD is shown for each gene. (CD55 and CD81 were collected 27 days postelectroporation; n = 2 donors) (FAS, PTPN2, RASA2, RC3H1, MED12, and SUV39H1 were collected on day 7 post-electroporation; n = 4 donors). FIG. 16B: Nuclease-active Cas9 editing efficiency at each of the therapeutically relevant target loci collected for RNA-seq as measured by TIDE indel analysis. FIGS. 16C-16M: Transcriptomic assessment by RNA-seq of CRISPRoff or Cas9 activity upon silencing or KO, respectively, of (FIG. 16C) FAS, (FIGS.PATENT Attorney Docket No. 048536-774001WO16D-16E) RC3H1, (FIGS. 16F-16G) SUV39H1, (FIGS. 16H-16I) PTPN2 or (FIGS. 16K-16J) RASA2 or (FIGS. 16L-16M) MED12 relative to NTC. For CRISPRoff treated samples, cells were electroporated with the most potent guide identified from RT-qPCR for each gene in panel FIG IK. For both Cas9 and CRISPRoff treated samples, cells were harvested for RNA extraction and bulk RNA sequencing seven days post-electroporation (n = 4 donors). The target gene for each panel is labeled in light gray with a larger dot, dark gray dots to the left of the dashed line indicate significantly downregulated DEGs, dark gray dots to the right of the dashed line indicate significantly upregulated DEGs, and unlabeled, light grey7dots have no significance (Empirical-Bayes moderated statistics with Benjamini-Hochberg FDR control, adjusted P < 0.05). FIG. 16N: Comparison of gene log2-fold change between CRISPRoff and CRISPR KO targeting MED12. CRISPR KO for MED12 RNA-sequencing data is from Arce et al 40. Non-significant hits are shown in grey and significant hits are colored by direction of effect between experiments (FDR <0.05) (CRISPR KO data: n = 2 donors. CRISPRoff data: n = 4 donors).

[0034] FIGS. 17A-17F show- off-target assessment of CRISPRoff. (A-F) Off-target assessment of CRISPRoff silencing as measured by RNA-seq when targeting (FIG. 17A) FAS, (FIG. 17B) RC3H1, or (FIG. 17C) SUV39H1, (FIG. 17D) PTPN2, (FIG. 17E) MED12, or (FIG. 17F) RASA2. Predicted off-target sites were generated through IDT’s CRISPR-Cas9 guide RNA design checker for each of the CRISPRoff KD guides, and loci were filtered for ±lkb around a gene promoter. Proximal genes within lOOkb of the target gene were also assessed. Genes are ordered by left to right by highest to lowest off-target score generated from IDT. Predicted off-target genes that met significance thresholds (adjusted P > 0.05 and absolute log2 fold change > 1) are denoted with a star (n = 4 donors). Predicted off-targets, proximal genes, and the target gene are annotated with gray boxes at the top of each panel.DETAILED DESCRIPTION DEFINITIONS

[0035] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understoodPATENT Attorney Docket No. 048536-774001WOthat various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0036] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0037] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0038] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g.. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed„ J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0039] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g.. deoxyribonucleosides or ribonucleosides). In embodiments, ‘“nucleic acid” does not include nucleosides. The terms "polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary' sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and doublePATENT Attorney Docket No. 048536-774001WOstranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary’ sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0040] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0041] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphorami date, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH. Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and ChaptersPATENT Attorney Docket No. 048536-774001WO6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety7of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the intemucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0042] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary7to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0043] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology7searching. Polynucleotides may7optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0044] The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly know n in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides ofPATENT Attorney Docket No. 048536-774001WOa complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0045] As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

[0046] The terms “single guide RNA,” “sgRNA,” “guide RNA,” or “gRNA” are used herein according to their plain ordinary meaning and refer to a RNA molecule that functions as a guide for a RNA- or a DNA-targeting protein (e.g. enzyme). In embodiments, the gRNA non-covalently binds the RNA- or DNA-target enzyme. In embodiments, the guide RNA is capable of binding to a non-double strand break-dependent gene editor protein thereby forming a nondouble strand break-dependent gene editor complex. In embodiments, the non-double strand break-dependent gene editor complex is capable of editing at least one base within a genomic nucleic acid sequence.

[0047] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxy proline, y-carboxy glutamate,PATENT Attorney Docket No. 048536-774001WOand O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms "non-natural ly occurring amino acid'’ and ‘‘unnatural amino acid’' refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0048] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0049] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A "fusion protein" is used in accordance with its plain and ordinary meaning and refers to a recombinant protein encoding two or more protein domains as a single amino acid sequence wherein the two or more protein domains are not naturally expressed as a single amino acid sequence.

[0050] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in thePATENT Attorney Docket No. 048536-774001WOreference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0051] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. One skilled in the art will immediately recognize the identity and location of residues corresponding to a specific position in a protein or a nucleic acid in other proteins or nucleic acids with different numbering systems. For example, by performing a simple sequence alignment with a protein or a nucleic acid the identity and location of residues corresponding to specific positions of the protein or the nucleic acid are identified in other protein sequences or nucleic acid sequences aligning to the protein or the nucleic acid. For example, a selected residue in a selected protein corresponds to glutamic acid at position 138 when the selected residue occupies the same essential spatial or other structural relationship as a glutamic acid at position 138. In some embodiments, where a selected protein is aligned for maximum homology with a protein, the position in the aligned selected protein aligning with glutamic acid 138 is the to correspond to glutamic acid 138. Instead of a primary sequence alignment, a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the glutamic acid at position 138, and the overall structures compared. In this case, an amino acid that occupies the same essential position as glutamic acid 138 in the structural model is the to correspond to the glutamic acid 138 residue.

[0052] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variantsPATENT Attorney Docket No. 048536-774001WOrefers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every' position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively- modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every' possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG. which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0053] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0054] The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);PATENT Attorney Docket No. 048536-774001WO6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)(see, e.g., Creighton, Proteins (1984)).

[0055] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see. e.g., NCBI web site http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0056] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.PATENT Attorney Docket No. 048536-774001WO

[0057] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology' alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443. by the search for similarity' method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0058] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity' are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query' sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; alway s > 0) and N (penalty’ score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximumPATENT Attorney Docket No. 048536-774001WOachieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0059] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0060] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0061] The phrase "specifically (or selectively) binds to" when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassayPATENT Attorney Docket No. 048536-774001WOconditions, the specified proteins bind to a particular protein at least two times the background and more ty pically more than 10 to 100 times background.

[0062] For specific proteins described herein, the named protein includes any of the protein’s naturally occurring forms, variants or homologs that maintain the protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference, homolog or functional fragment thereof.

[0063] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary’ during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0064] A “detectable agent” or “detectable moiety” is a composition detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable agents include18F.32p 33p 45Ti47S (,, 52Fe, 59^ 62^ 64^ 67^ 67^ 68^ 77^ 86y, 90y 89^ 89^ 94^ 94^ "Ho. Er, Tm, Yb, Lu,32P, fluorophore (e.g. fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, monochrystalline iron oxide nanoparticles, monochrystalline iron oxide,PATENT Attorney Docket No. 048536-774001WOnanoparticle contrast agents, liposomes or other delivery’ vehicles containing Gadolinium chelate ("Gd-chelate") molecules. Gadolinium, radioisotopes, radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emitting radionuclides, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g. including microbubble shells including albumin, galactose, lipid, and / or polymers; microbubble gas core including air, heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.), iodinated contrast agents (e.g., iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores. or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide.

[0065] Radioactive substances (e.g., radioisotopes) that may be used as imaging and / or labeling agents in accordance with the aspects of the disclosure include, but are not limited to,18F,32P,33P,45Ti,47Sc,52Fe,59Fe,62Cu,64Cu,67Cu,67Ga,68Ga,77As,86Y,90Y.89Sr,89Zr,94Tc,211Pb,212Bi,212Pb,213Bi,223Ra and223Ac. Paramagnetic ions that may be used as additional imaging agents in accordance with the aspects of the disclosure include, but are not limited to, ions of transition and lanthanide metals (e.g., metals having atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

[0066] When the label or detectable moiety is a radioactive metal or paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding to these ions. The long tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which the metals or ions may be added for binding. Examples of chelating groups that may be used according to the disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), DOTA, NOTA. NETA. TETA. porphyrins, polyamines, crown ethers, bis-thiosemicarbazones,PATENT Attorney Docket No. 048536-774001WOpolyoximes, and like groups. The chelate is normally linked to the PSMA antibody or functional antibody fragment by a group, which enables the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies and carriers described herein. Macrocyclic chelates such as NOTA, DOT A, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively. Other ring-type chelates such as macrocyclic poly ethers, which are of interest for stably binding nuclides, such as22?Ra for RAIT may be used. In certain embodiments, chelating moieties may be used to attach a PET imaging agent, such as an A1-18F complex, to a targeting molecule for use in PET analysis.

[0067] The term "recombinant" when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0068] The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0069] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typicallyPATENT Attorney Docket No. 048536-774001WOrecombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0070] The term "exogenous" refers to a molecule or substance (e.g. , a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the cell or organism it is expressed by. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to. or originates within, a given cell or organism.

[0071] The term "‘expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The level of expression of non-coding nucleic acid molecules (e.g., sgRNA) may be detected by standard PCR or Northern blot methods well known in the art. See. Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88. The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.Expression can be detected using conventional techniques for detecting protein (e.g, ELISA. Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0072] The term “transcriptional regulatory sequence” as provided herein refers to a segment of DNA that is capable of increasing or decreasing transcription (e.g., expression) of a specific gene within an organism. Non-limiting examples of transcriptional regulatory sequences include promoters, enhancers, and silencers.

[0073] The terms “transcription start site” and transcription initiation site” may be used interchangeably to refer herein to the 5’ end of a gene sequence (e.g., DNA sequence) where RNA polymerase (e g., DNA-directed RNA polymerase) begins synthesizing the RNA transcript. The transcription start site may be the first nucleotide of a transcribed DNA sequencePATENT Attorney Docket No. 048536-774001WOwhere RNA polymerase begins synthesizing the RNA transcript. A skilled artisan can determine a transcription start site via routine experimentation and analysis, for example, by performing a run-off transcription assay or by definitions according to FANTOM5 database.

[0074] The term “promoter” as used herein refers to a region of DNA that initiates transcription of a particular gene. Promoters are typically located near the transcription start site of a gene, upstream of the gene and on the same strand (i . e. , 5’ on the sense strand) on the DNA. Promoters may be about 100 to about 1000 base pairs in length.

[0075] The term “enhancer” as used herein refers to a region of DNA that may be bound by¬ proteins (e.g., transcription factors) to increase the likelihood that transcription of a gene will occur. Enhancers may be about 50 to about 1500 base pairs in length. Enhancers may be located dow nstream or upstream of the transcription initiation site that it regulates and may be several hundreds of base pairs away from the transcription initiation site. In embodiments, the enhancer is an enhancer of a gene encoding a T cell receptor protein. In embodiments, the enhancer is an enhancer of a T cell receptor alpha constant (TRAC) gene. In embodiments, the enhancer is an enhancer of a forkhead box P3 (FOXP3) gene. In embodiments, the enhancer of the FOXP3 gene is aTreg specific demethylated region (TSDR) of the FOXP3 gene. In embodiments, the TSDR of the FOXP3 gene includes a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 148. In embodiments, the TSDR of the FOXP3 gene includes the nucleotide sequence of SEQ ID NO: 148. In embodiments, the TSDR of the FOXP3 gene is the nucleotide sequence of SEQ ID NO: 148.

[0076] The term “silencer” as used herein refers to a DNA sequence capable of binding transcription regulation factors known as repressors, thereby negatively effecting transcription of a gene. Silencer DNA sequences may be found at many different positions throughout the DNA, including, but not limited to, upstream of a target gene for which it acts to repress transcription of the gene (e.g., silence gene expression).

[0077] A "guide RNA" or "gRNA" as provided herein refers to any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In embodiments, the degree of complementarity between a guide sequence and itsPATENT Attorney Docket No. 048536-774001WOcorresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

[0078] In embodiments, the polynucleotide (e.g., gRNA) is a single-stranded ribonucleic acid. In embodiments, the polynucleotide (e g., gRNA) is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length. In embodiments, the polynucleotide (e.g., gRNA) is from 10 to 30 nucleic acid residues in length. In embodiments, the polynucleotide (e.g., gRNA) is 20 nucleic acid residues in length. In embodiments, the length of the polynucleotide (e.g., gRNA) can be at least 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, 99, 100 or more nucleic acid residues or sugar residues in length. In embodiments, the polynucleotide (e.g., gRNA) is from 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 5 to 75, 10 to 75, 15 to 75, 20 to 75, 25 to 75, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75, 70 to 75, 5 to 100, 10 to 100, 15 to 100, 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100. 65 to 100, 70 to 100. 75 to 100, 80 to 100, 85 to 100, 90 to 100, 95 to 100, or more residues in length. In embodiments, the polynucleotide (e.g., gRNA) is from 10 to 15, 10 to 20, 10 to 30, 10 to 40, or 10 to 50 residues in length.

[0079] For specific proteins described herein (e.g., KRAB, dCas9, Dnmt3A, Dnmt3L), the named protein includes any of the protein's naturally occurring forms, or variants or homologs that maintain the protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In embodiments, variants or homologs have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity' across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In embodiments, the protein is the protein as identified by its NCBI sequence reference. In embodiments, the protein is the protein as identified by its NCBI sequence reference or functional fragment or homolog thereof.

[0080] The term “Kriippel associated box domain” or “KRAB domain” is used in accordancePATENT Attorney Docket No. 048536-774001WOwith its plain and ordinary meaning in the art and refers to a category of transcriptional repression domains present in approximately 400 human zinc finger protein-based transcription factors. KRAB domains typically include about 45 to about 75 amino acid residues. A description of KRAB domains, including their function and use, may be found, for example, in Ecco, G., Imbeault. M., Trono, D., KRAB zinc finger proteins, Development 144, 2017; Lambert et al. The human transcription factors. Cell 172, 2018; Gilbert et al., Cell (2013); and Gilbert et al., Cell (2014), all of which are incorporated herein by reference in their entirety. In embodiments, the KRAB domain is a KRAB domain of Kox 1. In embodiments, the KRAB domain is encoded by a nucleotide sequence including the sequence of SEQ ID NO: 51. In embodiments, the KRAB domain is encoded by the nucleotide sequence of SEQ ID NO:51. In embodiments, the KRAB domain is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:51. In embodiments, the KRAB domain is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:51. In embodiments, the KRAB domain is encoded by a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO:51. In embodiments, the KRAB domain is encoded by a nucleotide sequence that has at least 85% sequence identity to SEQ ID NO:51. In embodiments, the KRAB domain is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:51. In embodiments, the KRAB domain is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:51. In embodiments, the KRAB domain includes the amino acid sequence of any one of SEQ IDNO:177-180. In embodiments, the KRAB domain includes the amino acid sequence of SEQ ID NO: 177. In embodiments, the KRAB domain includes the amino acid sequence of SEQ ID NO: 178. In embodiments, the KRAB domain includes the amino acid sequence of SEQ ID NO: 179. In embodiments, the KRAB domain includes the amino acid sequence of SEQ ID NO: 180. In embodiments, the KRAB domain includes an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 177. In embodiments, the KRAB domain includes an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 178. InPATENT Attorney Docket No. 048536-774001WOembodiments, the KRAB domain includes an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 179. In embodiments, the KRAB domain includes an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 180.

[0081] The term "DNA methyltransferase'’ as provided herein refers to an enzyme that catalyzes the transfer of a methyl group to DNA. Non-limiting examples of DNA methyltransferases include Dnmtl, Dnmt3A, Dnmt3B, and Dnmt3L. In embodiments, the DNA methyltransferase is a bacterial cytosine methyltransferase and / or a bacterial non-cytosine methyltransferase. Depending on the specific DNA methyltransferase, different regions of DNA are methylated. For example, Dnmt3A typically targets CpG dinucleotides for methylation. Through DNA methylation, DNA methyltransferases can modify the activity of a DNA segment (e.g., gene expression) without altering the DNA sequence. In embodiments, DNA methylation results in repression of gene transcription and / or modulation of methylation sensitive transcription factors or CTCF. As described herein, fusion proteins may include one or more (e.g., two) DNA metyltransferases. When a DNA methyltransferase is included as part of a fusion protein, the DNA methyltransferase may be referred to as a “DNA methyltransferase domain.” In embodiments, a DNA methyltransferase domain includes one or more DNA methyltransferases. In embodiments, a DNA methyltransferase domain includes two DNA methyltransferases .

[0082] In embodiments, the DNA methyltransferase includes Dnmt3A. In embodiments, the DNA methyltransferase domain is Dnmt3A. In embodiments, the DNA methyltransferase domain is encoded by the nucleotide sequence of SEQ ID NO:43. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 43. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:43. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 80% sequence identify to SEQ ID NO: 43. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 85% sequence identify to SEQ IDPATENT Attorney Docket No. 048536-774001WONO:43. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:43. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:43. In embodiments, the Dnmt3A includes the amino acid sequence of SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 50%, 55%, 60%. 65%, 70%. 75%. 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%, 99% or 100% sequence identity to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 75% sequence identity7to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 90% sequence identity7to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 97% sequence identity7to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 173. In embodiments, the Dnmt3A includes an amino acid sequence that has at least 100% sequence identity to SEQ ID NO: 173.

[0083] In embodiments, the DNA methyltransferase domain includes Dnmt3L. In embodiments, the DNA methyltransferase domain is Dnmt3L. In embodiments, the DNA methyltransferase domain is encoded by the nucleotide sequence of SEQ ID NO:45. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:45. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:45. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO:45. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 85%PATENT Attorney Docket No. 048536-774001WOsequence identity to SEQ ID NO: 45. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:45. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 45. In embodiments, the Dnmt3L includes the amino acid sequence of SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 91%. 92%. 93%.94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 75% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 175. In embodiments, the Dnmt3L includes an amino acid sequence that has at least 100% sequence identity to SEQ ID NO: 175.

[0084] In embodiments, the DNA methyltransferase domain includes Dnmt3A and Dnmt3L. In embodiments, the DNA methyltransferase domain includes a Dnmt3A-3L domain. In embodiments, the DNA methyltransferase encodes a Dnmt3A-3L domain. In embodiments, the DNA methyltransferase domain includes Dnmt3A or a portion or a fragment thereof and Dnmt3L or a portion or a fragment thereof. In embodiments, the DNA methyltransferase domain includes a Dnmt3A comprising the nucleotide sequence of SEQ ID NO:43 and a Dnmt3L comprising the nucleotide sequence of SEQ ID NO:45. In embodiments, the DNA methyltransferase domain includes the catalytic domain of Dnmt3A and the C-terminus of Dnmt3L. In embodiments, the Dnmt3A or portion or fragment thereof is attached to the Dnmt3LPATENT Attorney Docket No. 048536-774001WOor portion or fragment thereof via a linker. In embodiments, the linker includes the nucleotide sequence of SEQ ID NO:44. In embodiments, the DNA methyltransferase domain includes the nucleotide sequence of SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45. In embodiments, the DNA methyltransferase domain is encoded by the nucleotide sequence of SEQ ID NO: 53. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%. 94%. 95%. 96%.97%, 98%, 99% or 100% sequence identity to SEQ ID NO:53. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:3. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO:53. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 85% sequence identity to SEQ ID NO: 53. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:53. In embodiments, the DNA methyltransferase domain is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:53. In embodiments, the Dnmt3A-3L domain includes the amino acid sequence of SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 75% sequence identity to SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 80% sequence identity7to SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 176. In embodiments, the Dnmt3 A-3L domain includes an amino acid sequence that has at least 98% sequence identity' to SEQ ID NO: 176. InPATENT Attorney Docket No. 048536-774001WOembodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 176. In embodiments, the Dnmt3A-3L domain includes an amino acid sequence that has at least 100% sequence identity to SEQ ID NO: 176. A description of Dnmt3A-3L domain structure and use may be found, for example, in Siddique et al, Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3A-Dnmt3L single-chain fusion protein with increased DNA methylation activity, J. Mol. Biol. 425, 2013 and Stepper et al, Efficient targeted DNA methylation with chimeric dCas9-Dnmt3A-Dnmt3L methyltransferase, Nucleic Acids Res. 45, 2017, which are incorporated herein by reference in their entirety and for all purposes.

[0085] A "Dnmt3A". “Dnmt3a,” "DNA (cytosine-5)-methyltransferase 3A" or "DNA methyltransferase 3a" protein as referred to herein includes any of the recombinant or naturally-occurring forms of the Dnmt3A enzyme or variants or homologs thereof that maintain Dnmt3A enzyme activity (e.g. within at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Dnmt3A). In embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Dnmt3A protein. In embodiments, the Dnmt3A protein is substantially identical to the protein identified by the UniProt reference number Q9Y6K1 or a variant or homolog having substantial identity thereto. In embodiments, the Dnmt3A polypeptide is encoded by a nucleic acid sequence identified by the NCBI reference sequence Accession number NM_022552, homologs or functional fragments thereof. In embodiments, Dnmt3A is encoded by a nucleotide sequence including the sequence of SEQ ID NO:43. In embodiments, Dnmt3A is encoded by the nucleotide sequence of SEQ ID NO:43. In embodiments, Dnmt3A is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:43. In embodiments, Dnmt3A is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:43. In embodiments, Dnmt3A is encoded by a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO:43. In embodiments, Dnmt3A is encoded by a nucleotide sequence that has at least 85% sequence identity to SEQ ID NO:43. In embodiments,PATENT Attorney Docket No. 048536-774001WODnmt3A is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:43. In embodiments Dnmt3A is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 43.

[0086] A "Dnmt3L", "DNA (cytosine-5)-methyltransferase 3L" or "DNA methyltransferase 3L" protein as referred to herein includes any of the recombinant or naturally -occurring forms of the Dnmt3L enzyme or variants or homologs thereof that maintain Dnmt3L enzyme activity (e.g., within at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Dnmt3L). In embodiments, the variants or homologs have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Dnmt3L protein. In embodiments, the Dnmt3L protein is substantially identical to the protein identified by the UniProt reference number Q9CWR8 or a variant or homolog having substantial identity thereto. In embodiments, the Dnmt3L protein is identical to the protein identified by the UniProt reference number Q9CWR8. In embodiments, the Dnmt3L protein has at least 75% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9CWR8. In embodiments, the Dnmt3L protein has at least 80% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9CWR8. In embodiments, the Dnmt3L protein has at least 85% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9CWR8. In embodiments, the Dnmt3L protein has at least 95% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9CWR8.

[0087] In embodiments, the Dnmt3L protein is substantially identical to the protein identified by the UniProt reference number Q9UJW or a variant or homolog having substantial identity thereto. In embodiments, the Dnmt3L protein is identical to the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3L protein has at least 50% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3L protein has at least 55% sequence identity to the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3L protein has atPATENT Attorney Docket No. 048536-774001WOleast 60% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3L protein has at least 65% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3L protein has at least 70% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3L protein has at least 75% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3E protein has at least 80% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3E protein has at least 85% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3L protein has at least 90% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3E protein has at least 95% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q9UJW. In embodiments, the Dnmt3E polypeptide is encoded by a nucleic acid sequence identified by the NCBI reference sequence Accession number NM_001081695, or homologs or functional fragments thereof. In embodiments, Dnmt3U is encoded by a nucleotide sequence including the sequence of SEQ ID NO:28. In embodiments, Dnmt3L is encoded by the nucleotide sequence of SEQ ID NO:28. In embodiments, Dnmt3E is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 45. In embodiments, Dnmt3L is encoded by a nucleotide sequence that has at least 50% sequence identity to SEQ ID NO:45. In embodiments, Dnmt3L is encoded by a nucleotide sequence that has at least 55% sequence identity to SEQ ID NO:45. In embodiments, Dnmt3L is encoded by a nucleotide sequence that has at least 60% sequence identity to SEQ ID NO:45. In embodiments, Dnmt3L is encoded by a nucleotide sequence that has at least 65% sequence identity7to SEQ ID NO:45. In embodiments, Dnmt3L is encoded by a nucleotide sequence that has at least 97% sequence identity to SEQ ID NO:45. In embodiments, Dnmt3L is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:45. In embodiments, Dnmt3L is encoded by a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO:45. In embodiments, Dnmt3L is encoded by a nucleotidePATENT Attorney Docket No. 048536-774001WOsequence that has at least 85% sequence identity to SEQ ID NO:45. In embodiments. Dnmt3L is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:45. In embodiments, Dnmt3L is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:45.

[0088] The term “nuclease-deficient RNA-guided DNA endonuclease domain” and the like refer, in the usual and customary sense, to an RNA-guided DNA endonuclease (e.g. a mutated form of a naturally occurring RNA-guided DNA endonuclease) that targets a specific phosphodiester bond within a DNA polynucleotide, wherein the recognition of the phosphodiester bond is facilitated by7a separate polynucleotide sequence (for example, a RNA sequence (e.g., single guide RNA (sgRNA)), but is incapable of cleaving the target phosphodi ester bond to a significant degree (e.g. there is no measurable cleavage of the phosphodi ester bond under physiological conditions or the amount of cleavage is reduced (e.g. by 50%, 60%, 60%, 90%, 90%, 95%, 96%, 97%, 98%, 99% or more) relative to the wild type sequence). A nuclease-deficient RNA-guided DNA endonuclease thus retains DNA-binding ability7(e.g. specific binding to a target sequence) when complexed with a polynucleotide (e.g., sgRNA), but may lack significant endonuclease activity (e.g. any amount of detectable endonuclease activity). In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a CRISPR-associated protein. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a dCas9, dCpfl, ddCpfl, Cas-phi, a nuclease-deficient Cas9 variant, a nuclease-deficient Class II CRISPR endonuclease, a zinc finger domain, a transcription activator-like effector (TALE), a leucine zipper domain, a winged helix domain, a hehx-tum-helix motif, a helix-loop-helix domain, an HMB-box domain, a Wor3 domain, an OB-fold domain, an immunoglobulin domain, or a B3 domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a zinc finger domain, a leucine zipper domain, a winged helix domain, a helix-tum-helix motif, a helix-loop-helix domain, an HMB-box domain, a Wor3 domain, an OB-fold domain, an immunoglobulin domain, or a B3 domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a leucine zipper domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a winged helix domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a helix-tum-helix motif. In embodiments, the nuclease-deficient RNA-guided DNAPATENT Attorney Docket No. 048536-774001WOendonuclease domain is a helix-loop-helix domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is an HMB-box domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a Wor3 domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is an OB-fold domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is an immunoglobulin domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a B3 domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas9, ddCpfl, Cas-phi, a nuclease-deficient Cas9 variant, or a nuclease-deficient Class II CRISPR endonuclease. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas9. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas9 from S’, pyogenes. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas9 from S', aureus. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas!2a from Lachnospiracea (dLbCas!2a). In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCasl2a from Lachnospiracea bacterium. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCasl2a. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas!2. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is ddCas!2a. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is Cas-phi.

[0089] The term ■ €’ RIS PR-associated protein" or “CRISPR protein” refers to any CRISPR protein that functions as a nuclease-deficient RNA-guided DNA endonuclease enzyme, i.e., a CRISPR protein in which catalytic sites for endonuclease activity are defective or lack activity. Exemplary7CRISPR-associated proteins include dCas9, dCpfl, dCas!2, Cas-phi, a nuclease-deficient Cas9 variant, a nuclease-deficient Class II CRISPR endonuclease, and the like.

[0090] A “CRISPR associated protein 9,” “Cas9,” “Csnl” or “Cas9 protein” as referred to herein includes any of the recombinant or naturally-occurring forms of the Cas9 endonuclease or variants or homologs thereof that maintain Cas9 endonuclease enzyme activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas9). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100,PATENT Attorney Docket No. 048536-774001WO150 or 200 continuous amino acid portion) compared to a naturally occurring Cas9 protein. In embodiments, the Cas9 protein is substantially identical to the protein identified by the UniProt reference number Q99ZW2 or a variant or homolog having substantial identity thereto. In embodiments, the Cas9 protein has at least 75% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 80% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 85% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 90% sequence identity' to the amino acid sequence of the protein identified by the UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 95% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number Q99ZW2.

[0091] The terms “dCas9” or “dCas9 protein” as referred to herein is a Cas9 protein in which both catalytic sites for endonuclease activity' are defective or lack activity'. In embodiments, the dCas9 protein has mutations at positions corresponding to D10A and H840A of S. pyogenes Cas9. In embodiments, the dCas9 protein lacks endonuclease activity due to point mutations at both endonuclease catalytic sites (RuvC and HNH) of wild type Cas9. The point mutations can be D10A and H840A. In embodiments, the dCas9 has substantially no detectable endonuclease (e.g., endodeoxyribonuclease) activity. In embodiments, dCas9 is encoded by a nucleotide sequence including the sequence of SEQ ID NO:48 or SEQ ID NO:49. In embodiments, dCas9 is encoded by the nucleotide sequence of SEQ ID NO:48 or SEQ ID NO:49. In embodiments, dCas9 includes the amino acid sequence of SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 75% sequence identity to SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 80% sequence identity' to SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 176. In embodiments, dCas9 includes an amino acid sequence that has at least 95% sequence identity toPATENT Attorney Docket No. 048536-774001WOSEQ ID NO: 169. In embodiments. dCas9 includes an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 169. In embodiments, dCas9 includes an amino acid sequence that has at least 100% sequence identity to SEQ ID NO: 169.

[0092] In embodiments, dCas9 is encoded by a nucleotide sequence including the sequence of SEQ ID NO:48. In embodiments, dCas9 is encoded by the nucleotide sequence of SEQ ID NO:48. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:48. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:48. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 80% sequence identity7to SEQ ID NO:48. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 85% sequence identity to SEQ ID NO:48. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:48. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:48. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas9 from S', pyogenes. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas9 from S. aureus.

[0093] In embodiments, dCas9 is encoded by a nucleotide sequence including the sequence of SEQ ID NO:49. In embodiments, dCas9 is encoded by a nucleotide sequence that has the sequence of SEQ ID NO:49. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:49. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:49. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 80% sequence identity7to SEQ ID NO:49. In embodiments, dCas9 is encoded by a nucleotide sequence that has at least 85% sequence identity to SEQ ID NO:49. In embodiments, dCas9 is encoded by a nucleotidePATENT Attorney Docket No. 048536-774001WOsequence that has at least 90% sequence identity to SEQ ID NO:49. In embodiments. dCas9 is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:49. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas9 from S. pyogenes. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas9 from S. aureus.

[0094] The terms “DNAse-dead Cpfl" or “ddCpfl” refer to mutated Acidaminococcus sp. Cpfl (AsCpfl) resulting in the inactivation of Cpfl DNAse activity. In embodiments, ddCpfl includes an E993A mutation in the RuvC domain of AsCpfl. In embodiments, the ddCpfl has substantially no detectable endonuclease (e.g., endodeoxyribonuclease) activity. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is dCas!2a from Lachnospiracea bacterium.

[0095] The term “dLbCpfT’ refers to mutated Cpfl from Lachnospiraceae bacterium ND2006 (LbCpfl) that lacks DNAse activity. In embodiments, dLbCpfl includes a D832A mutation. In embodiments, the dLbCpfl has substantially no detectable endonuclease (e.g., endodeoxyribonuclease) activity.

[0096] The term “dFnCpfl” refers to mutated Cpfl from Francisella novicida U112 (FnCpfl) that lacks DNAse activity'. In embodiments, dFnCpfl includes aD917A mutation. In embodiments, the dFnCpfl has substantially no detectable endonuclease (e.g., endodeoxyribonuclease) activity.

[0097] A "Cpfl" or " Cpfl protein" as referred to herein includes any of the recombinant or naturally-occurring forms of the Cpfl (CRISPR from Prevotella and Francisella 1) endonuclease or variants or homologs thereof that maintain Cpfl endonuclease enzyme activity' (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity' compared to Cpfl). In embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Cpfl protein. In embodiments, the Cpfl protein is substantially identical to the protein identified by the UniProt reference number U2UMQ6 or a variant or homolog having substantial identity thereto. In embodiments, the Cpfl protein is identical to the protein identified by the UniProtPATENT Attorney Docket No. 048536-774001WOreference number U2UMQ6. In embodiments, the Cpfl protein has at least 75% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number U2UMQ6. In embodiments, the Cpfl protein has at least 80% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number U2UMQ6. In embodiments, the Cpfl protein is identical to the protein identified by the UniProt reference number U2UMQ6. In embodiments, the Cpfl protein has at least 85% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number U2UMQ6. In embodiments, the Cpfl protein is identical to the protein identified by the UniProt reference number U2UMQ6. In embodiments, the Cpfl protein has at least 90% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number U2UMQ6. In embodiments, the Cpfl protein is identical to the protein identified by the UniProt reference number U2UMQ6. In embodiments, the Cpfl protein has at least 95% sequence identity to the amino acid sequence of the protein identified by the UniProt reference number U2UMQ6.

[0098] The term “nuclease-deficient Cas9 variant” refers to a Cas9 protein having one or more mutations that increase its binding specificity' to PAM compared to wild type Cas9 and further include mutations that render the protein incapable of or having severely impaired endonuclease activity. Without wishing to be bound by theory, it is believed that the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex. The precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). The binding specificity of nuclease-deficient Cas9 variants to PAM can be determined by any method known in the art. Descriptions and uses of known Cas9 variants may be found, for example, in Shmakov et al., Diversity' and evolution of class 2 CRISPR-Cas systems. Nat. Rev. Microbiol. 15, 2017 and Cebrian-Serrano et al, CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools. Mamm. Genome 7-8, 2017. Exemplary Cas9 variants are listed in the Table 1 below.

[0099] Table 1PATENT Attorney Docket No. 048536-774001WO>>

[0100] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a nuclease-deficient Class II CRISPR endonuclease. The term “nuclease-deficient Class II CRISPR endonuclease'’ as used herein refers to any Class II CRISPR endonuclease having mutations resulting in reduced, impaired, or inactive endonuclease activity.

[0101] The term “nuclease-deficient DNA endonuclease enzy me” refers to a DNA endonuclease (e.g. a mutated form of a naturally occurring DNA endonuclease) that targets a specific phosphodi ester bond within a DNA polynucleotide, but that does not require an RNA guide. In embodiments, the “nuclease-deficient DNA endonuclease enzyme” is a zinc finger domain or a TALE.

[0102] In embodiments, the nuclease-deficient DNA endonuclease enzyme is a “zinc finger domain.” The term “zinc finger domain” or “zinc finger binding domain” or “zinc finger DNA binding domain” are used interchangeably and refer to a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. In embodiments, the zinc finger domain is non-naturallyPATENT Attorney Docket No. 048536-774001WOoccurring in that it is engineered to bind to a target site of choice. In embodiments, the zinc finger binding domain refers to a protein, a domain within a larger protein, or a nuclease-deficient RNA-guided DNA endonuclease domain that is capable of binding to any zinc finger known in the art, such as the C2H2 type, the CCHC ty pe, the PHD type, or the RING ty pe of zinc fingers.

[0103] As used herein, a “zinc finger” is a polypeptide structural motif folded around a bound zinc cation. In embodiments, the polypeptide of a zinc finger has a sequence of the formX3-Cys-X2-4-Cys-Xi2-His-X3-5-His-X4, wherein X is any amino acid (e.g., X2-4 indicates an oligopeptide 2-4 amino acids in length). There is generally a wide range of sequence variation in the 28-31 amino acids of the known zinc finger polypeptides. Only the two consensus histidine residues and two consensus cysteine residues bound to the central zinc atom are invariant. Of the remaining residues, three to five are highly conserved, while there may be significant variation among the other residues. Despite the wide range of sequence variation in the polypeptide, zinc fingers of this type have a similar three dimensional structure. How ever, there is a wide range of binding specificities among the different zinc fingers, i.e. different zinc fingers bind double stranded polynucleotides having a wide range of nucleotides sequences. In embodiments, the zinc finger is the C2H2 type. In embodiments, the zinc finger is the CCHC type. In embodiments, the zinc finger is the PHD type. In embodiments, the zinc finger is the RING type.

[0104] The term “TALE” or “transcription activator-like effector” refers to artificial restriction enz mes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain. TALEs enable efficient, programmable, and specific DNA cleavage and represent powerful tools for genome editing in situ. Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence. The term TALE, as used herein, is broad and includes a monomeric TALE that can cleave double stranded DNA without assistance from another TALE. The term TALE is also used to refer to one or both members of a pair of TALEs that are engineered to work together to cleave DNA at the same site. TALEs that work together may be referred to as a left-TALE and a right-TALE, which references the handedness of DNA. TALE are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a highly conserved 33-34 amino acid sequence with the exception of the 12th and 13th amino acids. These two locations are highly variable (repeat variable diresidue (RVD)) and show7PATENT Attorney Docket No. 048536-774001WOa strong correlation with specific nucleotide recognition. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

[0105] The term “Class II CRISPR endonuclease” refers to endonucleases that have similar endonuclease activity as Cas9 and participate in a Class II CRISPR system. An example Class II CRISPR system is the type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Casl, Cas2, and Csnl, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30 bp each). The Cpfl enzyme belongs to a putative ty pe V CRISPR-Cas system. Both type II and ty pe V sy stems are included in Class II of the CRISPR-Cas system.

[0106] A “nuclear localization sequence” or “nuclear localization signal” or “NLS” is a peptide that directs proteins to the nucleus. In embodiments, the NLS includes five basic, positively charged amino acids. The NLS may be located anywhere on the peptide chain. In embodiments, the NLS is an NLS derived from SV40. In embodiments, the NLS is encoded by the nucleotide sequence of SEQ ID NO: 47. In embodiments, the NLS is encoded by a nucleotide sequence including the sequence of SEQ ID NO:47. In embodiments, the NLS is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 47. In embodiments, the NLS is encoded by a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO:47. In embodiments, the NLS is encoded by a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO:47. In embodiments, the NLS is encoded by a nucleotide sequence that has at least 85% sequence identity to SEQ ID NO:47. In embodiments, the NLS is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:47. In embodiments, the NLS is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:47. In embodiments, the NLS has a nucleotide sequence of SEQ ID NO:47.

[0107] The term “5' cap structure” or “5' cap” are used herein according to its plain ordinary meaning and refers to an altered nucleotide on the 5' end of a nucleotide sequence. In embodiments, the nucleotide sequence is messenger RNA (mRNA). Non-limiting examples ofPATENT Attorney Docket No. 048536-774001WOtranscriptional regulatory sequences include an anti-reverse cap analog (ARCA) mRNA cap, a N7-methylgunosme (m7G) mRNA cap, a 3'-(9-methylation on m7G mRNA cap, a 2'-O-methylation of the first base (CAP1) mRNA cap, a / / ’-methyladenosine (m6A) mRNA cap, an alphavirus 5' mRNA cap, a symmetric two-m7G-headed mRNA cap, a 2',4'-locked nucleic acid-modified mRNA cap, a 3'-(?-benzyl-modified mRNA cap. an S mRNA cap, and a 2S mRNA cap.

[0108] The term “BCMA” or “B-cell maturation antigen” as used herein refers to any of the recombinant or naturally-occurring forms of the cell surface receptor B-cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), or variants or homologs thereof that maintain BCMA activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to BCMA). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring BCMA protein. In embodiments, the BCMA protein is substantially identical to the protein identified by UniProt No. Q02223 or a variant or homolog having substantial identity thereto.

[0109] The term “CD19” or “Cluster of Differentiation 19” as used herein refers to any of the recombinant or naturally-occurring forms of the transmembrane protein B-lymphocyte antigen CD19, also known as B-lymphocyte surface antigen B4, or variants or homologs thereof that maintain CD19 activity (e.g.. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD 19). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD 19 protein. In embodiments, the CD 19 protein is substantially identical to the protein identified by UniProt No. Pl 5391 or a variant or homolog having substantial identity thereto.

[0110] The term “CD151” or “Cluster of Differentiation 151” as used herein refers to any of the recombinant or naturally-occurring forms of the tetraspanin transmembrane protein CD151, or variants or homologs thereof that maintain CD151 activity (e.g., within at least 50%, 80%,PATENT Attorney Docket No. 048536-774001WO90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD151). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD151 protein. In embodiments, the CD151 protein is substantially identical to the protein identified by UniProt No. P48509 or a variant or homolog having substantial identity thereto.

[0111] The term “CD55” or ‘'Cluster of Differentiation 55” as used herein refers to any of the recombinant or naturally-occurring forms of the complement decay-accelerating factor, also known as decay-accelerating factor (DAF), or variants or homologs thereof that maintain CD55 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD55). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD55 protein. In embodiments, the CD55 protein is substantially identical to the protein identified by UniProt No. P08174 or a variant or homolog having substantial identity thereto.

[0112] The term “CD81” or “Cluster of Differentiation 81” as used herein refers to any of the recombinant or naturally-occurring forms of the target of the antiproliferative antibody 1 (TAPA-1), also known as tetraspanin-28 (Tspan-28), or variants or homologs thereof that maintain CD81 activity (e.g, within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD81). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD81 protein. In embodiments, the CD81 protein is substantially identical to the protein identified by UniProt No. P60033 or a variant or homolog having substantial identity thereto.

[0113] The term “CD45” or “Cluster of Differentiation 45” as used herein refers to any of the recombinant or naturally-occurring forms of protein tyrosine phosphatase, receptor type, C (PTPRC), also known as leukocyte common antigen (LCA), or variants or homologs thereof thatPATENT Attorney Docket No. 048536-774001WOmaintain CD45 activity (e.g.. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD45). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD45 protein. In embodiments, the CD45 protein is substantially identical to the protein identified by UniProt No. P08575 or a variant or homolog having substantial identity thereto.

[0114] The term “PD1” or “programmed cell death protein 1” as used herein refers to any of the recombinant or naturally-occurring forms of programmed cell death protein 1 (PD-1), also known as cluster of differentiation 279 (CD279), or variants or homologs thereof that maintain PD1 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PD1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g, a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD1 protein. In embodiments, the PD1 protein is substantially identical to the protein identified by UniProt No. Q15116 or a variant or homolog having substantial identity’ thereto.

[0115] The term “R ASA2" or “Ras p21 protein activator 2” as used herein refers to any of the recombinant or naturally-occurring forms of the GTPase-activating protein Ras p21 protein activator 2, or variants or homologs thereof that maintain RASA2 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to RASA2). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring RASA2 protein. In embodiments, the RASA2 protein is substantially identical to the protein identified by UniProt No. Q15283 or a variant or homolog having substantial identity thereto.

[0116] The term “CBL-B” as used herein refers to any of the recombinant or naturally-occurring forms of the E3 ubiquitin-protein ligase CBL-B, or variants or homologs thereof that maintain CBL-B activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CBL-B). In some aspects, the variants or homologs have at leastPATENT Attorney Docket No. 048536-774001WO90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g.. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CBL-B protein. In embodiments, the CBL-B protein is substantially identical to the protein identified by UniProt No. Q13191 or a variant or homolog having substantial identity thereto.

[0117] The term ’ GISH'’ or "‘cytokine-inducible SH2-containing protein” as used herein refers to any of the recombinant or naturally-occurring forms of the cytokine-inducible SH2-containing protein, or variants or homologs thereof that maintain CISH actin ty (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity' compared to CISH). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g, a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CISH protein. In embodiments, the CISH protein is substantially identical to the protein identified by UniProt No. Q9NSE2 or a variant or homolog having substantial identity thereto.

[0118] The term “Fas receptor” or “Fas” as used herein refers to any of the recombinant or naturally-occurring forms of the Fas cell surface death receptor, also known as apoptosis antigen 1 (APO-1), cluster of differentiation 95 (CD95), or tumor necrosis factor receptor superfamily member 6 (TNFRSF6) or variants or homologs thereof that maintain Fas activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Fas). In some aspects, the variants or homologs have at least 90%. 95%. 96%. 97%. 98%. 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Fas protein. In embodiments, the Fas protein is substantially identical to the protein identified by UniProt No. P25445 or a variant or homolog having substantial identity thereto.

[0119] The term “MED12” or “mediator of RNA polymerase II transcription subunit 12 homolog” as used herein refers to any of the recombinant or naturally-occurring forms of the mediator of RNA polymerase II transcription subunit 12 homolog (A cerevisiae), or variants or homologs thereof that maintain MED12 activity (e.g, within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity’ compared to MED12). In some aspects, the variants orPATENT Attorney Docket No. 048536-774001WOhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g.. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MED 12 protein. In embodiments, the MED12 protein is substantially identical to the protein identified by UniProt No. Q93074 or a variant or homolog having substantial identity thereto.

[0120] The term “NFKB2” or "‘nuclear factor NF-kappa-B plOO subunit” as used herein refers to any of the recombinant or naturally-occurring forms of the nuclear factor NF-kappa-B pl 00 subunit, alot known as the nuclear factor kappa-B subunit 2, or variants or homologs thereof that maintain NFKB2 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to NFKB2). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring NFKB2 protein. In embodiments, the NFKB2 protein is substantially identical to the protein identified by UniProt No. Q00653 or a variant or homolog having substantial identity thereto.

[0121] The term “PTPN2” or “protein tyrosine phosphatase nonreceptor type 2” as used herein refers to any of the recombinant or naturally-occurring forms of the protein tyrosine phosphatase nonreceptor type 2, or variants or homologs thereof that maintain PTPN2 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PTPN2). In some aspects, the variants or homologs have at least 90%. 95%. 96%. 97%. 98%. 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PTPN2 protein. In embodiments, the PTPN2 protein is substantially identical to the protein identified by UniProt No. Pl 7706 or a variant or homolog having substantial identity thereto.

[0122] The term “RC3H1” or “ring finger and CCCH-type domains 1” as used herein refers to any of the recombinant or naturally-occurring forms of the ring finger and CCCH-type domains 1 protein, also known as Roquin-1, or variants or homologs thereof that maintain RC3H1 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to RC3H1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%,PATENT Attorney Docket No. 048536-774001WO99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50. 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring RC3H1 protein. In embodiments, the RC3H1 protein is substantially identical to the protein identified by UniProt No. Q5TC82 or a variant or homolog having substantial identity thereto.

[0123] The term "SOCS 1" or "‘suppressor of cytokine signaling 1” as used herein refers to any of the recombinant or naturally-occurring forms of the suppressor of cytokine signaling 1 protein, or variants or homologs thereof that maintain SOCS1 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity' compared to SOCS1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g, a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring SOCS1 protein. In embodiments, the SOCS1 protein is substantially identical to the protein identified by UniProt No. Q15524 or a variant or homolog having substantial identity thereto.

[0124] The term “SUV39H 1 ” or “suppressor of variegation 3-9 homolog 1” as used herein refers to any of the recombinant or naturally-occurring forms of the suppressor of variegation 3-9 homolog 1, also known as SUV39H1 histone lysine methyltransferase or histone-lysine N-methyltransferase SUV39H1, or variants or homologs thereof that maintain SUV39H1 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to SUV39H1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring SUV39H1 protein. In embodiments, the SUV39H1 protein is substantially identical to the protein identified by UniProt No. 043463 or a variant or homolog having substantial identity thereto.

[0125] The term “TNFAIP3’’ or “tumor necrosis factor alpha-induced protein 3” as used herein refers to any of the recombinant or naturally-occurring forms of the tumor necrosis factor alphainduced protein 3, also know n as A20, or variants or homologs thereof that maintain TNFAIP3 activity (e.g, within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activityPATENT Attorney Docket No. 048536-774001WOcompared to TNFAIP3). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring TNFAIP3 protein. In embodiments, the TNFAIP3 protein is substantially identical to the protein identified by UniProt No. P21580 or a variant or homolog having substantial identity thereto.

[0126] The term “ZC3H12A’’ or “zinc finger CCCH-type containing 12A” as used herein refers to any of the recombinant or naturally-occurring forms of the zinc finger CCCH-type containing 12A protein, or variants or homologs thereof that maintain ZC3H12A activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ZC3H12A). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g, a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring ZC3H12A protein. In embodiments, the ZC3H12A protein is substantially identical to the protein identified by UniProt No. Q5D1E8 or a variant or homolog having substantial identity thereto.

[0127] The term “CD5” or “cluster of differentiation 5” as used herein refers to any of the recombinant or naturally-occurring forms of the cluster of differentiation 5 protein, or variants or homologs thereof that maintain CD5 activity (e.g, within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD5). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity' across the whole sequence or a portion of the sequence (e.g, a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD5 protein. In embodiments, the CD5 protein is substantially identical to the protein identified by UniProt No. P06127 or a variant or homolog having substantial identity thereto.

[0128] The term “LAG3” or “lymphocyte-activation gene 3” as used herein refers to any of the recombinant or naturally-occurring forms of the lymphocyte-activation gene 3 protein, also known as lymphocyte activating 3 or LAG-3, or variants or homologs thereof that maintain LAG3 activity (e.g., within at least 50%. 80%. 90%. 95%. 96%. 97%. 98%, 99% or 100%PATENT Attorney Docket No. 048536-774001WOactivity compared to LAG3). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring LAG3 protein. In embodiments, the LAG3 protein is substantially identical to the protein identified by UniProt No. Q9BZS 1 or a variant or homolog having substantial identity thereto.

[0129] The term “forkhead box P3” or “FOXP3” as used herein refers to any of the recombinant or naturally-occurring forms of the FOXP3 protein, also know n as scurfin, or variants or homologs thereof that maintain FOXP3 activity' (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity’ compared to FOXP3). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity^ across the whole sequence or a portion of the sequence (e g, a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring FOXP3 protein. In embodiments, the FOXP3 protein is substantially identical to the protein identified by UniProt No. Pl 8627 or a variant or homolog having substantial identity thereto. In embodiments, the FOXP3 protein is encoded by a FOXP3 gene. In embodiments, the FOXP3 gene is substantially identical to the gene identified by Ensemble entry No. ENSG00000049768 or a variant or homolog having substantial identity7thereto.

[0130] The term “transgene’' is used herein according to its plain ordinary7meaning and refers to a polynucleotide that encodes an exogenous protein.

[0131] The term “synthetic Notch receptor protein” or “SynNotch receptor protein” is used herein according to its plain ordinary meaning and refers to a recombinant Notch receptor protein capable of regulated intramembrane proteolysis. In embodiments, the SynNotch receptor protein includes an extracellular antigen recognition domain, a Notch transmembrane regulatory region, and an intracellular transcriptional regulatory domain. In embodiments, the regulated intramembrane proteolysis includes protease-mediated shedding of the extracellular antigen recognition domain, secretase-mediated cleavage of the transmembrane domain, and release of the intracellular transcriptional regulatory7domain. In embodiments, the binding of an antigen to the extracellular antigen recognition domain results in a conformational change of the SynNotchPATENT Attorney Docket No. 048536-774001WOreceptor protein. In embodiments, the conformational change of the SynNotch receptor protein exposes a protease cleavage site in the extracellular antigen recognition domain. In embodiments, the protease-mediated cleavage of the SynNotch receptor protein releases the extracellular regulatory domain. In embodiments, the conformational change of the SynNotch receptor protein exposes a secretase cleavage site in the transmembrane domain. In embodiments, the secretase-mediated cleavage releases the intracellular transcriptional regulatory domain, thereby allowing the intracellular transcriptional regulatory domain to enter the nucleus and activate a target gene. In embodiments, the SynNotch receptor protein recognizes and binds to an antigen and subsequently induces transcriptional activation of a polynucleotide encoding a CAR protein. SynNotch receptor proteins and methods of use thereof are well known the art. See Morsut et al., Cell, 2016;164(4):780-91; Roybal et al.. Cell, 2016;164(4):770-9.

[0132] The term “regulated intramembrane proteolysis” or “RIP” is used herein according to its plain ordinary meaning and refers to a process by which a transmembrane protein may be activated. In embodiments, the RIP process includes protease-mediated shedding of an extracellular domain, secretase-mediated cleavage of a transmembrane domain, and release of an intracellular transcription factor.

[0133] The term “synthetic intramembrane proteolysis receptor protein” or “SNIPR protein” is used herein according to its plain ordinary meaning and refers to a recombinant transmembrane receptor capable of regulated intramembrane proteolysis. In embodiments, the SNIPR protein includes an extracellular regulator}' domain, a transmembrane domain, and an intracellular transcriptional regulator}' domain. In embodiments, the binding of a ligand to the extracellular regulator}' domain results in a conformational change of the SNIPR protein. In embodiments, the conformational change of the SNIPR protein exposes a protease cleavage site in the extracellular regulatory domain. In embodiments, the protease-mediated cleavage of the SNIPR protein releases the extracellular regulatory domain. In embodiments, the conformational change of the SNIPR protein exposes a secretase cleavage site in the transmembrane domain. In embodiments, the secretase-mediated cleavage releases the intracellular transcriptional regulatory domain, thereby allowing the intracellular transcriptional regulatory domain to enter the nucleus and activate a target gene. In embodiments, the SNIPR protein recognizes and binds to an antigenPATENT Attorney Docket No. 048536-774001WOand subsequently induces transcriptional activation of a polynucleotide encoding a CAR protein. SNIPR proteins and methods of use thereof are well known the art. See Zhu et al.. Cell, 2022;! 85(8): 1431-43.

[0134] The term “T cell receptor protein” or “TCR protein” is used herein according to its plain ordinary meaning and refers to a protein expressed on the surface of a T cell that is responsible for antigen recognition. In embodiments, the TCR protein binds an antigen peptide bound to a major histocompatibility complex. In embodiments, the TCR protein is an alpha (a) chain, a beta (P) chain, a gamma (y) chain, or a delta (5) chain. In embodiments, the binding of an antigen peptide to the TCR protein results in the activation of the T cell.

[0135] The term “T cell receptor alpha constant gene” or “TRAC gene” is used herein according to its plain ordinary meaning and refers to a gene that encodes the constant region of a T cell receptor alpha chain.

[0136] The term “chimeric antigen receptor protein” or “CAR protein” is used herein according to its plain ordinary meaning and refers to a recombinant protein that includes both antigen binding and T cell activating functions. In embodiments, the CAR protein includes a heavy chain variable domain and a light chain variable domain which binds antigens. In embodiments, the binding of a CAR to an antigen activates a T cell.

[0137] The term “HLA-independent T cell receptor protein” or “HIT receptor protein” is used herein according to its plain ordinary' meaning and refers to a recombinant T cell receptor protein that includes the heavy chain and light chain variable domains of a chimeric antigen receptor. HIT receptor proteins and methods of use thereof are well known the art. See Mansilla-Soto et al., Nat Med. 2022; 28(2): 345-352.

[0138] A "cell" as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability’ to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokary otic cells include but are not limited to bacteria. Eukaryotic cells include, but are notPATENT Attorney Docket No. 048536-774001WOlimited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.

[0139] As used herein, the term ‘'vector’’ refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a linear or circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. 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.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Additionally, some viral vectors are capable of targeting a particular cells type either specifically or non-specifically. Replicationincompetent viral vectors or replication-defective viral vectors refer to viral vectors that are capable of infecting their target cells and delivering their viral pay load, but then fail to continue the typical lytic pathway that leads to cell lysis and death.

[0140] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule and / or a protein to a cell. Nucleic acids may be introduced to a cell using non-viral or viral-based methods. The nucleic acid molecule can be a sequence encoding complete proteins or functional portions thereof. Typically, a nucleic acid vector, comprising the elements necessary for protein expression (e.g., a promoter, transcription start site, etc.). Non-viral methods of transfection include any appropriate method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfectionPATENT Attorney Docket No. 048536-774001WOmethods include nanoparticle encapsulation of the nucleic acids that encode the fusion protein (e.g., lipid nanoparticles, gold nanoparticles, and the like), calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. For viral-based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms "transfection" or "transduction" also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g.. Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0141] A “peptide linker’ as provided herein is a linker including a peptide moiety. In embodiments, the peptide linker is a divalent peptide, such as an amino acid sequence attached at the N-terminus and the C-terminus to the remainder of the compound (e.g., fusion protein provided herein. The peptide linker may be a peptide moiety (a divalent peptide moiety) capable of being cleaved (e.g., a P2A cleavable polypeptide). A peptide linker as provided herein may also be referred to interchangeably as an amino acid linker. In embodiments, the peptide linker includes 1 to about 80 amino acid residues. In embodiments, the peptide linker includes 1 to about 70 amino acid residues. In embodiments, the peptide linker includes 1 to about 60 amino acid residues. In embodiments, the peptide linker includes 1 to about 50 amino acid residues. In embodiments, the peptide linker includes 1 to about 40 amino acid residues. In embodiments, the peptide linker includes 1 to about 30 amino acid residues. In embodiments, the peptide linker includes 1 to about 25 amino acid residues. In embodiments, the peptide linker includes 1 to about 20 amino acid residues. In embodiments, the peptide linker includes about 2 to about 20 amino acid residues. In embodiments, the peptide linker includes about 2 to about 19 amino acid residues. In embodiments, the peptide linker includes about 2 to about 18 amino acid residues. In embodiments, the peptide linker includes about 2 to about 17 amino acid residues. In embodiments, the peptide linker includes about 2 to about 16 amino acid residues. In embodiments, the peptide linker includes about 2 to about 15 amino acid residues. InPATENT Attorney Docket No. 048536-774001WOembodiments, the peptide linker includes about 2 to about 14 amino acid residues. In embodiments, the peptide linker includes about 2 to about 13 amino acid residues. In embodiments, the peptide linker includes about 2 to about 12 amino acid residues. In embodiments, the peptide linker includes about 2 to about 11 amino acid residues. In embodiments, the peptide linker includes about 2 to about 10 amino acid residues. In embodiments, the peptide linker includes about 2 to about 9 amino acid residues. In embodiments, the peptide linker includes about 2 to about 8 amino acid residues. In embodiments, the peptide linker includes about 2 to about 7 amino acid residues. In embodiments, the peptide linker includes about 2 to about 6 amino acid residues. In embodiments, the peptide linker includes about 2 to about 5 amino acid residues. In embodiments, the peptide linker includes about 2 to about 4 amino acid residues. In embodiments, the peptide linker includes about 2 to about 3 amino acid residues. In embodiments, the peptide linker includes about 3 to about 19 amino acid residues. In embodiments, the peptide linker includes about 3 to about 18 amino acid residues. In embodiments, the peptide linker includes about 3 to about 17 amino acid residues. In embodiments, the peptide linker includes about 3 to about 1 amino acid residues. In embodiments, the peptide linker includes about 3 to about 15 amino acid residues. In embodiments, the peptide linker includes about 3 to about 14 amino acid residues. In embodiments, the peptide linker includes about 3 to about 13 amino acid residues. In embodiments, the peptide linker includes about 3 to about 12 amino acid residues. In embodiments, the peptide linker includes about 3 to about 11 amino acid residues. In embodiments, the peptide linker includes about 3 to about 10 amino acid residues. In embodiments, the peptide linker includes about 3 to about 9 amino acid residues. In embodiments, the peptide linker includes about 3 to about 8 amino acid residues. In embodiments, the peptide linker includes about 3 to about 7 amino acid residues. In embodiments, the peptide linker includes about 3 to about 6 amino acid residues. In embodiments, the peptide linker includes about 3 to about 5 amino acid residues. In embodiments, the peptide linker includes about 3 to about 4 amino acid residues. In embodiments, the peptide linker includes about 10 to about 20 amino acid residues. In embodiments, the peptide linker includes about 15 to about 20 amino acid residues. InPATENT Attorney Docket No. 048536-774001WOembodiments, the peptide linker includes about 2 amino acid residues. In embodiments, the peptide linker includes about 3 amino acid residues. In embodiments, the peptide linker includes about 4 amino acid residues. In embodiments, the peptide linker includes about 5 amino acid residues. In embodiments, the peptide linker includes about 6 amino acid residues. In embodiments, the peptide linker includes about 7 amino acid residues. In embodiments, the peptide linker includes about 8 amino acid residues. In embodiments, the peptide linker includes about 9 amino acid residues. In embodiments, the peptide linker includes about 10 amino acid residues. In embodiments, the peptide linker includes about 11 amino acid residues. In embodiments, the peptide linker includes about 12 amino acid residues. In embodiments, the peptide linker includes about 13 amino acid residues. In embodiments, the peptide linker includes about 14 amino acid residues. In embodiments, the peptide linker includes about 15 amino acid residues. In embodiments, the peptide linker includes about 16 amino acid residues. In embodiments, the peptide linker includes about 17 amino acid residues. In embodiments, the peptide linker includes about 18 amino acid residues. In embodiments, the peptide linker includes about 19 amino acid residues. In embodiments, the peptide linker includes about 20 amino acid residues. In embodiments, the peptide linker includes about 21 amino acid residues. In embodiments, the peptide linker includes about 22 amino acid residues. In embodiments, the peptide linker includes about 23 amino acid residues. In embodiments, the peptide linker includes about 24 amino acid residues. In embodiments, the peptide linker includes about 25 amino acid residues.

[0142] In embodiments, the peptide linker is encoded by a nucleotide sequence that includes the sequence of SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:50. In embodiments, the peptide linker is encoded by a nucleotide sequence that includes the sequence of SEQ ID NO:44. In embodiments, the peptide linker is encoded by a nucleotide sequence that includes the sequence of SEQ ID NO:46. In embodiments, the peptide linker is encoded by a nucleotide sequence that includes the sequence of SEQ ID NO: 50. In embodiments, the peptide linker is encoded by the nucleotide sequence of SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:50. In embodiments, the peptide linker is encoded by the nucleotide sequence of SEQ ID NO:44. In embodiments, the peptide linker is encoded by the nucleotide sequence of SEQ ID NO:46. In embodiments, the peptide linker is encoded by the nucleotide sequence of SEQ ID NO:50. In embodiments, thePATENT Attorney Docket No. 048536-774001WOpeptide linker is an XTEN polypeptide. In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:44, 46, or 50. In embodiments, the peptide linker is encoded by the nucleotide sequence of SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:50. In embodiments, the peptide linker is encoded by the nucleotide sequence of SEQ ID NO:44. In embodiments, the peptide linker is encoded by the nucleotide sequence of SEQ ID NO:46. In embodiments, the peptide linker is encoded by the nucleotide sequence of SEQ ID NO:50.

[0143] In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:44, 46, or 50. In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:44. In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 46. In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:50.

[0144] In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:44, 46, or 50. In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:44. In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 46. In embodiments, the peptide linker is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:50.

[0145] The terms ‘“XTEN,” “XTEN linker,” or “XTEN polypeptide” as used herein refer to a recombinant polypeptide (e.g. unstructured recombinant peptide) lacking hydrophobic amino acid residues. The development and use of XTEN can be found in, for example, Schellenberger et al., Nature Biotechnology 27, 1186-1190 (2009), which is incorporated herein by reference in its entirety' and for all purposes. In embodiments, the XTEN linker is an XTEN 16 linker, an XTEN80 linker, or an XTEN 100 linker. In embodiments, the XTEN linker is an XTEN 16 linker. In embodiments, the XTEN linker is an XTEN80 linker. In embodiments, the XTEN linker is an XTEN100 linker. In embodiments, the XTEN linker is encoded by a nucleotide sequence that includes the sequence of SEQ ID NO:46. In embodiments, the XTEN linker is encoded by thePATENT Attorney Docket No. 048536-774001WOnucleotide sequence of SEQ ID NO:46. In embodiments, the XTEN linker is encoded by a nucleotide sequence that includes the sequence of SEQ ID NO:50. In embodiments, the XTEN linker is encoded by the nucleotide sequence of SEQ ID NO: 0. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:46. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:46. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:46. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NO:50. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:50. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:50. In embodiments, the XTEN linker is encoded by a nucleotide sequence that includes the sequence of SEQ ID NO: 143. In embodiments, the XTEN linker is encoded by the nucleotide sequence of SEQ ID NO: 143. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 143. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 143. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 143. In embodiments, the XTEN linker is encoded by a nucleotide sequence that includes the sequence of SEQ ID NO: 144. In embodiments, the XTEN linker is encoded by the nucleotide sequence of SEQ ID NO: 144. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 144. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 144. In embodiments, the XTEN linker is encoded by a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 144. In embodiments, the XTEN linker includes the amino acid sequence of any one of SEQ ID NOs: 166-168. In embodiments, the XTEN linker includes anPATENT Attorney Docket No. 048536-774001WOamino acid sequence that has at least 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 91%.92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 166. In embodiments, the XTEN linker includes an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 167. In embodiments, the XTEN linker includes an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%. 91%.92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 168.

[0146] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0147] The term ■’contacting" may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a fusion protein as provided herein and a nucleic acid sequence (e.g., target DNA sequence).

[0148] As defined herein, the term "inhibition", "inhibit", "inhibiting," "repression." repressing,” “silencing,” “silence” and the like when used in reference to a composition as provided herein (e.g., fusion protein, complex, nucleic acid, vector) refer to negatively affecting (e.g., decreasing) the activity (e.g.. transcription) of a nucleic acid sequence (e.g.. decreasing transcription of a gene) relative to the activity of the nuclei acid sequence (e.g., transcription of a gene) in the absence of the composition (e.g., fusion protein, complex, nucleic acid, vector). In embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer). Thus, inhibition includes, at least in part, partially or totally blocking activation (e.g., transcription), or decreasing, preventing, or delaying activation (e.g., transcnption) of the nucleic acid sequence. The inhibited activity (e.g., transcription) may be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than that in a control. In embodiments, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold. 5-fold, 10-fold, or more in comparison to a control.

[0149] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsyPATENT Attorney Docket No. 048536-774001WOsamples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluidjoint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0150] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, etc).

[0151] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.PATENT Attorney Docket No. 048536-774001WO

[0152] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirely for all purposes.NUCLEIC ACID COMPOSITIONS

[0153] Provided herein are, inter alia, nucleic acid compositions (i.e. polynucleotides) including nucleotide sequences described herein including embodiments thereof. Thus, in an aspect is provided a polynucleotide including the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136.

[0154] In embodiments, the polynucleotide includes the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide includes the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide is the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135. or SEQ ID NO: 136. In embodiments, the polynucleotide is the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide is the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide is the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide is the nucleotide sequence of SEQ ID NO: 136.

[0155] In embodiments, the polynucleotide includes a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 90% sequence identity to thePATENT Attorney Docket No. 048536-774001WOnucleotide sequence of SEQ ID NOT, SEQ ID NO:2, SEQ ID NO: 135. or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NOT, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 100% sequence identity' to the nucleotide sequence of SEQ ID NOT, SEQ ID NO:2, SEQ ID NO: 135. or SEQ ID NO: 136.

[0156] In embodiments, the polynucleotide includes a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide includes a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide includes a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide includes a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide includes a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide includes a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide includes a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide includes a nucleotide sequence having at least 97% sequence identity' to the nucleotide sequence of SEQ ID NOT. In embodiments, the polynucleotide includes a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NOT. In embodiments, the polynucleotide includes a nucleotide sequence having at least 99% sequence identity to the nucleotide sequencePATENT Attorney Docket No. 048536-774001WOof SEQ ID NO: 1. In embodiments, the polynucleotide includes a nucleotide sequence having at least 100% sequence identity to the nucleotide sequence of SEQ ID NO: 1.

[0157] In embodiments, the polynucleotide includes a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 98% sequence identity7to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:2. In embodiments, the polynucleotide includes a nucleotide sequence having at least 100% sequence identity to the nucleotide sequence of SEQ ID NO:2.

[0158] In embodiments, the polynucleotide includes a nucleotide sequence having at least 70% sequence identity7to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 80% sequence identity7to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 85% sequence identity7to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ IDPATENT Attorney Docket No. 048536-774001WONO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the polynucleotide includes a nucleotide sequence having at least 100% sequence identity to the nucleotide sequence of SEQ ID NO: 135.

[0159] In embodiments, the polynucleotide includes a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the polynucleotide includes a nucleotide sequence having at least 100% sequence identity to the nucleotide sequence of SEQ ID NO: 136.

[0160] In embodiments, the polynucleotide is messenger RNA(mRNA).PATENT Attorney Docket No. 048536-774001WOEXPRESSION VECTOR COMPOSITIONS

[0161] The compositions provided herein including embodiments thereof include expression vector compositions. The expression vectors include nucleic acid compositions provided herein including embodiments thereof as described in detail throughout this application (including the description above and in the examples section). Thus, in an aspect is provided a vector including the polynucleotide provided herein including embodiments thereof.

[0162] In embodiments, the vector includes a polynucleotide including the nucleotide sequence of SEQ ID NOT, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the vector includes a polynucleotide including the nucleotide sequence of SEQ ID NO:1. In embodiments, the vector includes a polynucleotide including the nucleotide sequence of SEQ ID NO:2. In embodiments, the vector includes a polynucleotide including the nucleotide sequence of SEQ ID NO: 135. In embodiments, the vector includes a polynucleotide including the nucleotide sequence of SEQ ID NO: 136. In embodiments, the vector includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the vector includes a polynucleotide having the nucleotide sequence of SEQ ID NOT. In embodiments, the vector includes a polynucleotide having the nucleotide sequence of SEQ ID NO:2. In embodiments, the vector includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 135. In embodiments, the vector includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 136.

[0163] In embodiments, the expression vector is a viral expression vector. In embodiments, the viral expression vector is an adeno-associated virus vector.CELLULAR COMPOSITIONS

[0164] The compositions provided herein including embodiments thereof include cellular compositions. The cells include nucleic acid compositions provided herein including embodiments thereof or expression vector compositions provided herein including embodiments thereof as described in detail throughout this application (including the description above and in the examples section). Thus, in an aspect is provided a cell including the polynucleotide provided herein including embodiments thereof or the vector provided herein including embodiments thereof.PATENT Attorney Docket No. 048536-774001WO

[0165] In embodiments, the cell includes a polynucleotide including the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the cell includes a polynucleotide including the nucleotide sequence of SEQ ID NO: 1. In embodiments, the cell includes a polynucleotide including the nucleotide sequence of SEQ ID NO: 2. In embodiments, the cell includes a polynucleotide including the nucleotide sequence of SEQ ID NO: 135. In embodiments, the cell includes a polynucleotide including the nucleotide sequence of SEQ ID NO: 136. In embodiments, the cell includes a polynucleotide having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the cell includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 1. In embodiments, the cell includes a polynucleotide having the nucleotide sequence of SEQ ID NO:2. In embodiments, the cell includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 135. In embodiments, the cell includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 136.

[0166] In embodiments, the cell includes a vector including a polynucleotide including the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 135. or SEQ ID NO: 136. In embodiments, the cell includes a vector including a polynucleotide including the nucleotide sequence of SEQ ID NO: 1. In embodiments, the cell includes a vector including a polynucleotide including the nucleotide sequence of SEQ ID NO:2. In embodiments, the cell includes a vector including a polynucleotide including the nucleotide sequence of SEQ ID NO: 135. In embodiments, the cell includes a vector including a polynucleotide including the nucleotide sequence of SEQ ID NO: 136. In embodiments, the cell includes a vector including a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 135, or SEQ ID NO: 136. In embodiments, the cell includes a vector including a polynucleotide having the nucleotide sequence of SEQ ID NO: 1. In embodiments, the cell includes a vector including a polynucleotide having the nucleotide sequence of SEQ ID NO:2. In embodiments, the cell includes a vector including a polynucleotide having the nucleotide sequence of SEQ ID NO: 135. In embodiments, the cell includes a vector including a polynucleotide having the nucleotide sequence of SEQ ID NO: 136.

[0167] In embodiments, the cell is a eukaryotic cell. In embodiments, the cell is a mammalian cell. In embodiments, the cell is an immune cell. In embodiments, the cell is a T cell.PATENT Attorney Docket No. 048536-774001WO

[0168] In another aspect is provided, a cell including a first polynucleotide, a first single guide RNA (sgRNA), a ribonucleoprotein, and a second polynucleotide, wherein the first polynucleotide encodes a fusion protein including a nuclease-deficient RNA-guided DNA endonuclease domain, a Kriippel associated box domain, and a DNA methyltransferase domain; wherein the first sgRNA binds the nuclease deficient RNA-guided DNA endonuclease domain; wherein the ribonucleoprotein includes an orthogonal RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; and wherein the second polynucleotide encodes a transgene.

[0169] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9, ddCpfl, a nuclease-deficient Cas9 variant, a nuclease-deficient Casl2 variant, or a nuclease-deficient Class II CRISPR endonuclease. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain encodes ddCpfl. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain encodes a nuclease-deficient Cas9 variant. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain encodes a nuclease-deficient Casl2 variant. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain encodes a nuclease-deficient Class II CRISPR endonuclease.

[0170] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a Staphylococcus aureus Cas9 (SaCas9) enzyme domain, a Staphylococcus pyogenes Cas9 (SpCas9) enzyme domain, an Act daminococcus sp. Casl2a (AsCasl2a) enzyme domain, or an Acidaminococcus sp. Casl2b (AsCasl2b) enzyme domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a Staphylococcus aureus Cas9 (SaCas9) enzyme domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is a Staphylococcus pyogenes Cas9 (SpCas9) enzy me domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is an Acidaminococcus sp. Cast 2a (AsCasl2a) enzyme domain. In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain is an Acidaminococcus sp. Cast 2b (AsCasl2b) enzyme domain.

[0171] In embodiments, the orthogonal RNA-guided DNA endonuclease domain is a Cas9 enzy me domain or a Casl2a enzy me domain. In embodiments, the orthogonal RNA-guidedPATENT Attorney Docket No. 048536-774001WODNA endonuclease domain is a Cas9 enzyme domain. In embodiments, the orthogonal RNA-guided DNA endonuclease domain is a Cast 2a enzyme domain.

[0172] In embodiments, the DNA methyltransferase domain encodes a Dnmt3A-3L domain.

[0173] In embodiments, the first polynucleotide includes, from N-terminus to C-terminus, the DNA methyltransferase domain, the nuclease-deficient RNA-guided DNA endonuclease domain, and the Kriippel associated box domain.

[0174] In embodiments, the fusion protein further includes a first XTEN linker and a second XTEN linker. In embodiments, the fusion protein further includes a first XTEN linker. In embodiments, the fusion protein further includes a second XTEN linker.

[0175] In embodiments, the fusion protein includes, from N-terminus to C-terminus, the DNA methyltransferase domain, the first XTEN linker, the nuclease-deficient RNA-guided DNA endonuclease domain, the second XTEN linker, and the Kriippel associated box domain.

[0176] In embodiments, the first XTEN linker includes from about 50 to about 864 amino acid residues, and the second XTEN linker includes from about 5 to about 864 amino acid residues. In embodiments, the first XTEN linker includes from about 50 to about 864 amino acid residues. In embodiments, the first XTEN linker includes from about 50 to about 100 amino acid residues. In embodiments, the first XTEN linker includes from about 60 to about 90 amino acid residues. In embodiments, the first XTEN linker includes from about 70 to about 90 ammo acid residues. In embodiments, the first XTEN linker includes about 80 amino acid residues (XTEN80). In embodiments, the first XTEN linker includes about 100 amino acid residues (XTEN100). In embodiments, the second XTEN linker includes from about 5 to about 864 amino acid residues. In embodiments, the second XTEN linker includes from about 5 to about 100 amino acid residues. In embodiments, the second XTEN linker includes from about 5 to about 80 amino acid residues. In embodiments, the second XTEN linker includes from about 10 to about 80 amino acid residues. In embodiments, the second XTEN linker includes about 16 amino acid residues (XTEN 16). In embodiments, the first XTEN linker includes about 80 amino acid residues (XTEN80), and the second XTEN linker includes about 16 amino acid residues (XTEN 16).PATENT Attorney Docket No. 048536-774001WO

[0177] In embodiments, the first XTEN linker is from about 50 to about 864 amino acid residues, and the second XTEN linker is from about 5 to about 864 amino acid residues. In embodiments, the first XTEN linker is from about 50 to about 864 amino acid residues. In embodiments, the first XTEN linker is about 80 amino acid residues (XTEN80). In embodiments, the first XTEN linker is about 100 amino acid residues (XTEN 100). In embodiments, the second XTEN linker is from about 5 to about 864 amino acid residues. In embodiments, the second XTEN linker is about 16 amino acid residues (XTEN 16). In embodiments, the first XTEN linker is about 80 amino acid residues (XTEN80), and the second XTEN linker is about 16 amino acid residues (XTEN16).

[0178] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9 and the DNA methyltransferase domain encodes a Dnmt3A-3L domain.

[0179] In embodiments, the first polynucleotide further includes a 5' cap structure. In embodiments, the 5' cap structure includes an anti-reverse cap analog (ARCA) mRNA cap, N1-methylgunosine (m7G) mRNA cap, a 3'-( -methylation on m7G mRNA cap, a 2'-<9-methylation of the first base (CAP1) mRNA cap, a A6-methyl adenosine (m6A) mRNA cap, an alphavirus 5' mRNA cap, a symmetric two-m7G-headed mRNA cap, a 2'.4'-locked nucleic acid-modified mRNA cap, a 3'-O-benzyl-modified mRNA cap, an S mRNA cap, or a 2S mRNA cap. In embodiments, the 5' cap structure includes an anti-reverse cap analog (ARCA) mRNA cap. In embodiments, the 5' cap structure includes a A'7-methylgunosine (m7G) mRNA cap. In embodiments, the 5' cap structure includes a 3'-< -methylation on m7G mRNA cap. In embodiments, the 5' cap structure includes a 2'-0-methylation of the first base (CAP1) mRNA cap. In embodiments, the 5' cap structure includes a Af’-methyladenosine (m6A) mRNA cap. In embodiments, the 5' cap structure includes an alphavirus 5' mRNA cap. In embodiments, the 5' cap structure includes a symmetric two-m7G-headed mRNA cap. In embodiments, the 5' cap structure includes a 2',4'-locked nucleic acid-modified mRNA cap. In embodiments, the 5' cap structure includes a 3'-0-benzyl-modified mRNA cap. In embodiments, the 5' cap structure includes an S mRNA cap. In embodiments, the 5' cap structure includes a 2S mRNA cap.

[0180] In embodiments, the first polynucleotide includes at least one uridine substitution, pseudouridine substitution. N1-methyl pseudouridine substitution, 2-thiouridine substitution, 5-PATENT Attorney Docket No. 048536-774001WOmethylcytidine substitution, A^’-melhyladenosine substitution. 2'-<9-methyluridme substitution, 2'-<9-methylcytidine substitution, 2'-(?-methyladenosine substitution, or 2'-<9-methylguanosine substitution. In embodiments, the first polynucleotide includes at least one uridine substitution. In embodiments, the first polynucleotide includes at least one pseudouridine substitution. In embodiments, the first polynucleotide includes at least one N1-methyl pseudouridine substitution. In embodiments, the first polynucleotide includes at least one 2-thiouridine substitution. In embodiments, the first polynucleotide includes at least one 5-methylcytidine substitution. In embodiments, the first polynucleotide includes at least one / V’-methy I adenosine substitution. In embodiments, the first polynucleotide includes at least one 2'-(9-methyluridine substitution. In embodiments, the first polynucleotide includes at least one 2'-(9-methylcylidine substitution. In embodiments, the first polynucleotide includes at least one 2'-0-methyladenosine substitution. In embodiments, the first polynucleotide includes at least one 2'-( -methylguanosine substitution.

[0181] In embodiments, the first sgRNA includes the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:3. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:4. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:5. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:6. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:7. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 8. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NON. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NOTO. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 11. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 12. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 13. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 14. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 15. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 16. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 17. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 18. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 19. InPATENT Attorney Docket No. 048536-774001WOembodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:20. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:21. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:22. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:23. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:24 In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:25. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:26. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:27. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:28. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:29. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:30.

[0182] In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:54. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:55. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:56. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:57. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:58. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:59. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:60. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:61. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:62. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:63. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:64. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:65. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:66. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:67. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:68. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:69.

[0183] In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:70. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:71. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:72. InPATENT Attorney Docket No. 048536-774001WOembodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:73. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:74. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:75. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:76. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:77. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:78. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:79. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 80. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:81. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 82. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:83. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 84. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:85. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 86. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 87. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 88. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 89.

[0184] In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:90. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:91. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:92. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:93. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:94. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:95. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:96. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:97. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:98. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO:99. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 100. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 101. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 102. InPATENT Attorney Docket No. 048536-774001WOembodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 103. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 104. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 105. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 106. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 107. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 108. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 109.

[0185] In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 110. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 111. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 112. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 113. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 114. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 115. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 116. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 117. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 118. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 119. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 120. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 121. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 122. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 123. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 124. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 125. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 126. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 127. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 128. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 129. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 130. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 131. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 132. InPATENT Attorney Docket No. 048536-774001WOembodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 133. In embodiments, the first sgRNA includes the nucleotide sequence of SEQ ID NO: 134.

[0186] In embodiments, the first sgRNA is the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:3. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:4. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:5. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:6. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:7. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 8. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:9. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NOTO. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 11. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 12. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 13. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 14. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 15. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 16. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 17. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 18. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 19. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:20. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:21. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:22. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:23. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 24 In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:25. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:26. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:27. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:28. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:29. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 30.

[0187] In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:54. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:55. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 56. In embodiments, the first sgRNAPATENT Attorney Docket No. 048536-774001WOis the nucleotide sequence of SEQ ID NO:57. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:58. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:59. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:60. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:61. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 62. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:63. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:64. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:65. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:66. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 67. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 68. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:69.

[0188] In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:70. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:71. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 72. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:73. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:74. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:75. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:76. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 77. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 78. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:79. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 80. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:81. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:82. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:83. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 84. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:85. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 86. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:87. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:88. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:89.

[0189] In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:90. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:91. In embodiments,PATENT Attorney Docket No. 048536-774001WOthe first sgRNA is the nucleotide sequence of SEQ ID NO: 92. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:93. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:94. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:95. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:96. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:97. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 98. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:99. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 100. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 101. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 102. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 103. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 104. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 105. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 106. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 107. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 108. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 109.

[0190] In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 110. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO:111. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 112. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 113. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 114. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 115. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 116. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 117. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 118. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 119. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 120. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 121. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 122. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 123. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 124. In embodiments, the first sgRNA is the nucleotide sequence of SEQ IDPATENT Attorney Docket No. 048536-774001WONO: 125. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 126. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 127. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 128. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 129. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 130. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 131. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 132. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 133. In embodiments, the first sgRNA is the nucleotide sequence of SEQ ID NO: 134.

[0191] In embodiments, the second sgRNA targets a gene encoding a T cell receptor protein. In embodiments, the T cell receptor protein is T cell receptor alpha constant (TRAC) protein.

[0192] In embodiments, the second sgRNA hybridizes to a second target nucleic acid sequence in a gene encoding a T cell receptor protein. In embodiments, the second target nucleic acid sequence is a portion of a T cell receptor alpha constant (TRAC) gene. In embodiments, the second target nucleic acid sequence is a portion of an exon of a TRAC gene or a portion of an intron of a TRAC gene. In embodiments, the second target nucleic acid sequence is a portion of an exon of a TRAC gene. In embodiments, the second target nucleic acid sequence is a portion of an intron of a TRAC gene. In embodiments, the second target nucleic acid sequence is a portion of exon 1 of a TRAC gene. In embodiments, the second sgRNA includes the nucleotide sequence of SEQ ID NO:31. In embodiments, the second sgRNA is the nucleotide sequence of SEQ ID NO:31. In embodiments, the second target nucleic acid sequence is a portion of an intergenic region of a genome. In embodiments, the second target nucleic acid sequence is a portion of a genomic safe harbor site of a genome.

[0193] In embodiments, the cell further includes: (a) a third sgRNA; (b) a third sgRNA and a fourth sgRNA; (c) a third sgRNA, a fourth sgRNA, and a fifth sgRNA; or (d) a third sgRNA. a fourth sgRNA, a fifth sgRNA, and a sixth sgRNA. In embodiments, the cell further includes a third sgRNA. In embodiments, the cell further includes a third sgRNA and a fourth sgRNA. In embodiments, the cell further includes a third sgRNA, a fourth sgRNA, and a fifth sgRNA. In embodiments, the cell further includes a third sgRNA, a fourth sgRNA, a fifth sgRNA, and a sixth sgRNA.PATENT Attorney Docket No. 048536-774001WO

[0194] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:3. In embodiments, the third sgRNA. the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 4. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:5. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:6. In embodiments, the third sgRNA. the fourth sgRNA. the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 7. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:8. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NON. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 10. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 11. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 12. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 13. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 14. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 15. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 16. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 17. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 18. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA,PATENT Attorney Docket No. 048536-774001WOand / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 19. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:20. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:21. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:22. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:23. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:24. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:25. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:26. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:27. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:28. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:29. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 30.

[0195] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:54. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 55. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 56. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:57. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:58. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include thePATENT Attorney Docket No. 048536-774001WOnucleotide sequence of SEQ ID NO:59. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:60. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:61. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:62. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:63. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:64. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:65. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:66. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:67. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:68. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:69.

[0196] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:70. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 71. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 72. In embodiments, the third sgRNA. the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:73. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:74. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA. and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:75. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQPATENT Attorney Docket No. 048536-774001WOID NO:76. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:77. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:78. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 79. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:80. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:81. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA. and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 82. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:83. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 84. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:85. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 86. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:87. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 88. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 89.

[0197] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:90. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 91. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:92. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:93. InPATENT Attorney Docket No. 048536-774001WOembodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:94. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:95. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:96. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:97. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:98. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO:99. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 100. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 101. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA. and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 102. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 103. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 104. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 105. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 106. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 107. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 108. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 109.

[0198] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 110. In embodiments, thePATENT Attorney Docket No. 048536-774001WOthird sgRNA. the fourth sgRNA. the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 111. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 112. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 113. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 114. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 115. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 116. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 117. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 118. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA. and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 119. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 120. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 121. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 122. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 123. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 124. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 125. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 126. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 127. In embodiments, the third sgRNA, the fourth sgRNA,PATENT Attorney Docket No. 048536-774001WOthe fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 128. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 129. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 130. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA. and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 131. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 132. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 133. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently include the nucleotide sequence of SEQ ID NO: 134.

[0199] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:3. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:4. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:5. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:6. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:7. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:8. In embodiments, the third sgRNA. the fourth sgRNA. the fifth sgRNA. and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:9. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NOTO. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 11. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 12. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and thePATENT Attorney Docket No. 048536-774001WOsixth sgRNA include the nucleotide sequence of SEQ ID NO: 13. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 14. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 15. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 16. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 17. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 18. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 19. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:20. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:21. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 22. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:23. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:24. In embodiments, the third sgRNA. the fourth sgRNA. the fifth sgRNA. and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:25. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:26. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:27. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:28. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 29. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NQ:30.

[0200] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:54. In embodiments, the third sgRNA,PATENT Attorney Docket No. 048536-774001WOthe fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:55. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:56. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:57. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 58. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:59. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 60. In embodiments, the third sgRNA. the fourth sgRNA. the fifth sgRNA. and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:61. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:62. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:63. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA. and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:64. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 65. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:66. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 67. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:68. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:69.

[0201] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:70. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:71. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:72. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotidePATENT Attorney Docket No. 048536-774001WOsequence of SEQ ID NO:73. In embodiments, the third sgRNA. the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 74. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:75. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 76. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:77. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:78. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:79. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 80. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 81. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 82. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 83. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:84. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 85. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 86. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 87. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 88. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 89.

[0202] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:90. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:91. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and thePATENT Attorney Docket No. 048536-774001WOsixth sgRNA include the nucleotide sequence of SEQ ID NO:92. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:93. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 94. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:95. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 96. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:97. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:98. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:99. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 100. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 101. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 102. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 103. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 104. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 105. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 106. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 107. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 108. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 109.

[0203] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO:110. In embodiments, the third sgRNA,PATENT Attorney Docket No. 048536-774001WOthe fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 111. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 112. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 113. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 114. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 115. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 116. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 117. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 118. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 119. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA. and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 120. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 121. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 122. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 123. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 124. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 125. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 126. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 127. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 128. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 129. In embodiments, the third sgRNA, thePATENT Attorney Docket No. 048536-774001WOfourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 130. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 131. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 132. In embodiments, the third sgRNA, the fourth sgRNA. the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 133. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, and the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 134.

[0204] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 3. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:4. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 5. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:6. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 7. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 8. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 9. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NOTO. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 11. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 12. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 13. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 14. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 15. In embodiments, the third sgRNA, the fourthPATENT Attorney Docket No. 048536-774001WOsgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 16. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 17. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 18. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 19. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 20. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:21. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 22. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:23. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 24. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:25. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 26. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:27. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 28. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:29. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 30.

[0205] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:54. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:55. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:56. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:57. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:58. In embodiments, the third sgRNA,PATENT Attorney Docket No. 048536-774001WOthe fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:59. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:60. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:61. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:62. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:63. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:64. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:65. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:66. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:67. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:68. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:69.

[0206] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:70. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:71. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:72. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:73. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:74. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:75. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:76. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:77. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixthPATENT Attorney Docket No. 048536-774001WOsgRNA include the nucleotide sequence of SEQ ID NO:78. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:79. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:80. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:81. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:82. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:83. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:84. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:85. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:86. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:87. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:88. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:89.

[0207] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:90. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:91. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:92. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:93. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:94. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:95. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:96. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQPATENT Attorney Docket No. 048536-774001WOID NO:97. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:98. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:99. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 100. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 101. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 102. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 103. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 104. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 105. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 106. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 107. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 108. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 109.

[0208] In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:110. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 111. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 112. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:113. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:114. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 115. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:116. In embodiments, the third sgRNA,PATENT Attorney Docket No. 048536-774001WOthe fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 117. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:118. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 119. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 120. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO:121. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 122. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 123. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 124. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 125. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 126. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 127. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 128. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 129. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 130. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 131. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 132. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 133. In embodiments, the third sgRNA, the fourth sgRNA, the fifth sgRNA, or the sixth sgRNA include the nucleotide sequence of SEQ ID NO: 134.

[0209] In embodiments, the third sgRNA includes the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:3. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ IDPATENT Attorney Docket No. 048536-774001WONO:4. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 5. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:6. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:7. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 8. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:9. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 10. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 11. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 12. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 13. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 14. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 15. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 16. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 17. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 18. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 19. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:20. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:21. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:22. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:23. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:24. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:25. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:26. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:27. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:28. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:29. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:30.

[0210] In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:54. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:55. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:56. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:57. InPATENT Attorney Docket No. 048536-774001WOembodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:58. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:59. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:60. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:61. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:62. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:63. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:64. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:65. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:66. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:67. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:68. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:69.

[0211] In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:70. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:71. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:72. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:73. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:74. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:75. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 76. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:77. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:78. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:79. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 80. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:81. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 82. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 83. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 84. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 85. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 86. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 87. InPATENT Attorney Docket No. 048536-774001WOembodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 88. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 89.

[0212] In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:90. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:91. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:92. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:93. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:94. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:95. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:96. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:97. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:98. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO:99. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 100. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 101. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 102. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 103. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 104. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 105. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 106. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 107. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 108. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 109.

[0213] In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 110. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 111. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 112. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 113. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 114. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 115. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 116. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 117. InPATENT Attorney Docket No. 048536-774001WOembodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 118. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 119. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 120. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 121. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 122. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 123. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 124. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 125. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 126. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 127. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 128. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 129. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 130. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 131. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 132. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 133. In embodiments, the third sgRNA includes the nucleotide sequence of SEQ ID NO: 134.

[0214] In embodiments, the fourth sgRNA includes the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 3. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:4. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:5. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:6. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:7. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 8. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NON. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NOTO. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 11. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 12. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 13. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 14. InPATENT Attorney Docket No. 048536-774001WOembodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 15. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 16. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 17. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 18. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 19. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:20. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:21. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:22. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:23. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:24. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:25. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:26. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:27. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:28. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:29. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:30.

[0215] In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:54. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:55. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:56. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:57. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:58. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:59. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:60. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:61. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:62. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:63. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:64. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:65. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:66. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:67. InPATENT Attorney Docket No. 048536-774001WOembodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:68. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:69.

[0216] In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:70. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:71. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 72. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:73. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:74. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:75. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:76. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:77. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:78. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:79. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 80. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:81. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 82. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:83. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 84. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:85. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 86. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 87. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 88. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 89.

[0217] In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:90. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:91. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:92. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:93. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:94. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:95. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:96. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:97. InPATENT Attorney Docket No. 048536-774001WOembodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:98. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:99. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 100. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 101. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 102. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 103. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 104. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 105. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 106. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 107. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 108. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 109.

[0218] In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 110. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 111. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:112. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:113. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 114. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 115. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 116. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO:117. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 118. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 119. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 120. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 121. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 122. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 123. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 124. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 125. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 126. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ IDPATENT Attorney Docket No. 048536-774001WONO: 127. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 128. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 129. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 130. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 131. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 132. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 133. In embodiments, the fourth sgRNA includes the nucleotide sequence of SEQ ID NO: 134.

[0219] In embodiments, the fifth sgRNA includes the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:3. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:4. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: . In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:6. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:7. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 8. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NON. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NOTO. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 11. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 12. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 13. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 14. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 15. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 16. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 17. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 18. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 19. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:20. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:21. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:22. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:23. InPATENT Attorney Docket No. 048536-774001WOembodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:24. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:25. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:26. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:27. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:28. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:29. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:30.

[0220] In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:54. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:55. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:56. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:57. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:58. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:59. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:60. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:61. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:62. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:63. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:64. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:65. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:66. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:67. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:68. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:69.

[0221] In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:70. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:71. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:72. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:73. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:74. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:75. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:76. InPATENT Attorney Docket No. 048536-774001WOembodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:77. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:78. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:79. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 80. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:81. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 82. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 83. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 84. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 85. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 86. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 87. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 88. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 89.

[0222] In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:90. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:91. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:92. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:93. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:94. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:95. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:96. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:97. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:98. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO:99. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 100. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 101. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 102. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 103. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 104. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 105. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 106. InPATENT Attorney Docket No. 048536-774001WOembodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 107. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 108. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 109.

[0223] In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 110. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 111. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 112. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 113. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 114. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 115. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 116. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 117. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 118. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 119. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 120. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 121. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 122. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 123. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 124. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 125. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 126. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 127. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 128. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 129. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 130. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 131. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 132. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 133. In embodiments, the fifth sgRNA includes the nucleotide sequence of SEQ ID NO: 134.

[0224] In embodiments, the sixth sgRNA includes the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134. In embodiments, the sixth sgRNA includes the nucleotide sequence ofPATENT Attorney Docket No. 048536-774001WOSEQ ID NO:3. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:4. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:5. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:6. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 7. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 8. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:9. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NOTO. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 11. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 12. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 13. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 14. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 15. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 16. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 17. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 18. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 19. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:20. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:21. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:22. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:23. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:24. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:25. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:26. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:27. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:28. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:29. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NOTO.

[0225] In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:54. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 55. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:56. InPATENT Attorney Docket No. 048536-774001WOembodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:57. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:58. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:59. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:60. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:61. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:62. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:63. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:64. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:65. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:66. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:67. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:68. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:69.

[0226] In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:70. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 71. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:72. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:73. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 74. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 75. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:76. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:77. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:78. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:79. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 80. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:81. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 82. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 83. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 84. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 85. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 86. InPATENT Attorney Docket No. 048536-774001WOembodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 87. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 88. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 89.

[0227] In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:90. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 91. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:92. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:93. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:94. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:95. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:96. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:97. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:98. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO:99. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 100. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 101. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 102. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 103. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 104. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 105. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 106. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 107. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 108. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 109.

[0228] In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 110. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 111. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 112. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 113. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 114. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 115. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ IDPATENT Attorney Docket No. 048536-774001WONO: 116. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 117. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 118. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 119. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 120. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 121. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 122. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 123. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 124. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 125. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 126. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 127. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 128. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 129. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 130. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 131. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 132. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 133. In embodiments, the sixth sgRNA includes the nucleotide sequence of SEQ ID NO: 134.

[0229] In embodiments, the transgene encodes a chimeric antigen receptor (CAR) protein, a T cell receptor (TCR) protein, a synthetic Notch (SynNotch) receptor protein, a synthetic intramembrane proteolysis receptor (SNIPR) protein, or an HLA-independent T cell (HIT) receptor protein. In embodiments, the transgene encodes the transgene encodes a chimeric antigen receptor (CAR) protein. In embodiments, the transgene encodes a T cell receptor (TCR) protein. In embodiments, the transgene encodes a synthetic Notch (SynNotch) receptor protein. In embodiments, the transgene encodes a synthetic intramembrane proteolysis receptor (SNIPR) protein. In embodiments, the transgene encodes an HLA-independent T cell (HIT) receptor protein.

[0230] In embodiments, the CAR protein is an anti-BCMA CAR protein, a TRAC CAR protein, or an anti-CD19 CAR protein. In embodiments, the CAR protein is an anti-BCMA CARPATENT Attorney Docket No. 048536-774001WOprotein. In embodiments, the CAR protein is a TRAC CAR protein. In embodiments, the CAR protein is an anti-CD19 CAR protein.

[0231] In embodiments, the fusion protein targets a target nucleic acid sequence encoding a RAS p21 protein activator 2 (RASA2) protein, a CD151 protein, a CD55 protein, a CD81 protein, a CD45 protein, a programmed cell death protein 1 (PD1) protein, a CBL-B protein, a cytokine-inducible SH2 domain containing protein (CISH), a DNA (cytosine-5)-methyltransferase 3A (DNMT3A) protein, a Fas receptor (Fas) protein, a mediator of RNA polymerase II transcription subunit 12 homolog (MED12) protein, a nuclear factor NF-kappa-B pl 00 subunit (NFKB2) protein, a protein tyrosine phosphatase nonreceptor type 2 (PTPN2) protein, a ring finger and CCCH-type...

Claims

PATENT Atorney Docket No. 048536-774001 WOWHAT IS CLAIMED IS:

1. A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:

135. or SEQ ID NO: 136.

2. The polynucleotide of claim 1, wherein the polynucleotide is messenger RNA (mRNA).

3. A vector comprising the polynucleotide of claim 1.

4. A cell comprising the polynucleotide of claim 1 or the vector of claim 3.

5. The cell of claim 4, wherein the cell is a eukaryotic cell.

6. The cell of claim 4, wherein the cell is a mammalian cell.

7. The cell of claim 4, wherein the cell is a T cell.

8. AT cell gene editing system, the system comprising:(i) a first polynucleotide encoding a fusion protein, wherein the fusion protein comprises a nuclease-deficient RNA-guided DNA endonuclease domain, a Kriippel associated box domain, a DNA methyltransferase domain, a first XTEN linker, and a second XTEN linker, wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO:1 or SEQ IDNO:2;(ii) a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guided DNA endonuclease domain;(iii) a ribonucleoprotein comprising an RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; and(iv) a third polynucleotide encoding a transgene.

9. The system of claim 8, wherein the RNA-guided DNA endonuclease enzyme domain is a Cas9 enzyme domain or a Casl2a enzyme domain.

10. The system of claim 8, wherein the first sgRNA comprises the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134.PATENT Attorney Docket No. 048536-774001 WO11. The system of claim 8, wherein the second sgRNA hybridizes to a second target nucleic acid sequence in a gene encoding a T cell receptor protein.

12. The system of claim 11 , wherein the second target nucleic acid sequence is a portion of a T cell receptor alpha constant (TRAC) gene.

13. The system of claim 11, wherein the second target nucleic acid sequence is a portion of an exon of a TRAC gene or a portion of an intron of a TRAC gene.

14. The system of claim 11, wherein the second target nucleic acid sequence is a portion of exon 1 of a TRAC gene.

15. The system of claim 8, wherein the second sgRNA comprises the nucleotide sequence of SEQ ID NO:31.

16. The system of claim 8, further comprising:(a) a third sgRNA;(b) a third sgRNA and a fourth sgRNA;(c) a third sgRNA, a fourth sgRNA, and a fifth sgRNA; or(d) a third sgRNA, a fourth sgRNA, a fifth sgRNA, and a sixth sgRNA.

17. The system of claim 16, wherein the third sgRNA, the fourth sgRNA, the fifth sgRNA. and / or the sixth sgRNA independently comprise the nucleotide sequence of any¬ one of SEQ ID NOs:3-30 or 54-134.

18. The system of claim 8, wherein the transgene encodes a chimeric antigen receptor (CAR) protein, a T cell receptor (TCR) protein, a synthetic Notch (SynNotch) receptor protein, a synthetic intramembrane proteolysis receptor (SNIPR) protein, or an HLA-independent T cell (HIT) receptor protein.

19. The system of claim 18, wherein the CAR protein is an anti-BCMA CAR protein, a TRAC CAR protein, or an anti-CD19 CAR protein.

20. The system of claim 8, wherein the fusion protein targets a target nucleic acid sequence encoding a RAS p21 protein activator 2 (RASA2) protein, a CD 151 protein, a CD55 protein, a CD81 protein, a CD45 protein, a programmed cell death protein 1 (PD1) protein, a CBL-B protein, a cytokine-inducible SH2 domain containing protein (CISH), a DNAPATENT Atorney Docket No. 048536-774001 WO (cytosine-5)-methyltransferase 3A (DNMT3 A) protein, a Fas receptor (Fas) protein, a mediator of RNA polymerase II transcription subunit 12 homolog (MED12) protein, a nuclear factor NF-kappa-B pl 00 subunit (NFKB2) protein, a protein tyrosine phosphatase nonreceptor type 2 (PTPN2) protein, a ring finger and CCCH-type domains 1 (RC3H1) protein, a suppressor of cytokine signaling 1 (SOCS1) protein, a suppressor of variegation 3-9 homolog 1 (SUV39H1) protein, a tumor necrosis factor alpha-induced protein 3 (TNFAIP3) protein, a zinc finger CCCH-ty pe containing 12A (ZC3H12A) protein, a CD5 protein, or a lymphocyte-activation gene 3 (LAG3) protein.

21. The system of claim 8, wherein the third polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 or SEQ ID NO:33.

22. The system of claim 8, wherein the first polynucleotide is messenger RNA (mRNA).

23. The system of claim 8, wherein the third polynucleotide is an adeno-associated virus vector.

24. A T cell gene editing system, the system comprising:(i) a first polynucleotide encoding a fusion protein, wherein the fusion protein comprises a nuclease-deficient RNA-guided DNA endonuclease domain, a Krtippel associated box domain, and a DNA methyltransferase domain;(ii) a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guided DNA endonuclease domain;(iii) a ribonucleoprotein comprising an orthogonal RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; and(iv) a third polynucleotide encoding a transgene.

25. The system of claim 24, wherein the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9, ddCpfl, a nuclease-deficient Cas9 variant, a nuclease-deficient Casl2 variant, or a nuclease-deficient Class II CRISPR endonuclease.

26. The system of claim 24, wherein the nuclease-deficient RNA-guided DNA endonuclease domain is a Staphylococcus aureus Cas9 (SaCas9) enzyme domain, aPATENT Atorney Docket No. 048536-774001 WO Staphylococcus pyogenes Cas9 (SpCas9) enzyme domain, an Acidaminococcus sp. Cast 2a (AsCasl2a) enzy me domain, or an Acidaminococcus sp. Casl2b (AsCasl2b) enzyme domain.

27. The system of claim 24, wherein the DNA methyltransferase domain encodes a Dnmt3A-3L domain.

28. The system of claim 24, wherein the first polynucleotide comprises, from N-terminus to C-terminus, the DNA methyltransferase domain, the nuclease-deficient RNA-guided DNA endonuclease domain, and the Kriippel associated box domain.29 . The system of claim 24, wherein the fusion protein further comprises a first XTEN linker and a second XTEN linker.

30. The system of claim 29, wherein the fusion protein comprises, from N-terminus to C-terminus, the DNA methyltransferase domain, the first XTEN linker, the nuclease-deficient RNA-guided DNA endonuclease domain, the second XTEN linker, and the Kriippel associated box domain.

31. The system of claim 29, wherein the first XTEN linker comprises from about 50 to about 864 amino acid residues, and the second XTEN linker comprises from about 5 to about 864 amino acid residues.

32. The system of claim 24, wherein the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9 and the DNA methyltransferase domain encodes a Dnmt3A-3L domain.

33. The system of claim 24, wherein the first polynucleotide further comprises a 5' cap structure.

34. The system of claim 33, wherein the 5' cap structure comprises an antireverse cap analog (ARCA) mRNA cap, a A7-methylgunosine (m7G) mRNA cap, a 3'-O-methylation on m7G mRNA cap, a 2'-(9-methylation of the first base (CAP 1) mRNA cap, a N6-methyladenosine (m6A) mRNA cap, an alphavirus 5' mRNA cap, a symmetric two-m7G-headed mRNA cap, a 2',4'-locked nucleic acid-modified mRNA cap, a 3'-O-benzyl-modified mRNA cap, an S mRNA cap, or a 2S mRNA cap.PATENT Atorney Docket No. 048536-774001 WO35. The system of claim 24, wherein the first polynucleotide comprises at least one uridine substitution, pseudouridine substitution, A1-methylpseudouridine substitution, 2-thiouridine substitution, 5-methylcytidine substitution, / V’-melhyladenosine substitution. 2'-O-methyluridine substitution, 2'-(?-methylcytidine substitution, 2'-O-methyladenosine substitution, or 2'-6>-methylguanosine substitution.

36. The system of claim 24, wherein the first sgRNA comprises the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134.

37. The system of claim 24, wherein the second sgRNA hybridizes to a second target nucleic acid sequence in a gene encoding a T cell receptor protein.

38. The system of claim 37, wherein the second target nucleic acid sequence is a portion of a T cell receptor alpha constant (TRAC) gene.

39. The system of claim 37, wherein the second target nucleic acid sequence is a portion of an exon of a TRAC gene or a portion of an intron of a TRAC gene.

40. The system of claim 37, wherein the second target nucleic acid sequence is a portion of exon 1 of a TRAC gene.

41. The system of claim 24, wherein the second sgRNA comprises the nucleotide sequence of SEQ ID NO:31.

42. The system of claim 24, further comprising:(a) a third sgRNA;(b) a third sgRNA and a fourth sgRNA;(c) a third sgRNA, a fourth sgRNA, and a fifth sgRNA; or(d) a third sgRNA, a fourth sgRNA, a fifth sgRNA, and a sixth sgRNA.

43. The system of claim 42, wherein the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently comprise the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134.

44. The system of claim 24, wherein the transgene encodes a chimeric antigen receptor (CAR) protein, a T cell receptor (TCR) protein, a synthetic Notch (SynNotch) receptorPATENT Atorney Docket No. 048536-774001 WO protein, a synthetic intramembrane proteolysis receptor (SNIPR) protein, or an HLA-independent T cell (HIT) receptor protein.

45. The system of claim 44, wherein the CAR protein is an anti-BCMA CAR protein, a TRAC CAR protein, or an anti-CD19 CAR protein.

46. The system of claim 24, wherein the fusion protein targets a target nucleic acid sequence encoding a RAS p21 protein activator 2 (RASA2) protein, a CD 151 protein, a CD55 protein, a CD81 protein, a CD45 protein, a programmed cell death protein 1 (PD1) protein, a CBL-B protein, a cytokine-inducible SH2 domain containing protein (CISH), a DNA (cytosine-5)-methyltransferase 3A (DNMT3A) protein, a Fas receptor (Fas) protein, a mediator of RNA polymerase II transcription subunit 12 homolog (MED 12) protein, a nuclear factor NF-kappa-B pl 00 subunit (NFKB2) protein, a protein tyrosine phosphatase nonreceptor type 2 (PTPN2) protein, a ring finger and CCCH-type domains 1 (RC3H1) protein, a suppressor of cytokine signaling 1 (SOCS1) protein, a suppressor of variegation 3-9 homolog 1 (SUV39H1) protein, a tumor necrosis factor alpha-induced protein 3 (TNFAIP3) protein, a zinc finger CCCH-type containing 12A (ZC3H12A) protein, a CD5 protein, or a lymphocyte-activation gene 3 (LAG3) protein.

47. The system of claim 24, wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

48. The system of claim 24, wherein the third polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 or SEQ ID NO:33.

49. The system of claim 24, wherein the first polynucleotide is messenger RNA (mRNA).

50. The system of claim 24, wherein the third polynucleotide is an adeno-associated virus vector.

51. A method of generating an engineered antigen-specific T cell, the method comprising:(i) delivering a first polynucleotide to a T cell, wherein the first polynucleotide encodes a fusion protein comprising a nuclease-deficient RNA-guided DNA endonuclease domain, a Kruppel associated box domain, and a DNA methyltransferase domain;PATENT Atorney Docket No. 048536-774001 WO (ii) delivering a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guided DNA endonuclease domain;(iii) delivering a ribonucleoprotein to the T cell, wherein the ribonucleoprotein comprises an orthogonal RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA;(iv) delivering a third polynucleotide to the T cell, wherein the third polynucleotide encodes a transgene; and(v) allowing the T cell to express the first polynucleotide, the ribonucleoprotein, and the third polynucleotide, thereby generating an engineered antigenspecific T cell.

52. The method of claim 51, wherein the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9, ddCpfl, a nuclease-deficient Cas9 variant, a nuclease-deficient Casl2 variant, or a nuclease-deficient Class II CRISPR endonuclease.

53. The method of claim 51, wherein the nuclease-deficient RNA-guided DNA endonuclease domain is a Staphylococcus aureus Cas9 (SaCas9) enzyme domain, a Staphylococcus pyogenes Cas9 (SpCas9) enzy me domain, an Acidaminococcus sp. Casl2a (AsCasl2a) enzyme domain, or an Aci daminococcus sp. Casl2b (AsCasl2b) enzyme domain.

54. The method of claim 51, wherein the orthogonal RNA-guided DNA endonuclease domain is a Cas9 enzy me domain or a Casl2a enzy me domain.

55. The method of claim 51 , wherein the DNA methyltransferase domain encodes a Dnmt3A-3L domain.

56. The method of claim 51, wherein the first polynucleotide comprises, from N-terminus to C-terminus, the DNA methyltransferase domain, the nuclease-deficient RNA-guided DNA endonuclease domain, and the Kriippel associated box domain.

57. The method of claim 51, wherein the fusion protein further comprises a first XTEN linker and a second XTEN linker.

58. The method of claim 57, wherein the fusion protein comprises, from N-terminus to C-terminus, the DNA methyltransferase domain, the first XTEN linker, thePATENT Atorney Docket No. 048536-774001 WO nuclease-deficient RNA-guided DNA endonuclease domain, the second XTEN linker, and the Kruppel associated box domain.

59. The method of claim 57, wherein the first XTEN linker comprises from about 50 to about 864 amino acid residues, and the second XTEN linker comprises from about 5 to about 864 amino acid residues.

60. The method of claim 51 , wherein the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9 and the DNA methyltransferase domain encodes a Dnmt3A-3L domain.

61. The method of claim 51 , wherein the first polynucleotide further comprises a 5' cap structure.

62. The method of claim 61, wherein the 5' cap structure comprises an antireverse cap analog (ARCA) mRNA cap. a A'7-methylgunosme (m7G) mRNA cap, a 3-0-methylation on m7GmRNA cap, a 2'-(9-methylation of the first base (CAP1) mRNA cap, a N6-methyladenosine (m6A) mRNA cap, an alphavirus 5' mRNA cap, a symmetric two-m7G-headed mRNA cap, a 2',4'-locked nucleic acid-modified mRNA cap, a 3'-O-benzyl-modified mRNA cap, an S mRNA cap, or a 2S mRNA cap.

63. The method of claim 51, wherein the first polynucleotide comprises at least one uridine substitution, pseudouridine substitution, N1-methylpseudouridine substitution, 2-thiouridine substitution, 5-methylcytidine substitution, M’-methyladenosine substitution, 2-O-methyluridine substitution, 2'-O-methylcytidine substitution, 2'-0-methyladenosine substitution, or 2'-O-methylguanosine substitution.

64. The method of claim 51, wherein the first sgRNA comprises the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134.

65. The method of claim 51, wherein the second sgRNA hybridizes to a second target nucleic acid sequence in a gene encoding a T cell receptor protein.

66. The method of claim 65, wherein the second target nucleic acid sequence is a portion of a T cell receptor alpha constant (TRAC) gene.PATENT Attorney Docket No. 048536-774001 WO67. The method of claim 65, wherein the second target nucleic acid sequence is a portion of an exon of a TRAC gene or a portion of an intron of a TRAC gene.

68. The method of claim 65, wherein the second target nucleic acid sequence is a portion of exon 1 of a TRAC gene.

69. The method of claim 51 , wherein the second sgRNA comprises the nucleotide sequence of SEQ ID NO:31.

70. The system of claim 51, further comprising:(a) a third sgRNA;(b) a third sgRNA and a fourth sgRNA;(c) a third sgRNA, a fourth sgRNA, and a fifth sgRNA; or(d) a third sgRNA, a fourth sgRNA, a fifth sgRNA, and a sixth sgRNA.

71. The system of claim 70, wherein the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently comprise the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134.

72. The method of claim 51, wherein the transgene encodes a chimeric antigen receptor (CAR) protein, a T cell receptor (TCR) protein, a synthetic Notch (SynNotch) receptor protein, a synthetic intramembrane proteolysis receptor (SNIPR) protein, or an HLA-independent T cell (EIIT) receptor protein.

73. The method of claim 72, wherein the CAR protein is an anti-BCMA CAR protein, a TRAC CAR protein, or an anti-CD19 CAR protein.

74. The method of claim 51 , wherein the fusion protein targets a target nucleic acid sequence encoding a RAS p21 protein activator 2 (RASA2) protein, a CD 151 protein, a CD55 protein, a CD81 protein, a CD45 protein, a programmed cell death protein 1 (PD1) protein, a CBL-B protein, a cytokine-inducible SH2 domain containing protein (CISH), a DNA (cytosine-5)-methyltransferase 3A (DNMT3A) protein, a Fas receptor (Fas) protein, a mediator of RNA polymerase II transcription subunit 12 homolog (MED12) protein, a nuclear factor NF-kappa-B pl 00 subunit (NFKB2) protein, a protein tyrosine phosphatase nonreceptor type 2 (PTPN2) protein, a ring finger and CCCH-type domains 1 (RC3H1) protein, a suppressor of cytokine signaling 1 (SOCS1) protein, a suppressor of variegation 3-9 homolog 1 (SUV39H1)PATENT Atorney Docket No. 048536-774001 WO protein, a tumor necrosis factor alpha-induced protein 3 (TNFAIP3) protein, a zinc finger CCCH-type containing 12A (ZC3H12A) protein, a CD5 protein, or a lymphocyte-activation gene 3 (LAG3) protein.

75. The method of claim 51, wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

76. The method of claim 51 , wherein the third polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 or SEQ ID NO:33.

77. The method of claim 51, wherein the first polynucleotide is messenger RNA (mRNA).

78. The method of claim 51, wherein the third polynucleotide is an adeno-associated virus vector.

79. The method of claims 51 , wherein the first polynucleotide and the ribonucleoprotein are independently introduced into the cell by electroporation.

80. A method of treating a cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of an engineered antigen-specific T cell generated according to the method of claim 51 to the subject in need thereof, thereby treating the cancer.

81. A cell comprising a first polynucleotide, a first single guide RNA (sgRNA), a ribonucleoprotein, and a second polynucleotide,wherein the first polynucleotide encodes a fusion protein comprising a nuclease-deficient RNA-guided DNA endonuclease domain, a Krtippel associated box domain, and a DNA methyltransferase domain;wherein the first sgRNA binds the nuclease deficient RNA-guided DNA endonuclease domain;wherein the ribonucleoprotein comprises an orthogonal RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; andwherein the second polynucleotide encodes a transgene.PATENT Atorney Docket No. 048536-774001 WO82. The cell of claim 81, wherein the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9, ddCpfl, a nuclease-deficient Cas9 variant, a nuclease-deficient Cast 2 variant, or a nuclease-deficient Class II CRISPR endonuclease.

83. The cell of claim 81, wherein the nuclease-deficient RNA-guided DNA endonuclease domain is a Staphylococcus aureus Cas9 (SaCas9) enzy me domain, a Staphylococcus pyogenes Cas9 (SpCas9) enzyme domain, an Acidaminococcus sp. Cast 2a (AsCasl2a) enzyme domain, or an Acidaminococcus sp. Casl2b (AsCasl2b) enzyme domain.

84. The cell of claim 81, wherein the orthogonal RNA-guided DNA endonuclease domain is a Cas9 enzyme domain or a Casl2a enzyme domain.

85. The cell of claim 81, wherein the DNA methyltransferase domain encodes a Dnmt3 A-3L domain.

86. The cell of claim 81, wherein the first polynucleotide comprises, from N-terminus to C-terminus, the DNA methyltransferase domain, the nuclease-deficient RNA-guided DNA endonuclease domain, and the Kruppel associated box domain.

87. The cell of claim 81, wherein the fusion protein further comprises a first XTEN linker and a second XTEN linker.

88. The cell of claim 87, wherein the fusion protein comprises, from N-terminus to C-terminus, the DNA methyltransferase domain, the first XTEN linker, the nuclease-deficient RNA-guided DNA endonuclease domain, the second XTEN linker, and the Kruppel associated box domain.

89. The cell of claim 87, wherein the first XTEN linker comprises from about 50 to about 864 amino acid residues, and the second XTEN linker comprises from about 5 to about 864 amino acid residues.

90. The cell of claim 81, wherein the nuclease-deficient RNA-guided DNA endonuclease domain encodes dCas9 and the DNA methyltransferase domain encodes a Dnmt3A-3L domain.

91. The cell of claim 81, wherein the first polynucleotide further comprises a 5' cap structure.PATENT Atorney Docket No. 048536-774001 WO92. The cell of claim 91, wherein the 5' cap structure comprises an antireverse cap analog (ARCA) mRNA cap, a A7-methylgunosine (m7G) mRNA cap, a 3-0-methylation on m7G mRNA cap, a 2'-0-methylation of the first base (CAP1) mRNA cap, a N6-methyladenosine (m6A) mRNA cap, an alphavirus 5' mRNA cap, a symmetric two-m7G-headed mRNA cap, a 2',4'-locked nucleic acid-modified mRNA cap, a 3'-O-benzyl-modi fied mRNA cap, an S mRNA cap, or a 2S mRNA cap.

93. The cell of claim 81, wherein the first polynucleotide comprises at least one uridine substitution, pseudouridine substitution, A—methylpseudouridine substitution, 2-thiouridine substitution, 5-methylcytidine substitution, A6-methyladenosine substitution, 2'-O-methyluridine substitution, 2'-(?-methylcytidine substitution, 2'-G-methyladenosine substitution, or 2'-(9-methylguanosine substitution.

94. The cell of claim 81, wherein the first sgRNA comprises the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134.

95. The cell of claim 81, wherein the second sgRNA hybridizes to a second target nucleic acid sequence in a gene encoding a T cell receptor protein.

96. The cell of claim 95, wherein the second target nucleic acid sequence is a portion of a T cell receptor alpha constant (TRAC) gene.

97. The cell of claim 95, wherein the second target nucleic acid sequence is a portion of an exon of a TRAC gene or a portion of an intron of a TRAC gene.

98. The cell of claim 95, wherein the second target nucleic acid sequence is a portion of exon 1 of a TRAC gene.

99. The cell of claim 81, wherein the second sgRNA comprises the nucleotide sequence of SEQ ID NO:31.

100. The cell of claim 81, further comprising:(a) a third sgRNA;(b) a third sgRNA and a fourth sgRNA;(c) a third sgRNA, a fourth sgRNA, and a fifth sgRNA; or(d) a third sgRNA, a fourth sgRNA, a fifth sgRNA, and a sixth sgRNA.PATENT Atorney Docket No. 048536-774001 WO101. The cell of claim 100, wherein the third sgRNA, the fourth sgRNA, the fifth sgRNA, and / or the sixth sgRNA independently comprise the nucleotide sequence of any one of SEQ ID NOs:3-30 or 54-134.

102. The cell of claim 81, wherein the transgene encodes a chimeric antigen receptor (CAR) protein, a T cell receptor (TCR) protein, a synthetic Notch (SynNotch) receptor protein, a synthetic intramembrane proteolysis receptor (SNIPR) protein, or an HLA-independent T cell (HIT) receptor protein.

103. The cell of claim 102, wherein the CAR protein is an anti-BCMA CAR protein, a TRAC CAR protein, or an anti-CD 19 CAR protein.

104. The cell of claim 81, wherein the fusion protein targets a target nucleic acid sequence encoding a RAS p21 protein activator 2 (RASA2) protein, a CD 151 protein, a CD55 protein, a CD81 protein, a CD45 protein, a programmed cell death protein 1 (PD1) protein, a CBL-B protein, a cytokine-inducible SH2 domain containing protein (CISH), a DNA (cytosine-5)-methyltransferase 3A (DNMT3 A) protein, a Fas receptor (Fas) protein, a mediator of RNA polymerase II transcription subunit 12 homolog (MED 12) protein, a nuclear factor NF-kappa-B pl 00 subunit (NFKB2) protein, a protein tyrosine phosphatase nonreceptor type 2 (PTPN2) protein, a ring finger and CCCH-type domains 1 (RC3H1) protein, a suppressor of cytokine signaling 1 (SOCS1) protein, a suppressor of variegation 3-9 homolog 1 (SUV39H1) protein, a tumor necrosis factor alpha-induced protein 3 (TNFAIP3) protein, a zinc finger CCCH-ty pe containing 12A (ZC3H12A) protein, a CD5 protein, or a lymphocyte-activation gene 3 (LAG3) protein.

105. The cell of claim 81, wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

106. The cell of claim 81, wherein the third polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 or SEQ ID NO:33.

107. The cell of claim 81, wherein the first polynucleotide is messenger RNA (mRNA).

108. The cell of claim 81, wherein the second polynucleotide is an adeno-associated virus vector.PATENT Atorney Docket No. 048536-774001 WO109. The cell of claim 81, wherein the first polynucleotide and the ribonucleoprotein are independently introduced into the cell by electroporation.

110. The cell of claim 81, wherein the cell is a eukaryotic cell.

111. The cell of claim 81, wherein the cell is a mammalian cell.

112. The cell of claim 81, wherein the cell is a T cell.

113. AT cell gene editing system, the system comprising:(i) a first polynucleotide encoding a fusion protein, wherein the fusion protein comprises from N-terminus to C-terminus a DNA demethylation domain, an XTEN linker, and a nuclease-deficient RNA-guided DNA endonuclease domain; and(ii) a second polynucleotide encoding a first single guide RNA (sgRNA), wherein the first sgRNA binds to the nuclease-deficient RNA-guided DNA endonuclease domain, wherein the first sgRNA hybridizes to a first target nucleic acid sequence in a forkhead box P3 (FOXP3) gene.

114. The system of claim 113, further comprising:(iii) a ribonucleoprotein comprising an RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA; and(iv) a third polynucleotide encoding a transgene.

115. The system of claim 113, wherein the first target nucleic acid sequence is anon-coding region of the FOXP3 gene.

116. The system of claim 115, wherein the non-coding region is an enhancer region of the FOXP3 gene.

117. The system of claim 115, wherein the non-coding region is a Treg specific demethylated region (TSDR) of the FOXP3 gene.

118. The system of claim 116, wherein the first sgRNA hybridizes with a portion of the enhancer region or a portion of the TSDR.

119. The system of claim 115, wherein the non-coding region comprises the nucleotide sequence of SEQ ID NO: 148.PATENT Attorney Docket No. 048536-774001 WO120. The system of claim 113, wherein the first sgRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 137-139.

121. The system of claim 113, wherein the second sgRNA hybridizes to a second target nucleic acid sequence in a gene encoding a T cell receptor protein.

122. The system of claim 121. wherein the second target nucleic acid sequence is a portion of a T cell receptor alpha constant (TRAC) gene.

123. The system of claim 121, wherein the second target nucleic acid sequence is a portion of an exon of a TRAC gene or a portion of an intron of a TRAC gene.

124. The system of claim 121, wherein the second target nucleic acid sequence is a portion of exon 1 of a TRAC gene.

125. The system of claim 113, wherein the second sgRNA comprises the nucleotide sequence of SEQ ID NO:31.

126. The system of claim 114. further comprising:(a) a third sgRNA; or(b) a third sgRNA and a fourth sgRNA.

127. The system of claim 126. wherein the third sgRNA and / or the fourth sgRNA independently comprise the nucleotide sequence of any one of SEQ ID NOs: 137-139.

128. The system of claim 114, wherein the transgene encodes a chimeric antigen receptor (CAR) protein, a T cell receptor (TCR) protein, a synthetic Notch (SynNotch) receptor protein, a synthetic intramembrane proteolysis receptor (SNIPR) protein, or an HLA-independent T cell (HIT) receptor protein.

129. The cell of claim 128, wherein the CAR protein is an anti-BCMA CAR protein, a TRAC CAR protein, or an anti-CD19 CAR protein.

130. The system of claim 114, wherein the third polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 or SEQ ID NO:33.PATENT Atorney Docket No. 048536-774001 WO131. The system of claim 113, wherein the first polynucleotide is messenger RNA (mRNA).

132. The system of claim 114, wherein the third polynucleotide is an adeno-associated virus vector.

133. A method of increasing expression of a forkhead box P3 (FOXP3) gene in a T cell, the method comprising transfecting the T cell with the T cell gene editing system of claim 113, thereby demethylating a portion of the FOXP3 gene within the T cell and increasing expression of the FOXP3 gene relative to the absence of the T cell gene editing system.

134. The method of claim 133, wherein the method further comprises transfecting the T cell with a ribonucleoprotein comprising an RNA-guided DNA endonuclease enzyme domain bound to a second sgRNA and a third polynucleotide encoding a transgene; and allowing the T cell to express the transgene.

135. A cell comprising the T cell gene editing system of claim 113.

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