Regulators of t cell exhaustion

Abstract

Disclosed herein are compositions and methods for modulating T cells. For example, the compositions and methods may be used to increase memory T cells. The compositions and methods may increase the expression or protein level of a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1, TWIST1, NKX6, and FEV, or a combination thereof. The compositions and method may be used in combination with Adoptive T Cell Therapy (ACT) to enhance the ACT.

Description

REGULATORS OF T CELL EXHAUSTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 520,320 filed August 17, 2023, U.S. Provisional Patent Application No. 63 / 579,777 filed August 30, 2023, U.S. Provisional Patent Application No. 63 / 582,396 filed September 13, 2023, U.S. Provisional Patent Application No. 63 / 622,490 filed January 18, 2024, and U.S. Provisional Patent Application No. 63 / 636,861 filed April 21 , 2024, the entire contents of each of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant UM1 HG012053 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] This disclosure relates to genes to target for activation to increase T cells such as memory T ceils and to reduce T cell exhaustion, as well as compositions and methods targeting the genes to improve immunotherapies for viral infections and cancer, including adoptive T cell therapy (ACT).INTRODUCTION

[0004] T cells are part of the immune system’s adaptive defense. They specifically target and kill both virally infected and cancerous ceils through antigen recognition. Unfortunately, cancer can exploit intrinsic T cell mechanisms to survive attack by the immune system. T ceil state and function are largely regulated by specific transcription factors (TFs) and epigenetic modifiers that process intrinsic and extrinsic signals into complex and tightly controlled gene expression programs. T cell exhaustion arises from chronic antigen stimulation, which shifts a portion of the T cell population to the exhausted state (TEX), resulting in diminished T cell proliferation and tumor / viral clearance. The transcription factor TOX drives and maintains the TEX cell state through epigenetic regulation of exhaustion-associated genetic programs.

[0005] Adoptive T cell therapy (ACT) holds tremendous potential for cancer treatment by redirecting T cells to cancer cells via expression of engineered receptors that recognize and bind to tumor-associated antigens. Receptor-antigen interactions can initiate complextranscriptional networks that drive multipotent T cell response and lead to cancer cell death. The potency and duration of T cell response are associated with defined T cell subsets, and cell products enriched in stem or memory T cells, provide superior tumor control in animal models and in the clinic. Given the association between defined T cell subsets and clinical outcomes, precise regulation or programming of T cell state may be one approach to improve the therapeutic potential of ACT.

[0006] T cell exhaustion drives dysfunction and impaired immune response to cancer and chronic viral infections. Modern genome engineering technologies have the potential to dramatically advance T cell therapy by programming exhausted T cells to desirable phenotypes. There is a need to find and develop regulators of T cell state and discover T cell gene networks and their corresponding phenotypes, in order to enhance T cel! phenotype and improve the efficacy of T cell therapies to help treat viral infections, kill cancer cells, and control solid tumors.SUMMARY

[0007] In an aspect, the disclosure relates to an isolated polynucleotide encoding a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof. In some embodiments, the isolated polynucleotide comprises a sequence selected from SEQ ID NOs: 75-84. In some embodiments, the transcription factor is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.[0(808] In a further aspect, the disclosure relates to a vector encoding a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST! , NKX6, and FEV, or a combination thereof. In some embodiments, the vector comprises a promoter operably linked to a polynucleotide sequence encoding the transcription factor. In some embodiments, the promoter is non-endogenous to the transcription factor. In some embodiments, the promoter is a constitutive promoter, or a ubiquitous promoter, or an inducible promoter, or a cell-specific promoter, or a tissue-specific promoter. In some embodiments, the vector comprises an open reading frame (ORF) of the transcription factor. In some embodiments, the vector comprises a sequence selected from SEQ ID NOs: 75-84 or encodes a polypeptide comprising a sequence selected from SEQ ID NOs: 85-94. In some embodiments, the transcription factor is selected from THAP6, DMRT3, and MEF2B, or a combination thereof, in some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is anadeno-associated virus (AAV) vector. In some embodiments, the AAV vector is selected from AAV1 , AAV2, AAV5, AAV6, AAV8, AAV9, and an engineered AAV vector.

[0009] Another aspect of the disclosure provides a method of moduiating T cells. The method may include administering to a T cell or a subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene. In some embodiments, moduiating T cells comprises increasing T cells, or increasing memory T cells, or preventing T cell exhaustions, or reversing T cell exhaustions, or a combination thereof,

[0910] Another aspect of the disclosure provides a method of increasing T cells. The method may include administering to a T cell or a subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.

[0911] Another aspect of the disclosure provides a method of enhancing adoptive T cell therapy (ACT) in a subject. The method may include administering to the subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.

[0912] Another aspect of the disclosure provides a method of treating cancer in a subject. The method may include administering to the subject or to a T cell of the subject, an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.

[0013] In some embodiments, the gene is selected from THAP6, DMRT3, and MEF2B, or a combination thereof. In some embodiments, the activator modulates T cells, and modulating T cells comprises increasing T cells, or increasing memory T cells, or increasing the lifetime of a T cell, or preventing T cell exhaustions, or reversing T cell exhaustions, or reducing T cell exhaustion, or enhancing the therapeutic potential of T cells, or a combination thereof, in some embodiments, the activator modulates gene expression within the T cell, in some embodiments, the activator decreases expression of TOX in the T cell.In some embodiments, the activator comprises a polypeptide, or a polynucleotide, or a small molecule, or a combination thereof. In some embodiments, the activator comprises apolynucleotide encoding the gene. In some embodiments, the activator comprises a polynucleotide comprising the open reading frame of the gene or a polynucleotide encoding a protein encoded by the gene. In some embodiments, the activator comprises a polynucleotide selected from SEQ ID NOs: 75-84 or a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 85-94. In some embodiments, the activator comprises a polypeptide comprising a protein encoded by the gene. In some embodiments, the activator comprises a polypeptide selected from SEQ ID NOs: 85-94. In some embodiments, the activator comprises a vector as detailed herein. In some embodiments, the activator or a polynucleotide encoding the activator is encapsulated within a lipid nanoparticle or polymeric carrier. In some embodiments, the method further includes administering at least one cancer therapy or at least one antiviral therapy.

[0014] Another aspect of the disclosure provides a vector comprising an isolated polynucleotide as detailed herein.

[0015] Another aspect of the disclosure provides a cell comprising an isolated polynucleotide as detailed herein, or a vector as detailed herein. In some embodiments, the cell is a CD8+ T cell.

[0016] Another aspect of the disclosure provides a pharmaceutical composition comprising an isolated polynucleotide as detailed herein, or a vector as detailed herein, or a combination thereof. In some embodiments, the pharmaceutical composition further includes at least one cancer therapy or at least one antiviral therapy.

[0017] Another aspect of the disclosure provides a composition for increasing T cells. The composition may include an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof. In some embodiments, the gene is selected from THAP6, DMRT3, and MEF2B, or a combination thereof. In some embodiments, the activator comprises a polynucleotide encoding the gene, or a polynucleotide encoding the open reading frame of the gene, or a polypeptide encoded by the gene, or a combination thereof. In some embodiments, the activator comprises a polynucleotide selected from SEQ ID NOs: 75-84 or a polypeptide selected from SEQ ID NOs: 85-94. In some embodiments, the composition further includes at least one cancer therapy or at least one antiviral therapy.

[0018] The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG, 1 is a diagram showing that the immune response to cancer is restrained by T cell exhaustion. Under acute stimulation, the adaptive immune system (e.g., reactive T ceils) can quickly eliminate any cancerous ceils identified (top). Whereas, under chronic stimulation, T cell exhaustion occurs through chronic stimulation of the T cell receptor and leads to decreased cancer cell killing and reduced T ceil proliferation (bottom).

[0020] FIG, 2 is a diagram of in vitro models of T cell exhaustion, showing protocols for in vitro acute (top) and chronic (bottom) stimulation of T cells.

[0021] FIGS, 3A-3G show that transcriptomic and epigenetic program of exhaustion is recapitulated by repeat stimulation. FIG, 3A is an RNA-seq volcano plot showing that gene pathways in primary human CD8+ T cells that are related to exhaustion are upregulated with repeat stimulations with anti-CD3 / CD28 Dynabeads in vitro (i.e., chronic stimulation;“Chronic”) as compared to acutely stimulated T cells (“Acute”). 5,705 differentially expressed genes were found. Upregulated exhaustion markers included TIGIT, PDCD1 , CLTA4, and LAG3. Downreguiated memory markers were SELL, TCF7, and IL7R. FIG, 3B is an ATAC-seq volcano plot showing that accesible / inaccessible chromatin in primary human CD8+ T cells that are related to exhaustion were upregulated with repeat stimulations with anti-CD3 / CD28 Dynabeads in vitro (i.e., chronic stimulation; “Dynabead”) as compared to acutely stimulated T cells (“Single-stim”). Chromatin regions near exhaustion genes were differentially accessible (LAG3, PD1 , CD39, TIM3, CTLA4, TIGIT, TOX) and regions near memory genes were differentially inaccessible (CCR7, IL7R, TCF7). FIG, 3C is a graph showing gene ontology (“GO”) biological pathways that are related to exhaustion are upregulated in primary human CD8+ T cells with repeat stimulations with anti-CD3 / CD28 Dynabeads in vitro based on the bulk RNA-seq data. FIG. 3D is a graph showing gene-set enrichment scores for exhaustion vs memory T cell gene programs, NES is normalized enrichment score, and FDR is false discovery rate. FIG. 3E is a graph showing gene-set enrichment scores for effector vs exhaustion T cell gene programs. NES is normalized enrichment score, and FDR is false discovery rate. FIG. 3F is a graph showing global ATAC peak analysis that reveals an increase in chromatin accessibility in regions associated with terminally exhausted t cells (i.e., chronic stimulation). Terminally dysfunctional chromatin regions were taken from Pritykin Y, et. al, Molecular Cell, 2021 , 81, 2477-2493. FIG. 3G is a readout of the ATAC track of the TOX locus and it reveals that TOX enhancers become differentially accessible with chronic stimulation.

[0022] FIG. 4 is a diagram showing a CRISPRa transcription factor (TF)-ome library design. The design is based on a list of all human TFs as defined by Lambert et al., Ce / / . 2018;172(4): 650-665. For all TFs that did not have 6 gRNAs in Calabrese and were present in Horlbeck et al., eUfe. 2016; 5:e1976Q, the TF gRNAs were supplemented to 6 with top gRNAs from Horlbeck et al.

[0023] FIGS. 5A-5D show TF-ome CRISPRa screening in chronically stimulated cells. FIG. SA is a diagram showing a protocol for generating a TF-ome CRISPRa with TOX protein expression as a reporter. FIG. 5B is a diagram showing a protocol for a TOX locus CRISPRi tiling screen. The TOX locus CRISPRi tiling screen library was designed to target the TOX gene locus ±100 kb upstream and downstream, which consisted of a total of 7,052 sgRNAs with ~5% of the library being non-targeting negative controls. FIG. 5C is a volcano plot showing the TF-ome CRISPRa with TOX protein expression as a reporter. FIG. 5D is a readout showing regulatory elements that positively and negatively regulate TOX expression identified by the tiling screen. The first 5 rows correspond to CRISPR-SURF analysis of the screen data, and the final two rows are a track visualization of the bulk ATAC-seq data previously generated when validating the model.

[0024] FIGS. 6A-6C shows diagrams for TF overexpression screening using the T cell exhaustion model described herein. FIGS. GA and 6B are diagrams showing a protocol for TF overexpression screening in chronic vs acute stimulation settings, CD8+ T cells were thawed and activated with CD3 / CD28 Dynabeads. A lentiviral library consisting of all TF open-reading frames in the human genome and constitutively expressed by a EF-1a promoter was generated from a DNA library (Joung J., et. al, Ce / / , 2023, incorporated herein by reference) and added to the cells one day later. Cells were puromycin selected.Transduced cells from the same donor were split into either acute or chronic stimulation conditions. T cells in the acute stimulation conditions were expanded until the final timepoint, and T cells in the chronic stimulation conditions were restimulated for two more times until the final timepoint. Cells were then stained for TOX expression and sorted based on high (top 10%) or low (bottom 10%) TOX expression. Genomic DNA was isolated, and the DNA was sequenced for differential TF enrichment analysis, based on whether the TOX expression was high or low, FIG. 6C is a diagram showing the lentiviral construct (Joung J,, et. al, Ce / / , 2023).

[0025] FIGS. 7A-7C show that screening reveals chronic-unique TF regulators of TOX. FIG. 7A is a plot of DeSeq2 differential analysis of two sorted bins (chronic TOX high and TOX low vs acute TOX high and TOX low) revealed many potential regulators of TOX. The x-axis refers to the z-score for the chronic stimulation screen, with positive values being TFsenriched in the TOX high bin and vice-versa. The y-axis is the z-score for the acute stimulation screen. TFs labeled are termed “chronic-unique” and represent TFs that were differentially enriched in the chronic screen (p adj. <= 0.001) but not enriched in the acute screen (p adj. > 0.05). FIG. 78 is a volcano plot showing the results of an acute stimulation screen of TF-ome open reading frame (ORF) with TOX readout and that differentially enriched TFs include both known and novel regulators of T cell exhaustion. FIG. 7C is a volcano plot showing the results of a chronic stimulation screen of TF-ome ORF with TOX readout and that differentially enriched TFs include both known and novel regulators of T cell exhaustion.

[0026] FIG. 8 is a Venn diagram showing the hit overlap from the acute vs chronic ORF screens. It can be seen that TFs regulate TOX in either chronic or acute stimulation settings, and there were many TFs regulating TOX uniquely in each stimulation setting.

[0027] FIGS. 9A-9C show that initial protein and RNA-level validations confirm modulation of exhaustion program. FIG. 9A is a diagram showing a protocol for phenotypic validation using FACS and qPCR with individual TF transductions. FIG. 9B is graphs showing FACS results of TOX protein expression validation of chronic-unique transcription factors after acute stimulation (left) and chronic stimulation (right). MFI is mean florescence intensity. FIG. 9C is graphs showing TOX RNA expression validation of chronic-unique transcription factors after acute stimulation (left) and chronic stimulation (right).

[0028] FIGS. 10A-10G show analyses of TF candidates that regulate exhaustion / memory protein and RNA expression. FIG. 10A is a diagram showing a protocol for a validation setup that focuses on individually transducing TFs and measuring TOX protein levels (via FACS), exhaustion / memory markers staining (via FACS), TOX transcript levels (via RT-qPCR), and transcriptomic changes (via bulk RNA-seq). FIG. 108 is a graph showing TOX MFI for most TFs validated in terms of TOX expression on the protein level via FACS. Each dot represents a different biological donor. Thy1.1 is an inert protein used as a control (dotted line represents the mean of this control) while the TOX condition is a positive control. The light grey color represents TFs that were enriched in the TOX high bin and the dark grey color represents TFs that were enriched in the TOX low bin. TFs that downregulated TOX were readily captured via FACS, but since baseline expression was high it was hard to capture upregulators. FIG. 10C is a graph showing results for most TFs validated in terms of TOX expression on the RNA level via qPCR. Thy1 .1 is an inert protein used as a control (dotted line represents the mean of this control) while the TOX condition is a positive control. The light grey color represents TFs that were enriched in the TOX high bin and the dark grey color represents TFs that were enriched in the TOX low bin. Nearly allTFs were validated and the effects of upregulators were effectively captured. FIG. 10D is a graph showing the percentage of PD-1+ expression in response to TFs as measured by FACS. TOX upregulated PD-1 as has been previously characterized which served as a positive control that the overexpression was functionally working. POU2F2 significantly upregulated PD-1 expression. FIG. WE is a graph showing the percentage of LAG3 expression in response to TFs as measured by FACS. MEF2B significantly downregulated expression of LAG3. FIG. 10F is a graph showing the percentage of TIGIT expression in response to TFs as measured by FACS. PAX2 significantly downregulated expression of TIGIT. FIG. 10G is a graph showing the percentage of IL7R expression in response to TFs as measured by FACS. MEF2B upregulated expression of the memory marker IL7R.

[0029] FIGS. 11 A-118 show that chronic-unique TFs modulate immune checkpoint protein expression, as shown for other TFs as in FIGS. 10D-10G. FIG. 11A is graphs showing FACS staining of exhaustion and memory markers in response to chronic-unique TFs after acute stimulation. FIG. 11B is graphs showing FACS staining of exhaustion and memory markers in response to chronic-unique TFs after chronic stimulation.

[0030] FIGS. 12A-12C show that chronic-unique TFs impact cancer killing in vitro. FIG. 12A is a diagram showing a chronic stimulation (top) and a TIL (bottom) protocol for functional validation with an in vitro CAR T cell cancer co-culture model. FIG. 12B is a graph showing cell counts of the SKBR3 human breast cancer cell line tagged with nuclear GFP over time that were co-cultured with CD8+ chronically stimulated CAR T cells expressing various TFs. FIG. 12C is a graph showing cell counts of the SKBR3 human breast cancer cell line overtime that were co-cultured with CD8+ CAR+ TILs expressing various TFs.

[0031] FIGS. 13A-13F show protocols and results for single-cell T ceil stimulation screens, FIGS. 13A and 138 are diagrams showing a general protocol for a chronic versus acute stimulation screen with a sub-pool of “chronic-unique” TFs and SHARE-seq as a readout (paired RNA+ATAC seq on top of TF capture). To add an additional layer of functional Information, a final PMA / IO stimulation was done in both conditions to dissect the functional capacity of T cells with specific TFs being overexpressed. A 770k single-cell atlas that contained matched RNA+ATAC+TF information with different stimulation conditions was successfully generated. As is represented by the UMAP, the cells clustered based on stimulation condition. FIGS. 13C and 13E are graphs showing RNA stimulation scores based on pseudo-bulked single-cell RNA-seq data in exhausted stimulated cells (TEX).THAP6 and DMRT3 pushed TEX away from exhaustion and improved TEX stimulation response. FIGS. 13D and 13F are graphs showing chromatin exhaustion scores based on pseudo-bulked single-cell ATAC-seq data in exhausted unstimulated cells.

[0032] FIGS. 14A-14H show that THAP6 reduces exhaustion marker expression and uniquely improves TEX function. FIG. 14A is a diagram showing a protocol for FACS analysis and cytokine analysis. To validate THAP6 phenotypically and functionally, the same exhaustion / memory markers were surface stained and cytokine expression was measured via 13-plex CD8 / NK cytokine panel. FIG. 14B is a graph showing the percentage of LAG3 expression in response to THAP6 as measured by FACS (Thy1 .1 is a control). A paired t-test was used for statistical significance testing. FIG. 14C is a graph showing the percentage of PD-1 expression in response to THAP6 as measured by FACS (Thy1.1 is a control). FiG. 14D is a graph showing the percentage of TIGIT expression in response to THAP6 as measured by FACS (Thy1 .1 is a control). FIG. 14E is a graph showing the percentage of PD-1 expression in acutely stimulated and chronically stimulated cells in response to THAP6 as measured by FACS (Thy1.1 is a control). FIG. 14F is a graph showing the percentage of LAG3 expression in acutely stimulated and chronically stimulated cells in response to THAP6 as measured by FACS (Thy1 .1 is a control). FIG. 14G is a graph showing the percentage of TIGIT expression in acutely stimulated and chronically stimulated cells in response to THAP6 as measured by FACS (Thy1.1 is a control). FACS analysis of exhaustion markers revealed that THAP6 reduces protein expression of LAG3 for all donors, and PD-1 and TIGIT for most donors. FiG. 14H is a graph showing results of multiplexed cytokine expression analysis (the entire data set is shown in FIGS. 1SF, 1SH, and 151). The analysis revealed that there was an improvement in IFN-gamma production, a drastic improvement in Granzyme A production, and an improvement in granzyme B production after a final PMA / IO stimulation.[0(8033] FIGS. 15A-15J show that THAP6 improves TEX effector function. FiG. 15A is a graph showing protein concentration of IFN-y from T cells transduced with Thy1.1 (control), THAP6, or DMRT3 and stimulated with an ethanol (EtOH) vehicle control. FIG. 15B is a graph showing protein concentration of sFasL from T cells transduced with Thyt .1 (control), THAP6, or DMRT3 and stimulated with an ethanol (EtOH) vehicle control. FiG. 15C is a graph showing protein concentration of granzyme A from T cells transduced with Thy1 .1 (control), THAP6, or DMRT3 and stimulated with an ethanol (EtOH) vehicle control. FIG. 15D Is a graph showing protein concentration of granzyme B from T cells transduced with Thy1.1 (control), THAP6, or DMRT3 and stimulated with an ethanol (EtOH) vehicle control. FIG. 15E is a graph showing protein concentration of perforin from T cells transduced with Thy1.1 (control), THAP6, or DMRT3 and stimulated with an ethanol (EtOH) vehicle control. FIG. 15F is a graph showing protein concentration of IFN-y from T cells transduced with Thy1.1 (control), THAP6, or DMRT3 and stimulated with phorbol myristate acetate (PMA) / ionomycin (IO), FiG. 15G is a graph showing protein concentration of sFasL from Tcells transduced with Thy1.1 (control), THAP6, or DMRT3 and stimulated with PMA / IO. FIG. 15H is a graph showing protein concentration of granzyme A from T cells transduced with Thy1.1 (control), THAP6, or DMRT3 and stimulated with PMA / IO. FIG. 1 SI is a graph showing protein concentration of granzyme B from T cells transduced with Thy1.1 (control), THAP6, or DMRT3 and stimulated with PMA / IO. FiG. 1SJ is a graph showing protein concentration of perforin from T cells transduced with Thy1 .1 (control), THAP6, or DMRT3 and stimulated with PMA / IO.

[0034] FIGS. 16A-16B show killing assay results from a co-cuiture of stimulated CD8+ T cells transduced with HERCAR-mCherry and TF lentivirus and SKBR3-GFP cells, showing that THAP6 was able to kill as effectively as the Thy1.1 control but also more potently. The different symbols correspond to different donors. FIG. 16A is a graph showing SKBR3-GFP cell counts overtime when co-cultured with stimulated CAR T cells expressing Thy1.1 (control), DMRT3, or THAP6. FIG. 1 SB is a graph showing T cell counts over time when cocultured with stimulated CAR T cells expressing Thy1.1 (control), DMRT3, or THAPS.

[0035] FIGS. 17A-17B show that the top TF candidates regulate exhaustion / memory genes. FIG. 17A is a graph showing the number of differentially expressed genes (DEGs) for various TF ORFs, FIG. 17B is a condensed gene expression heatmap showing the bulk RNA-seq data for various TF ORFs. TFs upregulating TOX are shown in the left three columns and TFs downregulating TOX are shown in the right two columns.DETAILED DESCRIPTION

[0036] Provided herein are compositions and methods for increasing or enhancing T cells, which may be used to enhance cancer therapies such as ACT. The compositions and methods may include increasing gene expression or a gene product such as transcription factors. Transcriptions factors (TFs) are central mediators of cellular reprogramming and differentiation. The gene may be selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV. The transcription factor TOX drives and maintains the TEX cell state through epigenetic regulation of exhaustion-associated genetic programs. As detailed herein, a T cell stimulation screen was designed using an Open Reading Frame (ORF) library of all the TFs in the human genome with TOX protein expression as a reporter for T cell exhaustion, to understand what TF regulators may be driving or preventing T cell exhaustion upstream of TOX. Comparing screen results in the context of acute versus chronic stimulation facilitated identification of factors specific to the TEX state. CD8+ T cells from three distinct donors were used in either an acute stimulation setting (one TOR stimulation with anti-CD3 / CD28 beads) and a chronic stimulation setting (two additionalstimulations). Over 100 TFs were enriched in TOX-high and TOX-low bins, including both known and novel regulators of TOX. individual validations of top enriched TFs unique to chronic stimulation confirmed modulation of TOX expression in the expected direction at the protein and transcript level, FACS staining of T cell exhaustion (LAG3, PD-1 , and TIGIT) and memory (IL7R) surface markers revealed significant differences in expression levels, suggesting phenotypic changes in T cell function. Genes were discovered that could be used to support specific features of memory T cells, counter T cell exhaustion, improve tumor control, and engineer T cells with enhanced durability and therapeutic potential. The gene targets identified and described herein may be used to improve the efficacy of ACT.1, Definitions

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0038] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0039] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0040] The term “about” or “approximately” as used herein as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in theart, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Alternatively, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-foid, and more preferably within 2- fold, of a value.

[0041] “Adeno-associated virus” or “AAV” as used interchangeably herein refers to a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.

[0042] “Allogeneic” refers to any material derived from another subject of the same species. Allogeneic cells are genetically distinct and immunologically incompatible yet belong to the same species. Typically, “allogeneic” is used to define cells, such as stem cells, that are transplanted from a donor to a recipient of the same species.

[0043] “Amino acid” as used herein refers to naturally occurring and non-natural 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. Amino acids can 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. Amino acids include the side chain and polypeptide backbone portions.

[0044] “Autologous" refers to any material derived from a subject and re-introduced to the same subject.

[0045] “Binding region” as used herein refers to the region within a target region that is recognized and bound by the CRISPR / Cas-based gene editing system.

[0046] The terms “cancer”, “cancer cell”, “tumor”, and “tumor cell” are used interchangeably herein and refer generally to a group of diseases characterized by uncontrolled, abnormal growth of cells (e.g., a neoplasia), in some forms of cancer, the cancer cells can spread locally or through the bloodstream and lymphatic system to otherparts of the body (“metastatic cancer"). “Cancer” refers to all types of cancer or neoplasm or malignant tumors found in animals, including carcinoma, adenoma, melanoma, sarcoma, lymphoma, leukemia, blastoma, glioma, astrocytoma, mesothelioma, or a germ cell tumor. Cancer may include cancer of, for example, the colon, rectum, stomach, bladder, cervix, uterus, skin, epithelium, muscle, kidney, liver, lymph, bone, blood, ovary, prostate, lung, brain, head and neck, and / or breast. Cancer may include medullablastoma, non-small cell lung cancer, and / or mesothelioma. In embodiments detailed herein, the cancer includes leukemia. The term “leukemia” refers to broadly progressive, malignant diseases of the hematopoietic organs / systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-ceil leukemia, aleukemic leukemia, a ieukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeiobiastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, and promyelocytic leukemia. In some embodiments, the leukemia is chronic myeloid leukemia (CML). In some embodiments, the leukemia is acute myeloid leukemia (AML).

[0047] “Clustered Regularly Interspaced Short Palindromic Repeats” and “CRISPRs”, as used interchangeably herein, refers to loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea.

[0048] “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The regulatory elements may include, for example, a promoter, an enhancer,an initiation codon, a stop codon, or a polyadenylation signal. The coding sequence may be codon optimized.

[0049] “Complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0050] The terms “control,” “reference level,” and “reference” are used herein interchangeably. The reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result. “Control group” as used herein refers to a group of control subjects. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group. Cutoff values (or predetermined cutoff values) may be determined by Adaptive Index Model (AIM) methodology. Cutoff values (or predetermined cutoff values) may be determined by a receiver operating curve (ROC) analysis from biological samples of the patient group. ROC analysis, as generally known in the biological arts, is a determination of the ability of a test to discriminate one condition from another, e.g,, to determine the performance of each marker in identifying a patient having CRC. A description of ROC analysis is provided in P.J. Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of which is hereby incorporated by reference in its entirety. Alternatively, cutoff values may be determined by a quartile analysis of biological samples of a patient group. For example, a cutoff value may be determined by selecting a value that corresponds to any value in the 25th-75th percentile range, preferably a value that corresponds to the 25th percentile, the 50th percentile or the 75th percentile, and more preferably the 75th percentile. Such statistical analyses may be performed using any method known in the art and can be implemented through any number of commercially available software packages (e.g., from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, TX; SAS institute Inc., Cary, NC.). The healthy or normal levels or ranges for a target or for a protein activity may be defined in accordance with standard practice. A control may be a subject or ceil without a composition as detailed herein. A control may be a subject, or a sample therefrom, whose disease state is known. The subject, or sample therefrom, may be healthy, diseased, diseased prior to treatment, diseased during treatment, or diseased after treatment, or a combination thereof.

[0051] “Correcting”, “gene editing,” and “restoring” as used herein refers to changing a mutant gene that encodes a dysfunctional protein or truncated protein or no protein at ail,such that a full-length functional or partially full-length functional protein expression is obtained. Correcting or restoring a mutant gene may include replacing the region of the gene that has the mutation or replacing the entire mutant gene with a copy of the gene that does not have the mutation with a repair mechanism such as homology-directed repair (HDR). Correcting or restoring a mutant gene may also include repairing a frameshift mutation that causes a premature stop codon, an aberrant splice acceptor site or an aberrant splice donor site, by generating a double stranded break in the gene that is then repaired using non-homologous end joining (NHEJ). NHEJ may add or delete at least one base pair during repair which may restore the proper reading frame and eliminate the premature stop codon. Correcting or restoring a mutant gene may also include disrupting an aberrant splice acceptor site or splice donor sequence. Correcting or restoring a mutant gene may also include deleting a non-essential gene segment by the simultaneous action of two nucleases on the same DNA strand in order to restore the proper reading frame by removing the DNA between the two nuclease target sites and repairing the DNA break by NHEJ.

[0052] “Donor DNA”, “donor template,” and “repair template” as used interchangeably herein refers to a double-stranded DNA fragment or molecule that includes at least a portion of the gene of interest. The donor DNA may encode a full-functional protein or a partially functional protein.

[0053] “Enhancer” as used herein refers to non-coding DNA sequences containing multiple activator and repressor binding sites. Enhancers range from 200 bp to 1 kb in length and may be either proximal, 5’ upstream to the promoter or within the first intron of the regulated gene, or distal, in introns of neighboring genes or intergenic regions far away from the locus. Through DNA looping, active enhancers contact the promoter dependently of the core DNA binding motif promoter specificity. 4 to 5 enhancers may interact with a promoter. Similarly, enhancers may regulate more than one gene without linkage restriction and may “skip” neighboring genes to regulate more distant ones. Transcriptional regulation may involve elements located in a chromosome different to one where the promoter resides. Proximal enhancers or promoters of neighboring genes may serve as platforms to recruit more distal elements.

[0054] “Frameshift” or “frameshift mutation” as used interchangeably herein refers to a type of gene mutation wherein the addition or deletion of one or more nucleotides causes a shift in the reading frame of the codons in the mRNA. The shift in reading frame may lead to the alteration in the amino acid sequence at protein translation, such as a missense mutation or a premature stop codon.

[0055] “Functional” and “full-functional” as used herein describes protein that has biological activity. A “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional protein.

[0056] “Fusion protein” as used herein refers to a chimeric protein created through the joining of two or more genes that originally coded for separate proteins. The translation of the fusion gene results in a single polypeptide with functional properties derived from each of the original proteins.

[0057] “Genetic construct" as used herein refers to the DNA or RNA molecules that comprise a polynucleotide that encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed. The regulatory elements may include, for example, a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal.

[0058] “Genome editing” or “gene editing” as used herein refers to changing the DNA sequence of a gene. Genome editing may include correcting or restoring a mutant gene or adding additional mutations. Genome editing may include knocking out a gene, such as a mutant gene or a normal gene. Genome editing may be used to treat disease or, for example, enhance muscle repair, by changing the gene of interest. In some embodiments, the compositions and methods detailed herein are for use in somatic cells and not germ line cells.

[0058] The term “heterologous” as used herein refers to nucleic acid comprising two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid that is recombinantly produced typically has two or more sequences from unrelated genes synthetically arranged to make a new functional nucleic acid, for example, a promoter from one source and a coding region from another source. The two nucleic acids are thus heterologous to each other in this context. When added to a cell, the recombinant nucleic acids would also be heterologous to the endogenous genes of the cell. Thus, in a chromosome, a heterologous nucleic acid would include a non-native (non- naturally occurring) nucleic acid that has integrated into the chromosome, or a non-native (non-naturally occurring) extrachromosomal nucleic acid. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in thesame relationship to each other in nature (for example, a “fusion protein,” where the two subsequences are encoded by a single nucleic acid sequence).

[0060] “Homology-directed repair” or “HDR” as used interchangeably herein refers to a mechanism in cells to repair double strand DNA lesions when a homologous piece of DNA is present in the nucleus, mostly in G2 and S phase of the cell cycle. HDR uses a donor DNA template to guide repair and may be used to create specific sequence changes to the genome, including the targeted addition of whole genes. If a donor template is provided along with the CRISPR / Cas9-based gene editing system, then the cellular machinery will repair the break by homologous recombination, which is enhanced several orders of magnitude in the presence of DNA cleavage. When the homologous DNA piece is absent, non-homologous end joining may take place instead.

[0061] “Identical” or “identity” as used herein in the context of two or more polynucleotide or polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical 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 specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0062] “Mutant gene” or “mutated gene” as used interchangeably herein refers to a gene that has undergone a detectable mutation. A mutant gene has undergone a change, such as the loss, gain, or exchange of genetic material, which affects the normal transmission and expression of the gene. A “disrupted gene” as used herein refers to a mutant gene that has a mutation that causes a premature stop codon. The disrupted gene product is truncated relative to a full-length undisrupted gene product.

[0063] “Non-homologous end joining (NHEJ) pathway” as used herein refers to a pathway that repairs double-strand breaks in DNA by directly ligating the break ends without the need for a homologous template. The template-independent re-ligation of DNA ends byNHEJ is a stochastic, error-prone repair process that introduces random micro-insertions and micro-deletions (indels) at the DNA breakpoint. This method may be used to intentionally disrupt, delete, or alter the reading frame of targeted gene sequences. NHEJ typically uses short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the end of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately, yet imprecise repair leading to loss of nucleotides may also occur, but is much more common when the overhangs are not compatible. “Nuclease mediated NHEJ” as used herein refers to NHEJ that is initiated after a nuclease cuts double stranded DNA.

[0064] “Normal gene” as used herein refers to a gene that has not undergone a change, such as a loss, gain, or exchange of genetic material. The normal gene undergoes normal gene transmission and gene expression. For example, a normal gene may be a wild-type gene.

[0065] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a polynucleotide also encompasses the complementary strand of a depicted single strand. Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions. Polynucleotides may be single stranded or double stranded or may contain portions of both double stranded and single stranded sequence. The polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, mRNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including, for example, uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.

[0066] “Open reading frame” refers to a stretch of codons that begins with a start codon and ends at a stop codon. In eukaryotic genes with multiple exons, introns are removed, and exons are then joined together after transcription to yield the final mRNA for protein translation. An open reading frame may be a continuous stretch of codons. In some embodiments, the open reading frame only applies to spliced mRNAs, not genomic DNA, for expression of a protein.

[0067] “Operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function. Nucleic acid or amino acid sequences are “operably linked” (or “operatively linked”) when placed into a functional relationship with one another. For instance, a promoter or enhancer is operably linked to a coding sequence if it regulates, or contributes to the modulation of, the transcription of the coding sequence. Operably linked DNA sequences are typically contiguous, and operably linked amino acid sequences are typically contiguous and in the same reading frame. However, since enhancers generally function when separated from the promoter by up to several kilobases or more and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.Similarly, certain amino acid sequences that are non-contiguous in a primary polypeptide sequence may nonetheless be operably linked due to, for example folding of a polypeptide chain. With respect to fusion polypeptides, the terms “operatively linked” and “operably linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.

[0068] “Partially-functional” as used herein describes a protein that is encoded by a mutant gene and has less biological activity than a functional protein but more than a nonfunctional protein.

[0069] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide”, “protein,” and “peptide” are used interchangeably herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, for example, enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. “Domains” are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alphahelices. “Tertiary structure” refers to the complete three-dimensional structure of apolypeptide monomer. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units. A “motif is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length. In some embodiments, a motif includes 3, 4, 5, 6, or 7 sequential amino acids. A domain may be comprised of a series of the same type of motif.

[0070] “Premature stop codon” or “out-of-frame stop codon” as used interchangeably herein refers to nonsense mutation in a sequence of DNA, which results in a stop codon at location not normally found in the wild-type gene. A premature stop codon may cause a protein to be truncated or shorter compared to the full-length version of the protein.

[0071] “Promoter” as used herein means a synthetic or naturally derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a ceil. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, human U6 (hU6) promoter, and CMV IE promoter. Promoters that target muscle-specific stem cells may include the CK8 promoter, the Spc5-12 promoter, and the MHCK7 promoter.

[0072] The term “recombinant” when used with reference to, for example, 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 (naturally occurring) form of the cell or express a second copy of a native gene that is otherwise normally or abnormally expressed, under expressed, or not expressed at all.

[0073] The term “shRNA” stands for short hairpin RNA or small hairpin RNA. A shRNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in ceils may be facilitated by delivery of plasmids or viral or bacterial vectors. The shRNA is processed by Dicer into siRNA.

[0074] The term “siRNA” stands for small interfering RNA siRNA, sometimes also known as short interfering RNA or silencing RNA. A siRNA is a class of double-stranded RNA molecule. The siRNA may be natural or artificial. The siRNA forms a complex with the RNA-induced silencing complex (RISC), The antisense (guide) strand of siRNA directs RISC to mRNA that has a complementary sequence, and then the mRNA is cleaved by RISC or its translation is repressed.[0007S] “Sample” or “test sample” as used herein can mean any sample in which the presence and / or level of a target is to be detected or determined or any sample comprising a DNA targeting or gene editing system or component thereof as detailed herein. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include a medical sample. Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof. In some embodiments, the sample comprises an aliquot. In other embodiments, the sample comprises a biological fluid. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0076] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal that wants or is in need of the herein described compositions or methods. The subject may be a human or a non-human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a nonprimate. The mammal can be a non-primate such as, for example, cow, pig, camel, llama, hedgehog, anteater, platypus, elephant, alpaca, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, and mouse. The mammal can be a primate such as a human. The mammal can be a non-human primate such as, for example, monkey, cynomolgous monkey,rhesus monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, a child, such as age 0-2, 2-4, 2-6, or 6-12 years, or an infant, such as age 0-1 years. The subject may be male. The subject may be female. In some embodiments, the subject has a specific genetic marker. The subject may be undergoing other forms of treatment. The subject may have a disease or condition. In some embodiments, the subject has cancer. In some embodiments, the subject is human.

[0077] “Substantially identical” can mean that a first and second amino acid or polynucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or less than 100% over a region of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 amino acids or nucleotides, respectively.

[0078] “Target gene” as used herein refers to any nucleotide sequence encoding a known or putative gene product. The target gene may be a mutated gene involved in a genetic disease. The target gene may encode a known or putative gene product that is intended to be corrected or for which its expression is intended to be modulated. In certain embodiments, the target gene is a gene detailed herein as a modulator of T cells.

[0079] “Target region” as used herein refers to the region of the target gene to which the CRISPR / Cas9-based gene editing or targeting system is designed to bind.

[0080] “T cells” are a type of white blood cell of the immune system and play a central role in the adaptive immune response. T cells express a T-cell receptor (TCR) on their cell surface. The T cell receptor (TCR) of a T cell is able to interact with immunogenic peptides (epitopes) bound to major histocompatibility complex (MHC) molecules and presented on the surface of target cells. Specific binding of the TCR triggers a signal cascade inside the T cell leading to proliferation and differentiation into a maturated effector T cell. T cells may differentiate into different types of T cells. T cells may include, for example, CD8+ T cells (“killer T cells” or “cytotoxic T cells) and CD4+ T cells (“helper T cells”). CD8+ T cells and CD4+ T cells may further differentiate into other types of T cells including, for example, regulatory T cells (“suppressor T cells”) and memory T cells. In some embodiments herein, the T cell is a memory T cell. An antigen-naive T cell expands and differentiates into a memory T cell after encountering the cognate antigen within the context of a major histocompatibility complex (MHC) molecule on the surface of an antigen presenting cell.Memory T cells may be CD8+ or CD4+. Memory T cells are long-lived and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen.

[0081] “Transgene” as used herein refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code. The introduction of a transgene has the potential to change the phenotype of an organism.

[0082] “Transcriptional regulatory elements” or “regulatory elements” refers to a genetic element which can control the expression of nucleic acid sequences, such as activate, enhancer, or decrease expression, or alter the spatial and / or temporal expression of a nucleic acid sequence. Examples of regulatory elements include, for example, promoters, enhancers, splicing signals, polyadenylation signals, and termination signals. A regulatory element can be “endogenous,” “exogenous,” or “heterologous” with respect to the gene to which it is operably linked. An “endogenous” regulatory element is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” regulatory element is one which is not normally linked with a given gene but is placed in operable linkage with a gene by genetic manipulation.

[0083] “Treatment” or “treating” or “therapy” when referring to protection of a subject from a disease, means suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Treatment may result in a reduction in the incidence, frequency, severity, and / or duration of symptoms of the disease. Preventing the disease involves administering a composition of the present invention to a subject prior to onset of the disease. Suppressing the disease involves administering a composition of the present invention to a subject after induction of the disease but before its clinical appearance. Repressing or ameliorating the disease involves administering a composition of the present invention to a subject after clinical appearance of the disease.

[0084] As used herein, the term “gene therapy” refers to a method of treating a patient wherein polypeptides or nucleic acid sequences are transferred into cells of a patient such that activity and / or the expression of a particular gene is modulated. In certain embodiments, the expression of the gene is suppressed. In certain embodiments, theexpression of the gene is enhanced. In certain embodiments, the temporal or spatial pattern of the expression of the gene is modulated.

[0085] “Variant” used herein with respect to a polynucleotide means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequence substantially identical thereto. A variant can be a polynucleotide sequence that is substantially identical over the full length of the full polynucleotide sequence or a fragment thereof. The polynucleotide sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or less than 100% identical over the full length of the polynucleotide sequence or a fragment thereof.

[0086] “Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or polypeptide or to promote an immune response. Variant can mean a functional fragment thereof. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker. A conservative substitution of an amino acid, for example, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (Kyte et al., J. Mol. Biol. 1982, 157, 105-132). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge, it is known in the art that amino acids of similar hydropathic indexes may be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids may also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatiblewith biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or less than 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0087] “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector may be capable of directing the delivery or transfer of a polynucleotide sequence to target cells, where it can be replicated or expressed. A vector may contain an origin of replication, one or more regulatory elements, and / or one or more coding sequences. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome, plasmid, cosmid, or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a seif-replicating extrachromosomai vector. Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector, or ientivirus vector. A vector may be an adeno-associated virus (AAV) vector. The vector may encode a Cas9 protein and at least one gRNA molecule.

[0088] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.2, Modulators of T Cells

[0089] Provided herein are modulators of T cells. Modifying or modulating may include increasing or decreasing, for example. In some embodiments, the compositions and methods comprise an agent that increases T cells, increasing T ceils may include increasing the number of T cells and / or increasing the number of memory T ceils and / or increasing the lifetime of a T ceil and / or preventing T ceil exhaustion and / or reducing T cell exhaustion and / or reversing T ceil exhaustion and / or enhancing the therapeutic potential ofT cells. Modifying a T cell may include modifying the expression of a target gene within the T cell. The compositions and methods detailed herein may engineer or modify the gene expression programs within T cells by engineering the T cells directly. In some embodiments, the compositions and methods comprise an agent that decreases expression or activity of TOX in T cells. TOX may comprise an amino acid sequence of SEQ ID NO: 73, encoded by a polynucleotide comprising the sequence of SEQ ID NO: 74. TOX expression or activity may be reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. TOX expression or activity may be reduced by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. TOX expression or activity may be reduced by about 5-95%, 10-90%, 15-85%, 20-80%, or 1 ,5-fold to 10-fold, relative to a control, in some embodiments, the compositions and methods comprise an agent that decreases expression of CCR7 and / or increases expression of IL7RA in T cells. Expression of a marker such as TOX or CCR7 or IL7RA may be done by any suitable means in the art, including, for example, ELISA, immunohistochemistry, flow cytometry, FACS, DNA or RNA sequencing, and hybridization of reporters or probes to RNA transcripts.

[0090] The modulator of T cells may target a gene or a regulatory element thereof. Regulatory elements include, for example, promoters and enhancers. Regulatory elements may be within 1000 base pairs of the transcription start site. Regulatory elements may be within 600 base pairs of the transcription start site. The agent, or the composition or the method comprising the agent, may modify the expression of a gene. For example, the agent, or the composition or the method comprising the agent, may reduce, inhibit, decrease, activate, increase, or enhance the expression or activity of a gene or its gene protein product. The agent, or the composition or the method comprising the agent, may directly or indirectly modulate the activity of the gene’s protein product. For example, the modulator of T cells may increase or decrease the binding or enzymatic activity of the gene’s protein product, or inhibit the binding of the gene’s protein product to another molecule or ligand, or increase the binding of the gene's protein product to another molecule or ligand, or increase or decrease the degradation of the gene's protein product, or a combination thereof.

[0091] The modulator of T cells may be an activator of a gene. Provided herein is an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a regulatory element thereof, or a region thereof, or acombination thereof. In some embodiments, the gene is THAP6, DMRT3, or MEF2B. The gene may encode a transcription factor. Further provided herein is an activator of a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST 1 , NKX6, and FEV, or a combination thereof. In some embodiments, the transcription factor is TRAPS, DMRT3, or MEF2B, or a combination thereof.

[0092] As an activator, an agent may activate or enhance expression or activity of a gene or gene protein product to increase T cells. The activator may increase the level of polynucleotide encoding the gene or encoding the gene product. The activator may increase the level of transcription of a polynucleotide encoding the gene or encoding the gene product. The activator may increase the level of translation of a mRNA encoding the gene product. The activator may increase the expression of protein encoded by the gene or the open reading frame thereof. The activator may increase the level or amount of protein expressed from the gene. The activator may increase the level or amount of protein expressed from the open reading frame of the gene.

[0093] The agent may comprise, for example, a polynucleotide, a polypeptide, a small molecule, a lipid, a carbohydrate, or a combination thereof. In some embodiments, the agent comprises a polynucleotide. The agent may comprise a polynucleotide encoding the gene or a fragment thereof. The agent may comprise a polynucleotide comprising a cDNA of the gene or a fragment thereof. For example, the polynucleotide may comprise a sequence selected from SEQ ID NOs: 75-84, or a fragment thereof. The polynucleotide may comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or at least 98%, or greater identity to a sequence selected from SEQ ID NOs: 75- 84, or a fragment thereof. The polynucleotide may comprise a sequence having one, two, three, four, five or more changes selected from nucleotide substitutions, insertions, or deletions, relative to a sequence selected from SEQ ID NOs: 75-84, or a fragment thereof.

[0094] In some embodiments, the agent comprises a protein. The agent may comprise a polypeptide comprising a protein product of the gene or a fragment thereof. For example, the polypeptide may comprise a sequence selected from SEQ ID NOs: 85-94, or a fragment thereof. The polypeptide may comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or at least 98%, or greater identity to a sequence selected from SEQ ID NOs: 85-94, or a fragment thereof. The polypeptide may comprise a sequence having one, two, three, four, five or more changes selected from amino acid substitutions, insertions, or deletions, relative to a sequence selected from SEQ ID NOs: 85- 94, or a fragment thereof. In some embodiments, the agent comprises a DNA targeting composition as detailed herein or at least one component thereof.

[0095] Examples of genes for modulating T cells are shown in TABLE 1 and TABLE 2.

[0096] T cells may be modulated by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1 .5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. T cells may be modulated by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, or 10-fold, relative to a control. T cells may be modulated by about 5- 95%, 10-90%, 15-85%, 20-80%, or 1 ,5-fold to 10-fold, relative to a control. T cells may be reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, or 10-fold, relative to a control. T cells may be reduced by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 1 .5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. T cells may be reduced by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control. T cells may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. T cells may be increased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. T cells may be increased by about 5-95%, 10-90%, 15-85%, 20-80%, or 1 .5-fold to 10-fold, relative to a control.

[0097] In some embodiments, the modulator of T cells is administered with or as a cancer therapy. The cancer therapy may include chemotherapy or immunotherapy. The cancer therapy may include adoptive T cell therapy (ACT) therapy. The cancer therapy may include a chimeric antigen receptor (CAR). A chimeric antigen receptor (CAR) may also be known as chimeric immunoreceptor, chimeric T cell receptor, or artificial T cell receptor. CARs are receptor proteins that have been engineered to give T ceils the new ability to target a specific antigen. CARs are chimeric in that they may combine both antigen-binding and T cell activating functions into a single receptor. CARs may include an antigen binding domain specific for an antigen on a cancer cell. The premise of CAR-T immunotherapy is to modify T cells to recognize cancer cells in order to target and destroy them. T cells are harvested from a subject, the T cells are genetically altered to add a chimeric antigen receptor (CAR) that specifically recognizes cancer cells, and the resulting CAR-T cells may be administered to the subject to attack their tumors. CAR-T cell therapy and modification to T cells are described in, for example, W02012 / 079000 and WO2012 / 129514 andWO2018 / 005712, each of which is incorporated herein by reference in its entirety. In some embodiments, the modulator of T cells is administered concurrently with a cancer therapy, or subsequent to a cancer therapy, or prior to a cancer therapy, or as a cancer therapy. a. DMA Targeting Systems

[0998] In some embodiments, the agent comprises a DNA targeting composition or at least one component thereof. A “DNA Targeting System” as used herein is a system capable of specifically targeting a particular region of DNA and modulating gene expression by binding to that region. Non-limiting examples of these systems are CRISPR-Cas-based systems, meganucleases, zinc finger (ZF)-based systems, and / or transcription activator-like effector (TALE)-based systems. The DNA Targeting System may be a nuclease system that acts through mutating or editing the target region (such as by insertion, deletion orsubstitution) or it may be a system that delivers a functional second polypeptide domain, such as an activator or repressor, to the target region.

[0099] Each of these systems comprises a DNA-binding portion or domain, such as a Cas protein and guide RNA, or a meganuciease, or a ZF, or a TALE, that specifically recognizes and binds to a particular target region of a target DNA. The DNA-binding portion (for example, Cas protein, ZF, or TALE) can be linked to a second protein domain, such as a polypeptide with transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, demethylase activity, acetylation activity, or deacetylation activity, to form a fusion protein. Exemplary second polypeptide domains are detailed further below (see “Cas Fusion Protein”). For example, the DNA-binding portion can be linked to an activator and thus guide the activator to a specific target region of the target DNA. Similarly, the DNA-binding portion can be linked to a repressor and thus guide the repressor to a specific target region of the target DNA.[090100] In some embodiments, the DNA targeting composition comprises a meganuciease. A meganuciease is an endodeoxyribonuclease characterized by a large recognition site, such as double-stranded DNA sequences of 12 to 40 base pairs. The recognition site may occur only once in any given genome. A meganuciease may be a homing endonuclease selected from an intron endonuclease or an intein endonuclease. Meganucleases may include, for example, the LAGLIDADG family of homing endonucleases.[900101] In some embodiments, the DNA-binding portion comprises a Cas protein, such as a Cas9 protein. Some CRISPR-Cas-based systems can operate to activate or repress expression using the Cas protein alone, not linked to an activator or repressor. For example, a nuclease-null Cas9 can act as a repressor on its own, a nuclease-active Cas9 can act as a repressor on its own, or a nuclease-active Cas9 can act as an activator when paired with an inactive (dead) guide RNA. In addition, RNA or DNA that hybridizes to a particular target region of the target DNA can be directly linked (covalently or non-covalently) to an activator or repressor. Some CRISPR-Cas-based systems can operate to activate or repress expression using the Cas protein linked to a second protein domain, such as, for example, an activator or repressor.i) DNA Binding Protein[000102] The DNA Targeting System may include a DNA binding protein. The DNA binding protein may comprise, for example, a zinc finger protein or a transcription activatorlike effector (TALE). The zinc finger protein or TALE may target a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a regulatory element thereof.(1) Zinc Finger Protein[000103] A zinc finger protein is a protein that includes one or more zinc finger domains. Zinc finger domains are relatively small protein motifs that contain multiple finger-like protrusions that make tandem contacts with their target molecule such as a DNA target molecule, A zinc finger domain may bind one or more zinc ions or other metal ion such as iron, or in some cases a zinc finger domain forms salt bridges to stabilize the finger-like folds. The zinc binding portion of a zinc finger protein may include one or more cysteine residues and / or one or more histidine residues to coordinate the zinc or other metal ion. A zinc finger protein recognizes and binds to a particular DNA sequence via the zinc finger domain. In some embodiments, a zinc finger protein is fused to or includes a nuclease domain and may be referred to as a zinc finger nuclease (ZFN). The nuclease domain may Include, for example, the endonuclease Fokl. ZFNs may recognize target sites that consist of two zlnc-finger binding sites that flank a 5- to 7-base pair (bp) spacer sequence recognized by the endonuclease Fokl cleavage domain.(2) Transcription Activator-like Effector (TALE)[000104] A TALE is another type of protein that recognizes and binds to a particular DNA sequence. The DNA-binding domain of a TALE includes an array of tandem 33-35 amino acid repeats, also known as RVD modules. Each RVD module specifically recognizes a single base pair of DNA. RVD modules may be arranged in any order to assemble an array that recognizes a defined DNA sequence. The binding specificity of a TALE DNA-binding domain is determined by the RVD array followed by a single truncated repeat of, for example, 20 amino acids, A TALE DNA-binding domain may have an array of 12 to 27 RVD modules, each RVD module recognizing a single base pair of DNA. Specific RVDs have been identified that recognize each of the four possible DNA nucleotides (A, T, C, and G). Because the TALE DNA-binding domains are modular, repeats that recognize the four different DNA nucleotides may be linked together to recognize any particular DNA sequence. These targeted DNA-binding domains may then be combined with catalytic domains tocreate functional enzymes, including artificial transcription factors and / or nucleases. In some embodiments, a TALE is fused to or includes a nuclease domain and may be referred to as a TALE nuclease (TALEN). The nuclease domain may include, for example, the endonuclease Fokl. TALENs may recognize target sites that consist of two TALE DNA- binding sites that flank a 12-bp to 20-bp spacer sequence recognized by the Fokl cleavage domain.(3) DNA Binding Fusion Protein[000105] Additionally or alternatively, a zinc finger protein or TALE can be fused to a polypeptide domain and referred to as a DNA binding fusion protein or fusion protein. The fusion protein may act as a synthetic transcription factor. The fusion protein comprises two heterologous polypeptide domains, including a first polypeptide domain comprising the zinc finger protein or the TALE or a Cas9 protein as further detailed below, and a second polypeptide domain having an activity selected from transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, methylase activity, and demethylase activity. A zinc finger protein or TALE can be fused to a polypeptide domain having epigenetic modifying activity to mediate targeted gene regulation, A fusion protein comprising a zinc finger protein or TALE, and a second polypeptide domain having transcription repression activity, may mediate targeted gene repression. A fusion protein comprising a zinc finger protein or TALE, and a second polypeptide domain having transcription activation activity, may mediate targeted gene activation. The second polypeptide domain is further detailed below (see “Cas Fusion Protein”). ii) CRISPR / Cas-based Gene Editing System[000106] Provided herein are CRISPR / Cas-based gene editing systems. The CRISPR / Cas-based gene editing system may be used to modulate T cells and / or enhance ACT. The CRISPR / Cas-based gene editing system may include a Cas protein or a fusion protein, and at least one gRNA, and may also be referred to as a “CRISPR-Cas system.”[000107] “Clustered Regularly Interspaced Short Palindromic Repeats” and “CRISPRs”, as used interchangeably herein, refers to loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. The CRISPR system is a microbial nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity. The CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRiSPR repeats, and serve as a “memory” of past exposures. Gas proteins include, for example, Cast 2a, Cas9, Cas13, and Cascade proteins. Cas12a may also be referred to as “Cpf1.” Cas12a causes a staggered cut in double stranded DNA, while Cas9 produces a blunt cut. In some embodiments, the Gas protein comprises Cas12a. Cast 2a is described in, for example, WO 2018 / 017754, which is incorporated herein by reference. Cas13 is an RNA-guided RNA endonuclease. Cas13 cleaves single-stranded RNA, and it does not cleave DNA. In some embodiments, the Gas protein comprises Cas13. In some embodiments, the Cas protein comprises Cas9. Cas9 forms a complex with the 3’ end of the sgRNA (which may be referred interchangeably herein as “gRNA”), and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5’ end of the gRNA sequence and a predefined 20 bp DNA sequence, known as the protospacer. This complex is directed to homologous loci of pathogen DNA via regions encoded within the crRNA, i.e., the protospacers, and protospacer-adjacent motifs (PAMs) within the pathogen genome. The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). By simply exchanging the 20 bp recognition sequence of the expressed gRNA, the Cas9 nuclease can be directed to new genomic targets. CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.[000108] Three classes of CRISPR systems (Types I, II, and III effector systems) are known. The Type II effector system carries out targeted DNA double-strand break in four sequential steps, using a single effector enzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type III effector systems, which require multiple distinct effectors acting as a complex, the Type II effector system may function in alternative contexts such as eukaryotic cells. The Type II effector system consists of a long pre-crRNA, which is transcribed from the spacer-containing CRISPR locus, the Cas9 protein, and a tracrRNA, which is involved in pre-crRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, thus initiating dsRNA cleavage by endogenous RNase III. This cleavage is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9, forming a Cas9:crRNA- tracrRNA complex. Cas12a systems include crRNA for successful targeting, whereas Cas9 systems include both crRNA and tracrRNA.[000109] The Cas9:crRNA-tracrRNA complex unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a “protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA. Cas9 mediates cleavage of target DNA if a correct protospacer-adjacent motif (PAM) is also present at the 3’ end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer- adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage. Different Cas and Cas Type II systems have differing PAM requirements. For example, Cast 2a may function with PAM sequences rich in thymine “T.”[000119] An engineered form of the Type II effector system of S. pyogenes was shown to function in human ceils for genome engineering. In this system, the Cas9 protein was directed to genomic target sites by a synthetically reconstituted “guide RNA” (“gRNA”, also used interchangeably herein as a chimeric single guide RNA (“sgRNA”)), which is a crRNA- tracrRNA fusion that obviates the need for RNase III and crRNA processing in general. Provided herein are CRISPR / Cas9-based engineered systems for use in gene editing and treating genetic diseases. The CRISPR / Cas9-based engineered systems can be designed to target any gene, including genes involved in, for example, a genetic disease, aging, tissue regeneration, or wound healing. The CRISPR / Cas9-based gene editing system can include a Cas9 protein or a Cas9 fusion protein. iii) Cas9 Protein[900111] Cas9 protein is an endonuclease that cleaves nucleic acid and is encoded by the CRISPR loci and is involved in the Type II CRISPR system. The Cas9 protein can be from any bacterial or archaea species, including, but not limited to, Streptococcus pyogenes, Staphylococcus aureus (S. aureus), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., oycliphiius denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula manna, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coll, Campylobacter jejuni, Campylobacter lari, Candidates Puniceispinllum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, llyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobil uncus mulieris, Neisseria bacilli formis,Neisseria cinerea, Neisseria flavescens, Neisseria iactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteureiia multocida, Phascolarctobacterium succinatutens, Raistonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensls, Streptococcus sp., Subdoligranulum sp., Tistreila mobllis, Treponema sp., or Verminephrobacter eiseniae. In certain embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule (also referred herein as “SpCas9”). SpCas9 may comprise an amino acid sequence of SEQ ID NO: 26. In certain embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule (also referred herein as “SaCas9”). SaCas9 may comprise an amino acid sequence of SEQ ID NO: 27.[000112] A Cas9 molecule or a Cas9 fusion protein can interact with one or more gRNA molecule(s) and, in concert with the gRNA molecule(s), can localize to a site which comprises a target domain, and in certain embodiments, a PAM sequence. The Cas9 protein forms a complex with the 3’ end of a gRNA. The ability of a Cas9 molecule or a Cas9 fusion protein to recognize a PAM sequence can be determined, for example, by using a transformation assay as known in the art.[000113] The specificity of the CRISPR-based system may depend on two factors: the target sequence and the protospacer-adjacent motif (PAM). The target sequence is located on the 5’ end of the gRNA and is designed to bond with base pairs on the host DNA at the correct DNA sequence known as the protospacer. By simply exchanging the recognition sequence of the gRNA, the Cas9 protein can be directed to new genomic targets. The PAM sequence is located on the DNA to be altered and is recognized by a Cas9 protein. PAM recognition sequences of the Cas9 protein can be species specific.[000114] In certain embodiments, the ability of a Cas9 molecule or a Cas9 fusion protein to interact with and cleave a target nucleic acid is PAM sequence dependent. A PAM sequence is a sequence in the target nucleic acid. In certain embodiments, cleavage of the target nucleic acid occurs upstream from the PAM sequence. Cas9 molecules from different bacterial species can recognize different sequence motifs (for example, PAM sequences). A Cas9 molecule of S. pyogenes may recognize the PAM sequence of NRG (5’-NRG-3!, where R is any nucleotide residue, and in some embodiments, R is either A or G, SEQ ID NO: 1). In certain embodiments, a Cas9 molecule of S. pyogenes may naturally prefer and recognize the sequence motif NGG (SEQ ID NO: 2) and directs cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, bp upstream from that sequence. In some embodiments, a Cas9 molecule of S. pyogenes accepts other PAM sequences, such as NAG (SEQ ID NO: 3) in engineered systems (Hsu et al., Nature Biotechnology 2013doi:10.1038 / nbt.2647, incorporated herein by reference), in certain embodiments, a Cas9 molecule of S. thermophiius recognizes the sequence motif NGGNG (SEQ ID NO: 4) and / or NNAGAAW (W = A or T) (SEQ ID NO: 5) and directs cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, bp upstream from these sequences. In certain embodiments, a Cas9 molecule of S. mutans recognizes the sequence motif NGG (SEQ ID NO: 2) and / or NAAR (R = A or G) (SEQ ID NO: 6) and directs cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5 bp, upstream from this sequence. In certain embodiments, a Cas9 molecule of S. aureus recognizes the sequence motif NNGRR (R = A or G) (SEQ ID NO: 7) and directs cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, bp upstream from that sequence. In certain embodiments, a Cas9 molecule of S. aureus recognizes the sequence motif NNGRRN (R = A or G) (SEQ ID NO: 8) and directs cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, bp upstream from that sequence. In certain embodiments, a Cas9 molecule of S. aureus recognizes the sequence motif NNGRRT (R = A or G) (SEQ ID NO: 9) and directs cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, bp upstream from that sequence. In certain embodiments, a Cas9 molecule of S. aureus recognizes the sequence motif NNGRRV (R ~ A or G; V = A or C or G) (SEQ ID NO: 10) and directs cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, bp upstream from that sequence. A Cas9 molecule derived from Neisseria meningitidis (NmCas9) normally has a native PAM of NNNNGATT (SEQ ID NO: 11), but may have activity across a variety of PAMs, including a highly degenerate NNNNGNNN PAM (SEQ ID NO: 12) (Esvelt et al. Nature Methods 2013 doi:10.1038 / nmeth.2681 , incorporated herein by reference). In the aforementioned embodiments, N can be any nucleotide residue, for example, any of A, G, C, or T. Cas9 molecules can be engineered to alter the PAM specificity of the Cas9 molecule.[000115] In some embodiments, the Cas9 protein recognizes a PAM sequence NGG (SEQ ID NO: 2) or NGA (SEQ ID NO: 13) or NNNRRT (R = A or G) (SEQ ID NO: 14) or ATTCCT (SEQ ID NO: 15) or NGAN (SEQ ID NO: 16) or NGNG (SEQ ID NO: 17). In some embodiments, the Cas9 protein is a Cas9 protein of S. aureus and recognizes the sequence motif NNGNO: 10). In the aforementioned embodiments, N can be any nucleotide residue, for example, any of A, G, C, or T.[000116] Additionally or alternatively, a nucleic acid encoding a Cas9 molecule or Cas9 polypeptide may comprise a nuclear localization sequence (NLS). Nuclear localizationsequences are known in the art, for example, SV40 NLS (Pro-Lys-Lys-Lys-Arg-Lys-Val; SEQ ID NO: 20).[000117] In some embodiments, the at least one Cas9 molecule is a mutant Cas9 molecule. The Cas9 protein can be mutated so that the nuclease activity is inactivated. An inactivated Cas9 protein (“iCas9”, also referred to as “dCas9”) with no endonuclease activity has been targeted to genes in bacteria, yeast, and human cells by gRNAs to silence gene expression through steric hindrance. Exemplary mutations with reference to the S. pyogenes Cas9 sequence to inactivate the nuclease activity include: D10A, E762A, H840A, N854A, N863A and / or D986A. A S. pyogenes Cas9 protein with the D10A mutation may comprise an amino acid sequence of SEQ ID NO: 28. A S. pyogenes Cas9 protein with D10A and H849A mutations may comprise an amino acid sequence of SEQ ID NO: 29. Exemplary mutations with reference to the S. aureus Cas9 sequence to inactivate the nuclease activity include D10A and N580A. In certain embodiments, the mutant S. aureus Cas9 molecule comprises a D10A mutation. The nucleotide sequence encoding this mutant S. aureus Cas9 is set forth in SEQ ID NO: 30. in certain embodiments, the mutant S. aureus Cas9 molecule comprises a N580A mutation. The nucleotide sequence encoding this mutant S. aureus Cas9 molecule is set forth in SEQ ID NO: 31 .[000118] In some embodiments, the Cas9 protein is a VQR variant. The VQR variant of Cas9 is a mutant with a different PAM recognition, as detailed in Kleinstiver, et al. (Nature 201S, 523, 481-485, incorporated herein by reference).[000119] A polynucleotide encoding a Cas9 molecule can be a synthetic polynucleotide. For example, the synthetic polynucleotide can be chemically modified. The synthetic polynucleotide can be codon optimized, for example, at least one non-common codon or less-common codon has been replaced by a common codon. For example, the synthetic polynucleotide can direct the synthesis of an optimized messenger mRNA, for example, optimized for expression in a mammalian expression system, as described herein. An exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S pyogenes is set forth in SEQ ID NO: 32. Exemplary codon optimized nucleic acid sequences encoding a Cas9 molecule of S. aureus, and optionally containing nuclear localization sequences (NLSs), are set forth in SEQ ID NOs: 33-39. Another exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. aureus comprises the nucleotides 1293-4451 of SEQ ID NO: 40.iv) Cas Fusion Protein[000120] Alternatively or additionally, the CRISPR / Cas-based gene editing system can include a fusion protein. The fusion protein can comprise two heterologous polypeptide domains. The first polypeptide domain comprises a Cas protein or a mutated Cas protein. The first polypeptide domain is fused to at least one second polypeptide domain. The second polypeptide domain has a different activity that what is endogenous to Cas protein. The second polypeptide domain may have any DNA editing activity. The second polypeptide domain may have an activity such as transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, histone methylase activity, DNA methylase activity, histone demethylase activity, DNA demethylase activity, acetylation activity, and / or deacetylation activity. The activity of the second polypeptide domain may be direct or indirect. The second polypeptide domain may have this activity itself (direct), or it may recruit and / or interact with a polypeptide domain that has this activity (indirect). In some embodiments, the second polypeptide domain has transcription activation activity. In some embodiments, the second polypeptide domain has transcription repression activity. In some embodiments, the second polypeptide domain comprises a synthetic transcription factor. The second polypeptide domain may be at the C-terminal end of the first polypeptide domain, or at the N-terminal end of the first polypeptide domain, or a combination thereof. The fusion protein may include one second polypeptide domain. In some embodiments, the fusion protein comprises more than one second polypeptide domain. The fusion protein may include two of the second polypeptide domains. For example, the fusion protein may include a second polypeptide domain at the N-terminal end of the first polypeptide domain as well as a second polypeptide domain at the C-terminal end of the first polypeptide domain. In other embodiments, the fusion protein may include a single first polypeptide domain and more than one (for example, two or three) second polypeptide domains in tandem.[000121] The linkage from the first polypeptide domain to the second polypeptide domain can be through reversible or irreversible covalent linkage or through a non-covalent linkage, as long as the linker does not interfere with the function of the second polypeptide domain. For example, a Cas polypeptide can be linked to a second polypeptide domain as part of a fusion protein. As another example, they can be linked through reversible non-covalent interactions such as avidin (or streptavidin)-biotin interaction, histidine-divalent metal ion interaction (such as, Ni, Co, Cu, Fe), interactions between multimerization (such as, dimerization) domains, or glutathione S-transferase (GST)-glutathione interaction. As yetanother example, they can be linked covalently but reversibly with linkers such as dlbromomaleimide (DBM) or amino-thiol conjugation.[000122] In some embodiments, the fusion protein includes at least one linker. A linker may be included anywhere in the polypeptide sequence of the fusion protein, for example, between the first and second polypeptide domains. A linker may be of any length and design to promote or restrict the mobility of components in the fusion protein. A linker may comprise any amino acid sequence of about 2 to about 100, about 5 to about 80, about 10 to about 60, or about 20 to about 50 amino acids. A linker may comprise an amino acid sequence of at least about 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids. A linker may comprise an amino acid sequence of less than about 100, 90, 80, 70, 60, 50, or 40 amino acids. A linker may include sequential or tandem repeats of an amino acid sequence that is 2 to 20 amino acids in length. Linkers may include, for example, a GS linker (Gly-Gly-Gly- Gly-Ser)n, wherein n is an integer between 0 and 10 (SEQ ID NO: 21). In a GS linker, n can be adjusted to optimize the linker length and achieve appropriate separation of the functional domains. Other examples of linkers may include, for example, Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 22), Gly-Gly-Ala-Gly-Gly (SEQ ID NO: 23), Gly / Ser rich linkers such as Gly-Gly-Gly-Gly- Ser-Ser-Ser (SEQ ID NO: 24), or Gly / Ala rich linkers such as Gly-Gly-Gly-Gly-Ala-Ala-Ala (SEQ ID NO: 25).[080123] In some embodiments, the agent and / or Cas protein and / or the Gas fusion protein and / or gRNAs detailed herein may be used in compositions and methods for modulating expression of gene. Modulating may include, for example, increasing or enhancing expression of the gene, or reducing or inhibiting expression of the gene. The expression of the gene may be modulated by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The expression of the gene may be modulated by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The expression of the gene may be modulated by about 5-95%, 10-90%, 15-85%, 20-80%, or 1 .5-fold to 10-foid, relative to a control. The expression of the gene may be reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-foid, relative to a control. The expression of the gene may be reduced by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold,relative to a control. The expression of the gene may be reduced by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control. The expression of the gene may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The expression of the gene maybe increased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1 .5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, or 10-fold, relative to a control. The expression of the gene may be increased by about 5-95%, 10-90%, 15-85%, 20-80%, or 1 .5-fold to 10-fold, relative to a control. In some embodiments, the expression of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV is increased. In some embodiments, the expression of a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV is increased. In some embodiments, the level of a transcription factor protein selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV is increased.(1) Transcription Activation Activity[000124] The second polypeptide domain can have transcription activation activity, for example, a transactivation domain. For example, gene expression of endogenous mammalian genes, such as human genes, can be achieved by targeting a fusion protein of a first polypeptide domain, such as dCas9, and a transactivation domain to mammalian promoters via combinations of gRNAs. The transactivation domain can include a VP16 protein, multiple VP16 proteins, such as a VP48 domain or VP64 domain, p65 domain of NF kappa B transcription activator activity, TET1 , VPR, VPH, Rta, and / or p300. For example, the fusion protein may comprise dCas9-p300. In some embodiments, p300 comprises a polypeptide having the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 42. In other embodiments, the fusion protein comprises dCas9-VP64. In other embodiments, the fusion protein comprises VP64-dCas9-VP64. VP64-dCas9-VP64 may comprise a polypeptide having the amino acid sequence of SEQ ID NO: 43, encoded by the polynucleotide of SEQ ID NO: 44. VPH may comprise a polypeptide having the amino acid sequence of SEQ ID NO: 53, encoded by the polynucleotide of SEQ ID NO: 54. VPR may comprise a polypeptide having the amino acid sequence of SEQ ID NO: 55, encoded by the polynucleotide of SEQ ID NO: 56.(2) Transcription Repression Activity[000125] The second polypeptide domain can have transcription repression activity. Nonlimiting examples of repressors include Kruppel associated box activity such as a KRAB domain or KRAB, MECP2, EED, ERF repressor domain (ERD), Mad mSIN3 interaction domain (SID) or Mad-SID repressor domain, SID4X repressor domain, Mxil repressor domain, SUV39H1 , SUV39H2, G9A, ESET / SETBD1 , Cir4, Su(var)3-9, Pr-SET7 / 8, SUV4- 20H1 , PR-set7, Suv4-20, Set9, EZH2, RIZ1 , JMJD2A / JHDM3A, JMJD2B, JMJ2D2C / GASC1 , JMJD2D, Rph1 , JARID1 A / RBP2, JARID1 B / PLU-1 , JARID1 C / SMCX, JARID1 D / SMCY, Lid, Jhn2, Jmj2, HDAC1 , HDAC2, HDAC3, HDAC8, Rpd3, Hos1 , Cir6, HDAC4, HDAC5, HDAC7, HDAC9, Hda1 , Cir3, SIRT1 , SIRT2, Sir2, Hst1 , Hst2, Hst3, Hst4, HDAC11 , DNMT1 , DNMT3a / 3b, DNMT3A-3L, MET1 , DRM3, ZMET2, CMT1 , CMT2, Laminin A, Laminin B, CTCF, and / or a domain having TATA box binding protein activity, or a combination thereof. In some embodiments, the second polypeptide domain has a KRAB domain activity, ERF repressor domain activity, Mxil repressor domain activity, SID4X repressor domain activity, Mad-SID repressor domain activity, DNMT3A or DNMT3L or fusion thereof activity, LSD1 histone demethyiase activity, or TATA box binding protein activity. In some embodiments, the polypeptide domain comprises KRAB. KRAB may comprise a polypeptide having the amino acid sequence of SEQ ID NO: 45, encoded by a polynucleotide comprising the sequence of SEQ ID NO: 46. For example, the fusion protein may be S. pyogenes dCas9-KRAB (protein sequence comprising SEQ ID NO: 47; polynucleotide sequence comprising SEQ ID NO: 48). The fusion protein may be S. aureus dCas9-KRAB (protein sequence comprising SEQ ID NO: 49; polynucleotide sequence comprising SEQ ID NO: 50).(3) Transcription Release Factor Activity[000126] The second polypeptide domain can have transcription release factor activity. The second polypeptide domain can have eukaryotic release factor 1 (ERF1) activity or eukaryotic release factor 3 (ERF3) activity.(4) Histone Modification Activity[000127] The second polypeptide domain can have histone modification activity. The second polypeptide domain can have histone deacetylase, histone acetyltransferase, histone demethyiase, or histone methyltransferase activity. The histone acetyltransferase may be p300 or CREB-binding protein (CBP) protein, or fragments thereof. For example,the fusion protein may be dCas9-p300. In some embodiments, p300 comprises a polypeptide of SEQ ID NO: 41 or SEQ ID NO: 42.(5) Nuclease Activity[0(80128] The second polypeptide domain can have nuclease activity that is different from the nuclease activity of the Cas9 protein, A nuclease, or a protein having nuclease activity, is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Nucleases are usually further divided into endonucleases and exonucleases, although some of the enzymes may fall in both categories. Well known nucleases include deoxyribonuclease and ribonuclease. In some embodiments, the second polypeptide domain includes a meganuclease, as detailed above. In some embodiments, the polypeptide domain having nuclease activity comprises Fokl.(6) Nucleic Acid Association Activity[000129] The second polypeptide domain can have nucleic acid association activity or nucleic acid binding protein-DNA-binding domain (DBD). A DBD is an independently folded protein domain that contains at least one motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence (a recognition sequence) or have a general affinity to DNA. A nucleic acid association region may be selected from helix-turn- helix region, leucine zipper region, winged helix region, winged helix-turn-helix region, helix- loop-helix region, immunoglobulin fold, B3 domain, Zinc finger, HMG-box, Wor3 domain, and TAL effector DNA-binding domain.(7) Base Editing Activity[000130] The second polypeptide domain may have base editing activity. Base editing enables the direct, irreversible conversion of a specific DNA base into another base at a targeted genomic locus without requiring double-stranded DNA breaks (DSB). A base editing domain has sequence requirements for activity. In a 20 nucleotide protospacer, the target base may be within 4-8 nucleotides from the PAM-distal end. An exemplary splice acceptor is an “AG” immediately before the exon, and an exemplary splice donor is a “GT” immediately following the exon. Cas9 molecules from different species may use different PAMs, and thereby provide some flexibility in selecting the base to edit. Disruption of canonical splice sites can lead to exon skipping or activation of cryptic splice sites. Both adenine and cytosine base editors may be capable of disrupting an “AG” splice acceptor, converting it to either a “GG” or “AA”, respectively. In some embodiments, the base-editing domain includes an adenine base editor (ABE). Adenine base editors may include, forexample, ecTadA, including wild-type and mutants thereof. The adenine base editor may be as described in Gaudelli et al. (Nature 2017, 551 , 464-471), Kobian et al. (Nature Biotech. 2018, 36, 843-846), Richter et al. (Nature Biotech. 2020, 38, 883-891), and Gaudelli et al. (Nature Biotech, 2020, 38, 892-900), each of which is incorporated herein by reference.The ABE may comprise a polypeptide selected from SEQ ID NOs: 57-64 and / or be encoded by a polynucleotide comprising a sequence selected from SEQ ID NOs: 65-72, respectively. In some embodiments, the base-editing domain includes a cytidine deaminase domain. A cytidine deaminase domain can convert the DNA base cytosine to uracil. In some embodiments, the cytidine deaminase domain can include an apolipoprotein B mRNA- editing enzyme, catalytic polypeptide-like (APOBEC) family deaminase. In some embodiments, the cytidine deaminase domain can include an APOBEC 1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, or a combination thereof. Base editing domains are detailed in, for example, WO 2020 / 210776 and WO 2022 / 081612, each of which is incorporated herein by reference.(8) Methylase Activity[000131] The second polypeptide domain can have methylase activity, which involves transferring a methyl group to DNA, RNA, protein, small molecule, cytosine, or adenine. In some embodiments, the second polypeptide domain includes a DNA methyltransferase.(9) Demethylase Activity[000132] The second polypeptide domain can have demethylase activity. The second polypeptide domain can include an enzyme that removes methyl (CH3-) groups from nucleic acids, proteins (in particular histones), and other molecules. Alternatively, the second polypeptide can convert the methyl group to hydroxymethylcytosine in a mechanism for demethylating DNA. The second polypeptide can catalyze this reaction. For example, the second polypeptide that catalyzes this reaction can be Tet1 , also known as TetICD (Ten- eleven translocation methylcytosine dioxygenase 1 ; amino acid sequence comprising SEQ ID NO: 51 ; polynucleotide sequence comprising SEQ ID NO: 52). In some embodiments, the second polypeptide domain has histone demethylase activity. In some embodiments, the second polypeptide domain has DNA demethylase activity.v) Guide RNA (gRNA)[000133] The CRISPR / Cas-based gene editing system includes at least one gRNA molecule. For example, the CRISPR / Cas-based gene editing system may include two gRNA molecules. The at least one gRNA molecule can bind and recognize a target region. The gRNA is the part of the CRISPR-Cas system that provides DNA targeting specificity to the CRISPR / Cas-based gene editing system. The gRNA is a fusion of two noncoding RNAs: a crRNA and a tracrRNA. gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system. This duplex, which may include, for example, a 42- nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide for the Cas9 to bind, and in some cases, cleave the target nucleic acid. The gRNA may target any desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. The Target region” or “target sequence” or “protospacer” refers to the region of the target gene to which the CRISPR / Cas9-based gene editing system targets and binds. The portion of the gRNA that targets the target sequence in the genome may be referred to as the “targeting sequence” or “targeting portion” or “targeting domain.” “Protospacer” or “gRNA spacer” may refer to the region of the target gene to which the CRISPR / Cas9-based gene editing system targets and binds; “protospacer” or “gRNA spacer” may also refer to the portion of the gRNA that is complementary to the targeted sequence in the genome. The gRNA may include a gRNA scaffold. A gRNA scaffold facilitates Cas9 binding to the gRNA and may facilitate endonuclease activity. The gRNA scaffold is a polynucleotide sequence that follows the portion of the gRNA corresponding to sequence that the gRNA targets. Together, the gRNA targeting portion and gRNA scaffold form one polynucleotide. The constant region of the gRNA may include the sequence of SEQ ID NO: 19 (RNA), which is encoded by a sequence comprising SEQ ID NO: 18 (DNA). The CRISPR / Cas9-based gene editing system may include at least one gRNA, wherein the gRNAs target different DNA sequences. The target DNA sequences may be overlapping. The gRNA may comprise at its 5’ end the targeting domain that is sufficiently complementary to the target region to be able to hybridize to, for example, about 10 to about 20 nucleotides of the target region of the target gene, when it is followed by an appropriate Protospacer Adjacent Motif (PAM). The target region or protospacer is followed by a PAM sequence at the 3’ end of the protospacer in the genome. Different Type II systems have differing PAM requirements, as detailed above.[000134] The targeting domain of the gRNA does not need to be perfectly complementary to the target region of the target DNA. In some embodiments, the targeting domain of the gRNA is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% complementary to(or has 1 , 2 or 3 mismatches compared to) the target region over a length of, such as, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. For example, the DNA-targeting domain of the gRNA may be at least 80% complementary over at least 18 nucleotides of the target region. The target region may be on either strand of the target DNA.[000135] The gRNA may target the Cas9 protein or fusion protein to a gene or a regulatory element thereof. The gRNA may target the Cas protein or fusion protein to a non-open chromatin region, an open chromatin region, a transcribed region of the target gene, a region upstream of a transcription start site of the target gene, a regulatory element of the target gene, an intron of the target gene, or an exon of the target gene, or a combination thereof. In some embodiments, the gRNA targets the Cas9 protein or fusion protein to a promoter of a gene. In some embodiments, the target region is located between about 1 to about WOO base pairs upstream of a transcription start site of a target gene, in some embodiments, the DNA targeting composition comprises two or more gRNAs, each gRNA binding to a different target region.[000136] The gRNA may target a region of a gene that modulates T cells. The gRNA may target a region of a gene encoding a transcription factor that modulates T cells. The gRNA may target a region of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKXS, or FEV, or a combination thereof, or a regulatory element thereof. The gRNA may target a region of a gene selected from THAP6, DMRT3, or MEF2B, or a combination thereof, or a regulatory element thereof. In some embodiments, the gRNA targets a gene and is used in combination with a Cas9 fusion protein wherein the second polypeptide domain has transcription activation activity, to activate or enhance expression of the gene to increase T cells. In some embodiments, the gRNA targets a gene and is used in combination with a Cas9 fusion protein wherein the second polypeptide domain has transcription repression activity, to inhibit or reduce or decrease expression of the gene to increase T cells. The gRNA may comprise a polynucleotide selected from at least one of SEQ ID NOs: 115-134, or a complement thereof, or a variant thereof, or a truncation thereof. The gRNA may be encoded by a polynucleotide sequence comprising at least one of SEQ ID NOs: 95-114, or a complement thereof, or a variant thereof, or a truncation thereof. The gRNA may bind and target a polynucleotide sequence comprising at least one of SEQ ID NOs: 95-114, or a complement thereof, or a variant thereof, or a truncation thereof. A truncation may be 1 , 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides shorter than the sequence of any one of SEQ ID NOs: 95-134. The gRNA may be used, for example, in a CRISPR / Cas-based gene editing system with SpCas9, such as a fusion protein comprising dSpCas9 with VP64 or p300. Exemplary gRNA sequences for modulating T ceils are shown in TABLE 3.[0(80137] As described above, the gRNA molecule comprises a targeting domain (also referred to as targeted or targeting sequence), which is a polynucleotide sequence complementary to the target DNA sequence. The gRNA may comprise a “G” at the 5’ end of the targeting domain or complementary polynucleotide sequence. The CRISPR / Cas9-basedgene editing system may use gRNAs of varying sequences and lengths. The targeting domain of a gRNA molecule may comprise at least a 10 base pair, at least a 11 base pair, at least a 12 base pair, at least a 13 base pair, at least a 14 base pair, at least a 15 base pair, at least a 16 base pair, at least a 17 base pair, at least a 18 base pair, at least a 19 base pair, at least a 20 base pair, at least a 21 base pair, at least a 22 base pair, at least a 23 base pair, at least a 24 base pair, at least a 25 base pair, at least a 30 base pair, or at least a 35 base pair complementary polynucleotide sequence of the target DNA sequence followed by a PAM sequence, in certain embodiments, the targeting domain of a gRNA molecule has 19-25 nucleotides in length. In certain embodiments, the targeting domain of a gRNA molecule is 20 nucleotides in length. In certain embodiments, the targeting domain of a gRNA molecule is 21 nucleotides in length, in certain embodiments, the targeting domain of a gRNA molecule is 22 nucleotides in length. In certain embodiments, the targeting domain of a gRNA molecule is 23 nucleotides in length.[000138] The number of gRNA molecules that may be included in the CRISPR / Cas9- based gene editing system can be at least 1 gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs, at least 6 different gRNAs, at least 7 different gRNAs, at least 8 different gRNAs, at least 9 different gRNAs, at least 10 different gRNAs, at least 11 different gRNAs, at least 12 different gRNAs, at least 13 different gRNAs, at least 14 different gRNAs, at least 15 different gRNAs, at least 16 different gRNAs, at least 17 different gRNAs, at least 18 different gRNAs, at least 18 different gRNAs, at least 20 different gRNAs, at least 25 different gRNAs, at least 30 different gRNAs, at least 35 different gRNAs, at least 40 different gRNAs, at least 45 different gRNAs, or at least 50 different gRNAs. The number of gRNA molecules that may be included in the CRISPR / Cas9-based gene editing system can be less than 50 different gRNAs, less than 45 different gRNAs, less than 40 different gRNAs, less than 35 different gRNAs, less than 30 different gRNAs, less than 25 different gRNAs, less than 20 different gRNAs, less than 19 different gRNAs, less than 18 different gRNAs, less than 17 different gRNAs, less than 16 different gRNAs, less than 15 different gRNAs, less than 14 different gRNAs, less than 13 different gRNAs, less than 12 different gRNAs, less than 11 different gRNAs, less than 10 different gRNAs, less than 9 different gRNAs, less than 8 different gRNAs, less than 7 different gRNAs, less than 6 different gRNAs, less than 5 different gRNAs, less than 4 different gRNAs, less than 3 different gRNAs, or less than 2 different gRNAs. The number of gRNAs that may be included in the CRISPR / Cas9-based gene editing system can be between at least 1 gRNA to at least 50 different gRNAs, at least 1 gRNA to af least 45 different gRNAs, at least 1 gRNA to at least 40 different gRNAs, at least 1 gRNA to at least 35 different gRNAs, at least 1 gRNA to at least 30 different gRNAs, atleast 1 gRNA to at least 25 different gRNAs, at least 1 gRNA to at least 20 different gRNAs, at least 1 gRNA to at least 16 different gRNAs, at least 1 gRNA to at least 12 different gRNAs, at least 1 gRNA to at least 8 different gRNAs, at least 1 gRNA to at least 4 different gRNAs, at least 4 gRNAs to at least 50 different gRNAs, at least 4 different gRNAs to at least 45 different gRNAs, at least 4 different gRNAs to at least 40 different gRNAs, at least 4 different gRNAs to at least 35 different gRNAs, at least 4 different gRNAs to at least 30 different gRNAs, at least 4 different gRNAs to at least 25 different gRNAs, at least 4 different gRNAs to at least 20 different gRNAs, at least 4 different gRNAs to at least 16 different gRNAs, at least 4 different gRNAs to at least 12 different gRNAs, at least 4 different gRNAs to at least 8 different gRNAs, at least 8 different gRNAs to at least 50 different gRNAs, at least 8 different gRNAs to at least 45 different gRNAs, at least 8 different gRNAs to at least 40 different gRNAs, at least 8 different gRNAs to at least 35 different gRNAs, 8 different gRNAs to at least 30 different gRNAs, at least 8 different gRNAs to at least 25 different gRNAs, 8 different gRNAs to at least 20 different gRNAs, at least 8 different gRNAs to at least 16 different gRNAs, or 8 different gRNAs to at least 12 different gRNAs. vi) Repair Pathways[000139] The CRISPR / Cas9-based gene editing system may be used to introduce sitespecific double strand breaks at targeted genomic loci, such as a gene for modulating T cells as detailed herein. Site-specific double-strand breaks are created when the CRISPR / Cas9- based gene editing system binds to a target DNA sequences, thereby permitting cleavage of the target DNA. This DNA cleavage may stimulate the natural DNA-repair machinery, leading to one of two possible repair pathways: homology-directed repair (HDR) or the non- homoiogous end joining (NHEJ) pathway.(1) Homology-Directed Repair (HDR)[000140] Restoration of protein expression from a gene may involve homology-directed repair (HDR). A donor template may be administered to a cell. A donor sequence comprises a polynucleotide sequence to be inserted into a genome. The donor template may include a nucleotide sequence encoding a full-functional protein or a partially functional protein. In such embodiments, the donor template may include fully functional gene construct for restoring a mutant gene, or a fragment of the gene that after homology-directed repair, leads to restoration of the mutant gene. In other embodiments, the donor template may include a nucleotide sequence encoding a mutated version of an inhibitory regulatory element of a gene. Mutations may include, for example, nucleotide substitutions, insertions, deletions, or a combination thereof. In such embodiments, introduced mutation(s) into theinhibitory regulatory element of the gene may reduce the transcription of or binding to the inhibitory regulatory element.(2) Non-Homoiogous End Joining (NHEJ)[000141] Restoration of protein expression from gene may be through template-free NHEJ- mediated DNA repair. In certain embodiments, NHEJ is a nuclease mediated NHEJ, which in certain embodiments, refers to NHEJ that is initiated a Cas9 molecule that cuts double stranded DNA. The method comprises administering a presently disclosed CRISPR / Cas9- based gene editing system or a composition comprising thereof to a subject for gene editing.[000142] Nuclease mediated NHEJ may correct a mutated target gene and offer several potential advantages over the HDR pathway. For example, NHEJ does not require a donor template, which may cause nonspecific insertional mutagenesis. In contrast to HDR, NHEJ operates efficiently in all stages of the cell cycle and therefore may be effectively exploited in both cycling and post-mitotic cells, such as muscle fibers. This provides a robust, permanent gene restoration alternative to oligonucleotide-based exon skipping or pharmacologic forced read-through of stop codons and could theoretically require as few as one drug treatment.3. Genetic Constructs[000143] The transcription factor, such as one selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, may be encoded by one or more genetic constructs. A polynucleotide encoding a transcription factor, such as one selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, may be comprised within one or more genetic constructs. The genetic construct may comprise an open reading frame (ORF) of the transcription factor. In some embodiments, the genetic construct comprises a sequence selected from SEQ ID NOs: 75-84 or encodes a polypeptide comprising a sequence selected from SEQ ID NOs: 85-94. The CRISPR / Cas9- based gene editing system may be encoded by or comprised within one or more genetic constructs. The CRISPR / Cas9-based gene editing system may comprise one or more genetic constructs. The genetic construct, such as a plasmid or expression vector, may comprise a nucleic acid that encodes the transcription factor or the CRISPR / Cas9-based gene editing system and / or at least one of the gRNAs. In certain embodiments, a genetic construct encodes one gRNA molecule, i.e., a first gRNA molecule, and optionally a Cas9 molecule or fusion protein. In some embodiments, a genetic construct encodes two gRNA molecules, i.e., a first gRNA molecule and a second gRNA molecule, and optionally a Cas9molecule or fusion protein. In some embodiments, a first genetic construct encodes one gRNA molecule, i.e., a first gRNA molecule, and optionally a Cas9 molecule or fusion protein, and a second genetic construct encodes one gRNA molecule, i.e., a second gRNA molecule, and optionally a Cas9 molecule or fusion protein. In some embodiments, a first genetic construct encodes one gRNA molecule and one donor sequence, and a second genetic construct encodes a Cas9 molecule or fusion protein. In some embodiments, a first genetic construct encodes one gRNA molecule and a Cas9 molecule or fusion protein, and a second genetic construct encodes one donor sequence.[000144] Genetic constructs may include polynucleotides such as vectors and plasmids. The genetic construct may be a linear minichromosome including centromere, telomeres, or plasmids or cosmids. The vector may be an expression vectors or system to produce protein by routine techniques and readily available starting materials including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated fully by reference. The construct may be recombinant. The genetic construct may be part of a genome of a recombinant viral vector, including recombinant ientivirus, recombinant adenovirus, and recombinant adenovirus associated virus. Viral vectors are further detailed below. The genetic construct may comprise regulatory elements for gene expression of the coding sequences of the nucleic acid. The regulatory elements may be a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal.[009145] The genetic construct may comprise heterologous nucleic acid encoding the transcription factor or the CRISPR / Cas-based gene editing system and may further comprise an initiation codon, which may be upstream of the transcription factor or the CRISPR / Cas- based gene editing system coding sequence, and a stop codon, which may be downstream of the transcription factor or the CRISPR / Cas-based gene editing system coding sequence. The genetic construct may include more than one stop codon, which may be downstream of the transcription factor or the CRISPR / Cas-based gene editing system coding sequence. In some embodiments, the genetic construct includes 1 , 2, 3, 4, or 5 stop codons. In some embodiments, the genetic construct includes 1 , 2, 3, 4, or 5 stop codons downstream of the sequence encoding the donor sequence. A stop codon may be in-frame with a coding sequence in the transcription factor or the CRISPR / Cas-based gene editing system. For example, one or more stop codons may be in-frame with the donor sequence. The genetic construct may include one or more stop codons that are out of frame of a coding sequence in the transcription factor or the CRISPR / Cas-based gene editing system. For example, one stop codon may be in-frame with the donor sequence, and two other stop codons may beincluded that are in the other two possible reading frames. A genetic construct may include a stop codon for all three potential reading frames. The initiation and termination codon may be in frame with the transcription factor or the CRISPR / Cas-based gene editing system coding sequence.[000146] The vector may also comprise a promoter that is operably linked to the transcription factor coding sequence or the CRISPR / Cas-based gene editing system coding sequence. In some embodiments, the vector comprises a promoter operably linked to a polynucleotide sequence encoding the transcription factor. The promoter may be a constitutive promoter, a ubiquitous promoter, an inducible promoter, a cell-specific promoter, a tissue-specific promoter, a repressible promoter, or a regulatable promoter. In some embodiments, the promoter is a ubiquitous promoter. The promoter may be non- endogenous to the transcription factor. The promoter may be non-native to the transcription factor. The promoter may be a cell-specific promoter. For example, the promoter may be a promoter specific for T cells. The promoter may be a tissue-specific promoter. The tissue specific promoter may be a muscle specific promoter. The tissue specific promoter may be a skin specific promoter. The transcription factor coding sequence or the CRISPR / Cas- based gene editing system may be under the light-inducible or chemically inducible control to enable the dynamic control of gene / genome editing in space and time. The promoter operably linked to the transcription factor coding sequence or the CRISPR / Cas-based gene editing system coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (Bi V) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. Examples of a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic, are described in U.S. Patent Application Publication No. US20040175727, the contents of which are incorporated herein in its entirety. The promoter may be a CK8 promoter, a Spc512 promoter, a MHCK7 promoter, for example.[0(80147] The genetic construct may also comprise a polyadenylation signal, which may be downstream of the transcription factor coding sequence or the CRISPR / Cas-based gene editing system. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenyiation signal, bovine growth hormone (bGH) polyadenylation signal, human growthhormone (hGH) polyadenyiation signal, or human p-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).[000148] Coding sequences in the genetic construct may be optimized for stability and high levels of expression. In some instances, codons are selected to reduce secondary structure formation of the RNA such as that formed due to intramolecular bonding.[000149] The genetic construct may also comprise an enhancer upstream of the transcription factor coding sequence or the CRISPR / Cas-based gene editing system or gRNAs. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV, or EBV. Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94 / 016737, the contents of each are fully incorporated by reference. The genetic construct may also comprise a mammalian origin of replication in order to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The genetic construct may also comprise a regulatory sequence, which may be well suited for gene expression in a mammalian or human cell into which the vector is administered. The genetic construct may also comprise a reporter gene, such as polynucleotide encoding a reporter protein and / or a selectable marker, such as hygromycin (“Hygro”). The reporter protein may include any protein or peptide that is suitably detectable, such as, by fluorescence, chemiluminescence, enzyme activity such as beta galactosidase or alkaline phosphatase, and / or antibody binding detection. The reporter protein may comprise a fluorescent protein. The reporter protein may comprise a protein or peptide detectable with an antibody. For example, the reporter protein may comprise green fluorescent protein (“GFP”), YFP, RFP, CFP, DsRed, luciferase, and / or Thy1.[000150] The genetic construct may be useful for transfecting cells with nucleic acid encoding the transcription factor coding sequence or the CRISPR / Cas-based gene editing system, which the transformed host cell is cultured and maintained under conditions wherein expression of the transcription factor coding sequence or the CRISPR / Cas-based gene editing system takes place. The genetic construct may be transformed or transduced into a cell. The genetic construct may be formulated into any suitable type of delivery vehicle including, for example, a viral vector, lentiviral expression, mRNA electroporation, and lipid- mediated transfection for delivery into a cell. The genetic construct may be part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. The genetic construct may be present in the cell as a functioning extrachromosomal molecule.[000151] Further provided herein is a ceil transformed or transduced with a system or component thereof as detailed herein. Suitable cell types are detailed herein. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell. The cell may be a CD8+ T cell. In some embodiments, the cell is a stem cell. The stem cell may be a human stem cell. In some embodiments, the cell is an embryonic stem cell. The stem cell may be a human pluripotent stem cell (IPSCs). Further provided are stem cell-derived neurons, such as neurons derived from iPSCs transformed or transduced with a DNA targeting system or component thereof as detailed herein. a. Viral Vectors[000152] A genetic construct may be a viral vector. Further provided herein is a viral delivery system. Viral delivery systems may include, for example, lentivirus, retrovirus, adenovirus, mRNA electroporation, or nanoparticles. In some embodiments, the vector is a lentiviral vector. Lentiviruses are a subclass of Retroviruses. Lentiviruses resemble y- retroviruses (y-RV) in their ability to stably integrate into the target cell genome, resulting in persistent expression of the gene of interest. Species of lentivirus include, for example, human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV). In some embodiments, the vector is a modified lentiviral vector. In some embodiments, the vector is an engineered lentiviral vector. Lentiviruses may include, for example, pseudo-type lentivirus, integrase-deficient lentivirus, and virus-like particles. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. The AAV vector is a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species.[000153] Viral vectors may be used to deliver the transcription factor coding sequence or the CRISPR / Cas9-based gene editing systems using various construct configurations. For example, AAV vectors may deliver Cas9 or fusion protein and gRNA expression cassettes on separate vectors or on the same vector. Alternatively, if the small Cas9 proteins or fusion proteins, derived from species such as Staphylococcus aureus or Neisseria meningitidis, are used then both the Cas9 and up to two gRNA expression cassettes may be combined in a single AAV vector. In some embodiments, the AAV vector has a 4.7 kb packaging limit.[000154] In some embodiments, the AAV vector is a modified AAV vector. In some embodiments, the AAV vector is an engineered AAV vector. The AAV vector may include an engineered AAV capsid. The AAV vector may be an engineered AAV vector for a specific cell type. For example, the AAV vector may be an engineered AAV vector for T cells. Themodified AAV vector may have enhanced cell type tropism. The modified AAV vector may have enhanced cardiac and / or skeletal muscle tissue tropism. The modified AAV vector may be capable of delivering and expressing the transcription factor coding sequence or the CRISPR / Cas9-based gene editing system in the cell of a mammal. For example, the modified AAV vector may be an AAV-SASTG vector (Piacentino et al. Human Gene Therapy 2012, 23, 635-646, incorporated herein by reference). The modified AAV vector may be based on one or more of several capsid types, including AAV1 , AAV2, AAV5, AAV6, AAV8, and AAV9. The modified AAV vector may be based on AAV2 pseudotype with alternative muscle-tropic AAV capsids, such as AAV2 / 1 , AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, and AAV / SASTG vectors that efficiently transduce skeletal muscle or cardiac muscle by systemic and local delivery (Seto et al. Current Gene Therapy 2012, 12, 139-151 , incorporated herein by reference). The modified AAV vector may be AAV2I8G9 (Shen et ai. J. Biol. Chem. 2013, 288, 28814-28823, incorporated herein by reference).4, Additional Therapies[000155] The compositions and methods detailed herein may further include at least one additional therapy, such as at least one cancer therapy or at least one antiviral therapy, or a combination thereof. As used herein, the term “standard of care treatment” or “additional therapy” or “additional treatment” are used interchangeably and refer to any other standard treatments / additional treatments that do not include the specific compositions detailed herein for modifying a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV. Additional therapies may comprise a small molecule, peptide, polypeptide, antibody, nucleotide, polynucleotide, lipid, or carbohydrate, or a combination thereof. Additional therapies may be synthesized and / or extracted and / or purified by any suitable means known in the art. Additional therapies may be commercially available. An effective amount of the additional therapy may be administered. a. Cancer Therapies[000156] The compositions and methods detailed herein may further include at least one cancer therapy. The term “standard of care treatment” or “additional therapy” or “additional treatment” are used interchangeably and refer to any other standard cancer treatments / additional cancer treatments that do not include the specific compositions detailed herein for modifying a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV. Additional cancer therapies may comprise a small molecule, peptide, polypeptide, antibody, nucleotide, polynucleotide, lipid, or carbohydrate, or a combination thereof. Additional cancer therapies may be synthesized and / or extractedand / or purified by any suitable means known in the art. Additional cancer therapies may be commercially available. Additional cancer therapies may include, for example, chemotherapy, immunotherapy, radiation therapy, hormone therapy, targeted drug therapy, cryoablation, antibody drug conjugates, and surgery, or a combination thereof. Hormone therapy, for example, may block hormone synthesis such as blocking estrogen synthesis. An effective amount of the additional cancer therapy may be administered.[000157] Chemotherapy may include, for example, an antimitotic agent, an alkylating agent, an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, a cytotoxic agent, a cell cycle inhibitor, a growth factor inhibitor, a histone deacetylase (HDAC) inhibitor, and an inhibitor of a pathway that cross-talks with and activates ER transcriptional activity, or a combination thereof,[000158] Alkylating agents may include, for example, cisplatin (PLATINOL®), oxaliplatin (ELOXATIN®), chlorambucil (LEUKERAN®), procarbazine (MATULANE®; NATULAN®), or carmustine (BiCNU®), or a combination thereof. Antimetabolites may include, for example, methotrexate (also known as amethopterin), 5-fluorouracil, cytarabine (also known as cytosine arabinoside or ara-C; CYTOSAR®), or gemcitabine (GEMZAR®), or a combination thereof. Antimicrotubule agents may include, for example, vinblastine (VELBAN®;VELBE®), or paclitaxel (TAXOL®), or a combination thereof. Topoisomerase inhibitors may include, for example, etoposide (VEPESID®), or doxorubicin (ADRIAMYCIN®; MYOCET®), or a combination thereof. Cytotoxic agents may include, for example, bleomycin (BLENOXANE®). Growth factor inhibitors may include, for example, human epidermal growth factor receptor 2 (HER2) inhibitors. HER2 inhibitors include, for example, trastuzumab (HERCEPTIN®), deruxtecan, sacitizumab, and / or ado-trastuzumab emtansine (KADCYLA®). HDAC inhibitors may include, for example, vorinostat (ZOLINZA®), romidepsin (ISTODAX®), chidamide (also known as tucidinostat; EPIDAZA®; HIYASTA™), panobinostat (FARYDAK®), belinostat (also known as BELEODAQ® or PXD101), valproic acid (DEPAKOTE®; DEPAKENE®; STAVZOR®)), mocetinostat (also known as MGCD0103), abexinostat (also known as PCI-24781), entinostat (also known as SNDX-275 or MS-275), pracinostat (also known as SB939), resminostat (also known as 4SC-201 or RAS2410), givinostat (also known as gavinostat or ITF2357), quisinostat (also known as JNJ-26481585), kevetrin, CUDC-101 , AR-42, tefinostat (also known as CHR-2845), nanatinostat (also known as CHR-3996), domatinostat (also known as 4SC-202), ivaltinostat (also known as CG-200745), rocilinostat (also known as ACY-1215), or sulforaphane, or a combination thereof. Inhibitors of a pathway that cross-talks with and activates ER transcriptional activity may include, for example, a phosphoinositide 3-kinase (PI3K)inhibitor, a heat shock protein 90 (HSP90) inhibitor, or a mammalian target of rapamycin (mTOR) inhibitor. mTOR inhibitors include, for example, everolimus (AFINITOR®; VOTUBIA®; ZORTRESS®). In some embodiments, the HDAC inhibitor comprises vorinostat (ZOLINZA®) and / or romidepsin (ISTODAX®).[000159] Immunotherapies may include, for example, a checkpoint inhibitor, or denosumab (PROLIA®; XGEVA®), or a combination thereof. “Checkpoint inhibitor” or “immune checkpoint inhibitor” may also be referred to as an immune checkpoint blockade (ICB) therapy. Checkpoint inhibitors may comprise an antibody. Checkpoint inhibitors may include, for example, an antibody to programmed ceil death protein 1 (PD1) (anti-PD1), or an antibody to cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (anti-CTLA4), or an antibody to programmed death-ligand 1 (PDL1) (anti-PDL1), or DMXAA (sting agonist; also known as ASA404, vadimezan, or dimethylxanthone acetic acid) or a combination thereof. “Anti-PD1” refers to an antibody that binds PD1 , “anti-CTLA4” refers to an antibody that binds CTLA4, and “anti-PDL1 ” refers to an antibody that binds PDL1 . In some embodiments, the PD-1 antibody comprises pembrolizumab (KEYTRUDA®) or nivolumab (OPDIVOo®). In some embodiments, the CTLA-4 antibody comprises ipilimumab (YERVOY®).[000160] Antibody drug conjugates may include, for example, gemtuzumab ozogamicin (MYLOTARG™), brentuximab vedotin (ADCETRIS®), ado-trastuzumab erntansine (KADCYLA®), inotuzumab ozogamicin (BESPONSA®), polatuzumab vedotin (POLIVY®), enfortumab vedotin (PADCEV®), fam-trastuzumab deruxtecan (ENHERTU®), sacituzumab govitecan (TRODELVY®), loncastuximab tesirine (ZYNLONTA®), tisotumab vedotin (TIVDAK®), mirvetuximab soravtansinegynx (ELAHERE™), moxetumomab pasudotox (LUMOXITI™), belantamab mafodotin-blmf (BLENREP®), cetuximab saratolacan (AKALUX®), or disitamab vedotin (AIDIXI®), or a combination thereof. b. Antiviral Therapies[000161] The compositions and methods detailed herein may further include at least one antiviral therapy. The term “standard of care treatment” or “additional therapy” or “additional treatment” are used interchangeably and refer to any other standard antiviral treatments / additional antiviral treatments that do not include the specific compositions detailed herein for modifying a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV. Additional antiviral therapies may comprise a small molecule, peptide, polypeptide, antibody, nucleotide, polynucleotide, lipid, or carbohydrate, or a combination thereof. Additional antiviral therapies may be synthesized and / or extractedand / or purified by any suitable means known in the art. Additional antiviral therapies may be commercially available. An effective amount of the additional antiviral therapy may be administered. Antiviral therapies and viruses targeted are described in, for example, US 20200165594, and De Clercq et al. (Clin. Microbiol. Rev. 2016, 29, 695-747), each of which is incorporated herein by reference. Antiviral therapies may include, for example, the antiviral therapies listed in TABLE 4 below.[000162] The antivirals described herein may target one or more viruses. The virus may be a DNA virus (single or double stranded, positive or negative sense, or ambisense) or an RNA virus (single or double stranded, positive or negative sense, or ambisense). In some embodiments, the virus may be Ebola, measles, SARS, Chikungunya, hepatitis, Marburg, yellow fever, MERS, Dengue, Lassa, influenza, rhabdovirus, COVID-19, or HIV. A hepatitis virus may include hepatitis A, hepatitis B, or hepatitis C. An influenza virus may include, for example, influenza A or influenza B. An HIV may include HIV 1 or HIV 2. In some embodiments, the virus may be a human respiratory syncytial virus, Sudan Ebola virus, Bundibugye virus, Tai Forest Ebola virus, Reston Ebola virus, Achimota, Aedes flavivirus, Aguacate virus, Akabane virus, Alethinophid reptarenavirus, Allpahuayo mammarenavirus, Amapari mmarenavirus, Andes virus, Apoi virus, Aravan virus, Aroa virus, Arumwot virus, Atlantic salmon paramyoxivirus, Australian bat lyssavirus, Avian bornavirus, Avian metapneumovirus, Avian paramyoxviruses, penguin or Falkland Islandsvirus, BK polyomavirus, Bagaza virus, Banna virus, Bat hepevirus, Bat sapovirus, Bear Canon mammarenavirus, Beilong virus, Betacoronoavirus, Betapapillomavirus 1-6, Bhanja virus, Bokeloh bat lyssavirus, Borna disease virus, Bourbon virus, Bovine hepacivirus, Bovine parainfluenza virus 3, Bovine respiratory syncytial virus, Brazoran virus, Bunyamwere virus, Caliciviridae virus. California encephalitis virus, Candiru virus, Canine distemper virus, Canaine pneumovirus, Cedar virus, Cell fusing agent virus, Cetacean morbillivirus, Chandipura virus, Chaoyang virus, Chapare mammarenavirus, Chikungunya virus, CoIobus monkey papillomavirus, Colorado tick fever virus, Cowpox virus, Crimean-Congo hemorrhagic fever virus, Culex flavivirus, Cupixi mammarenavirus, Dengue virus, Dobrava- Belgrade virus, Donggang virus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Entebbe bat virus, Enterovirus A-D, European bat lyssavirus 1-2, Eyach virus, Feline morbillivirus, Fer-de-Lance paramyxovirus, Fitzroy River virus, Flaviviridae virus, Flexal mammarenavirus, GB virus C, Gairo virus, Gemycircularvirus, Goose paramyoxiviurs SFQ2, Great Island virus, Guanarito mammarenavirus, Hantaan virus, Hantavirus Z10, Heartland virus, Hendra virus, Hepatitis A / B / C / E, Hepatitis delta virus, Human bocavirus, Human coronavirus, Human endogenous retrovirus K, Human enteric coronavirus, Human gential- associated circular DNA virus-1 , Human herpesvirus 1-8, Human immunodeficiency virus1 / 2, Huan mastadenovirus A-G, Human papillomavirus, Human parainfluenza virus 1-4, Human paraechovirus, Human picobirnavirus, Human smacovirus, ikoma lyssavirus, llheus virus, Influenza A-C, Ippy mammarenavirus, Irkut virus, J-virus, JC polyomavirus, Japanses encephalitis virus, Junin mammarenavirus, KI polyomavirus, Kadipiro virus, Kamiti River virus, Kedougou virus, Khujand virus, Kokobera virus, Kyasanur forest disease virus, Lagos bat virus, Langat virus, Lassa mammarenavirus, Latino mammarenavirus, Leopards Hill virus, Liao ning virus, Ljungan virus, Lloviu virus, Looping ill virus, Lujo mammarenavirus, Luna mammarenavirus, Lunk virus, Lymphocytic choriomeningitis mammarenavirus, Lyssavirus Ozernoe, MSSI2\.225 virus, Machupo mammarenavirus, Mamastrovirus 1 , Manzanilla virus, Mapuera virus, Marburg virus, Mayaro virus, Measles virus, Menangle virus, Mercadeo virus, Merkel cell polyomavirus, Middle East respiratory syndrome coronavirus, Mobala mammarenavirus, Modoc virus, Moijang virus, Mokolo virus, Monkeypox virus, Montana myotis leukoenchalitis virus, Mopeia lassa virus reassortant 29, Mopeia mammarenavirus, Morogoro virus, Mossman virus, Mumps virus, Murine pneumonia virus, Murray Valley encephalitis virus, Nariva virus, Newcastle disease virus, Nipah virus, Norwalk virus, Norway rat hepacivirus, Ntaya virus, O'nyong-nyong virus, Oliveros mammarenavirus, Omsk hemorrhagic fever virus, Oropouche virus, Parainfluenza virus 5, Parana mammarenavirus, Parramatta River virus, Peste-des-petits-ruminants virus, Pichande mammarenavirus, Picornaviridae virus, Pirital mammarenavirus, Piscihepevirus A, Procine parainfluenza virus 1 , porcine rubulavirus, Powassan virus, Primate T-lymphotropic virus 1-2, Primate erythroparvovirus 1 , Punta Toro virus, Puumala virus, Quang Binh virus, Rabies virus, Razdan virus, Reptile bornavirus 1 , Rhinovirus A-B, Rift Valley fever virus, Rinderpest virus, Rio Bravo virus, Rodent Torque Teno virus, Rodent hepacivirus, Ross River virus, Rotavirus A-l, Royal Farm virus, Rubella virus, Sabla mammarenavirus, Salem virus, Sandfly fever Naples virus, Sandfly fever Sicilian virus, Sapporo virus, Sathuperi virus, Seal anellovirus, Semliki Forest virus, Sendai virus, Seoul virus, Sepik virus, Severe acute respiratory syndrome-related coronavirus, Severe fever with thrombocytopenia syndrome virus, Shamonda virus, Shimon! bat virus, Shuni virus, Simbu virus, Simian torque teno virus, Simian virus 40-41 , Sin Nombre virus, Sindbis virus, Small anellovirus, Sosuga virus, Spanish goat encephalitis virus, Spondweni virus, St. Louis encephalitis virus, Sunshine virus, TTV-like mini virus, Tacaribe mammarenavirus, Taila virus, Tamana bat virus, Tamiami mammarenavirus, Tembusu virus, Thogoto virus, Thottapalayam virus, Tick-borne encephalitis virus, Tioman virus, Togaviridae virus, Torque teno canis virus, Torque teno douroucouli virus, Torque teno fells virus, Torque teno midi virus, Torque teno sus virus, Torque teno tamarin virus, Torque teno virus, Torque teno zalophus virus, Tuhoko virus, Tula virus, Tupaia paramyxovirus, Usutu virus, Uukuniemi virus, Vaccinia virus, Variola virus, Venezuelan equine encephalitis virus, Vesicular stomatitis Indiana virus, WU Polyomavirus,Wesselsbron virus, West Caucasian bat virus, West Nile virus, Western equine encephalitis virus, Whitewater Arroyo mammarenavirus, Yellow fever virus, Yokose virus, Yug Bogdanovac virus, Zaire ebolavirus, Zika virus, or Zygosaccharomyces bailii virus Z viral sequence. Examples of RNA viruses include one or more of (or any combination of) Coronaviridae virus, a Picomaviridae virus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus, a Bomaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, or a Deltavirus. In some embodiments, the virus is Coronavirus, SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zika virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Human respiratory syncytial virus, Rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, Influenza, or Hepatitis D virus.5, Pharmaceutical Compositions[000163] Further provided herein are pharmaceutical compositions comprising the abovedescribed modulator of T ceils or genetic constructs or gene editing systems, in some embodiments, the composition further includes at least one cancer therapy such as a chimeric antigen receptor (CAR). In some embodiments, the pharmaceutical composition may comprise about 1 ng to about 10 mg of DNA encoding the transcription factor or the CRISPR / Cas-based gene editing system. The systems or genetic constructs as detailed herein, or at least one component thereof, may be formulated into pharmaceutical compositions in accordance with standard techniques well known to those skilled in the pharmaceutical art. The pharmaceutical compositions can be formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free, and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation.[000164] The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules as vehicles, adjuvants, carriers, or diluents. The term “pharmaceutically acceptable carrier,” may be a non-toxlc, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Pharmaceutically acceptable carriers include, for example, diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives,glidants, solvents, suspending agents, wetting agents, surfactants, emollients, propellants, humectants, powders, pH adjusting agents, and combinations thereof. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent may be a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The transfection facilitating agent may be poly-L- glutamate, and more preferably, the poly-L-glutamate may be present in the composition for gene editing in skeletal muscle or cardiac muscle at a concentration less than 6 mg / mL.6, Administration[000165] The systems or genetic constructs as detailed herein, or at least one component thereof, may be administered or delivered to a cell. Methods of introducing a nucleic acid into a host cell are known in the art, and any known method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, polycation or lipldmuclelc acid conjugates, lipofection, electroporation, nucleofection, immunoliposomes, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle- mediated nucleic acid delivery, and the like. In some embodiments, the composition may be delivered by mRNA delivery and ribonucleoprotein (RNP) complex delivery. The system, genetic construct, or composition comprising the same, may be electroporated using BioRad Gene Pulser Xcell or Amaxa Nucleofector lib devices or other electroporation device.Several different buffers may be used, including BioRad electroporation solution, Sigma phosphate-buffered saline product #08537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (N.V.). Transfections may include a transfection reagent, such as Lipofectamine 2000. The activators detailed herein may be delivered or administered, for example, to a cell ex vivo or to a subject in vivo, by a method including viral delivery (such as, for example, lentivlrus or AAV vectors as detailed above), non-viral integrating methods (such as, for example, transposons, integrases, and gene editing), and non-viral transient methods (such as, for example, mRNA, plasmids, and minicircles as detailed above). The activators detailed herein may be delivered by any delivery method suitable for ex vivo engineered cell therapy. The activators detailed herein may be delivered by any suitabledelivery method, including, for example, lipid nanoparticles and micelles. In some embodiments, the activator or a polynucleotide encoding the activator is encapsulated within a lipid nanoparticle or polymeric carrier.[000166] The systems or genetic constructs as detailed herein, or at least one component thereof, or the pharmaceutical compositions comprising the same, may be administered to a subject or a cell. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The presently disclosed systems, or at least one component thereof, genetic constructs, or compositions comprising the same, may be administered to a subject by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, intranasal, intravaginal, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intradermally, epidermally, intramuscular, intranasal, intrathecal, intracranial, and intraarticular or combinations thereof. In certain embodiments, the system, genetic construct, or composition comprising the same, is administered to a subject intramuscularly, intravenously, or a combination thereof. The systems, genetic constructs, or compositions comprising the same may be delivered to a subject by several technologies including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, nanoparticle facilitated, recombinant vectors such as recombinant lentivirus, recombinant adenovirus, and recombinant adenovirus associated virus. The composition may be injected into the brain or other component of the central nervous system. The composition may be injected into the skeletal muscle or cardiac muscle. For example, the composition may be injected into the tibialis anterior muscle or tail. For veterinary use, the systems, genetic constructs, or compositions comprising the same may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian may readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The systems, genetic constructs, or compositions comprising the same may be administered by traditional syringes, needleless injection devices, “microprojectile bombardment gone guns,” or other physical methods such as electroporation (“EP”), “hydrodynamic method”, or ultrasound. Alternatively, transient in vivo delivery of CRISPR / Cas-based systems by non-viral or non-integrating viral gene transfer, or by direct delivery of purified proteins and gRNAs containing cell-penetrating motifs may enable highly specific correction and / or restoration in situ with minimal or no risk of exogenous DNA integration.[000167] Upon delivery of the presently disclosed modulator or T cells, a variety of effects may be elicited, such as, for example, T cells may be increased, T cell numbers may be increased, memory T cells may be increased, T cell exhaustion may be inhibited or prevented, T cell exhaustion may be reversed, cancer therapy may be enhanced or its effectiveness increased, or a combination thereof. Upon delivery of the presently disclosed systems or genetic constructs as detailed herein, or at least one component thereof, or the pharmaceutical compositions comprising the same, and thereupon the vector into the cells of the subject, the transfected cells may express the gRNA molecule(s) and the Cas9 molecule or fusion protein. a. Cell Types[000168] Any of the delivery methods and / or routes of administration detailed herein can be utilized with a myriad of ceil types. Further provided herein is a cell transformed or transduced with a system or component thereof as detailed herein. For example, provided herein is a cell comprising an isolated polynucleotide encoding the protein product from a gene as detailed herein. For example, provided herein is a cell comprising an isolated polynucleotide encoding a transcription factor or a CRISPR / Cas9 system as detailed herein. Suitable cell types are detailed herein. The cell may be isolated, as from a subject or tissue, or ex vivo, or in vivo. In some embodiments, the cell is an immune cell. Immune cells may include, for example, lymphocytes such as T cells and B cells and natural killer (NK) cells. In some embodiments, the cell is a T cell. T cells may be divided into cytotoxic T cells and helper T cells, which are in turn categorized as TH1 or TH2 helper T cells. Immune cells may further include innate immune cells, adaptive immune cells, tumor-primed T cells, NKT cells, IFN-y producing killer dendritic cells (IKDC), memory ! cells (TCMs), and effector ! cells (!Es). The cell may be a stem cell such as a human stem cell. In some embodiments, the cell is an embryonic stem cell or a hematopoietic stem cell. !he stem cell may be a human induced pluripotent stem cell (IPSCs). Further provided are stem cell-derived neurons, such as neurons derived from IPSCs transformed or transduced with a DNA targeting system or component thereof as detailed herein. !he cell may be a muscle cell. Cells may further include, but are not limited to, immortalized myoblast cells, dermal fibroblasts, bone marrow-derived progenitors, skeletal muscle progenitors, human skeletal myoblasts, CD 133+ cells, mesoangioblasts, cardiornyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, and MyoD- or Pax7-transduced cells, or other myogenic progenitor cells. In some embodiments, the cell is a ! cell. In some embodiments, the cell is a CD8+ ! cell. In some embodiments, the cell is a CD4+ ! cell.7. Kits[000169] Provided herein is a kit, which may be used to modulate, such as increase, T cells. The kit may be used in conjunction with ACT to enhance the ACT. The kit comprises an activator as detailed herein, or genetic constructs or a composition comprising the same, as described above, and instructions for using said composition. The kit includes an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof. In some embodiments, the kit comprises at least one polynucleotide sequence selected from SEQ ID NOs: 75-84 or 95- 134, a complement thereof, a variant thereof, or fragment thereof. In some embodiments, the kit comprises at least one polypeptide sequence selected from SEQ ID NOs: 85-94, a variant thereof, or fragment thereof. In some embodiments, the kit comprises at least one gRNA comprising a polynucleotide sequence selected from SEQ ID NOs: 115-134, a complement thereof, a variant thereof, or fragment thereof, or at least one gRNA targeting or encoded by a polynucleotide comprising a sequence selected from SEQ ID NOs: 95-114, a complement thereof, a variant thereof, or fragment thereof. The kit may further include instructions for using the activator or the CRISPR / Cas-based gene editing system.[000170] Instructions included in kits may be affixed to packaging material or may be included as a package insert. While the instructions are typically written on printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” may include the address of an internet site that provides the instructions.[000171] The genetic constructs or a composition comprising thereof for modulating T cells may include a modified AAV vector that includes a gRNA molecule(s) and a Cas9 protein or fusion protein, as described above, that specifically binds a region of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a regulatory element thereof.8. Methods a. Methods of Modulating T Cehs[000172] Provided herein are methods of modulating T cells. The methods may include administering to a ceil or a subject a composition as detailed herein, or an isolated polynucleotide sequence as detailed herein, or a vector as detailed herein, or a cell asdetailed herein, or a pharmaceutical composition as detailed herein, or a combination thereof, in some embodiments, modulating T cells comprises increasing T cells, or increasing memory T cells, or preventing T cell exhaustions, or reversing T cell exhaustions, or a combination thereof. In some embodiments, the methods decrease expression of TOX in a T cell. In some embodiments, the methods increase expression of IL7RA in a T cell, or decrease expression of CCR7 in a T cell, or a combination thereof. b. Methods of Increasing T Cells[000173] Provided herein are methods of increasing T ceils. The methods may include administering to a cell or a subject a composition as detailed herein, or an isolated polynucleotide sequence as detailed herein, or a vector as detailed herein, or a cell as detailed herein, or a pharmaceutical composition as detailed herein, or a combination thereof. In some embodiments, the methods decrease expression of TOX in a T cell. In some embodiments, the methods increase expression of IL7RA in a T cell, or decrease expression of CCR7 in a T cell, or a combination thereof. c. Methods of Enhancing Adoptive T Cell Therapy (ACT)[000174] Provided herein are methods of enhancing adoptive T ceil therapy (ACT) In a subject. The methods may Include administering to the subject a composition as detailed herein, or an isolated polynucleotide sequence as detailed herein, or a vector as detailed herein, or a cell as detailed herein, or a pharmaceutical composition as detailed herein, or a combination thereof. In some embodiments, the methods decrease expression of TOX in a T cell. In some embodiments, the methods increase expression of IL7RA in a T cell, or decrease expression of CCR7 in a T cell, or a combination thereof. d. Methods of Treating Cancer[000175] Provided herein are methods of treating cancer in a subject. The methods may include administering to the subject a composition as detailed herein, or an isolated polynucleotide sequence as detailed herein, or a vector as detailed herein, or a cell as detailed herein, or a pharmaceutical composition as detailed herein, or a combination thereof. In some embodiments, the methods decrease expression of TOX in a T cell, in some embodiments, the methods increase expression of IL7RA in a T ceil, or decrease expression of CCR7 in a T cell, or a combination thereof.9. Examples[000176] The foregoing may be better understood by reference to the foilowing examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention. The present disclosure has multiple aspects and embodiments, illustrated by the appended non-limiting examples.Example 1Materials and Methods[000177] to vitro CDS* T cell exhaustion mode. To recapitulate T cell exhaustion in vitro, we thawed CD8+ T cells from three different human donors, splitting each donor into two conditions: acute and chronic stimulation. For acute stimulation, CD8* T cells were activated once with a 3:1 anti CD3 / CD28 bead to T cell ratio and expanded for 8 days. For chronic stimulation, T cells were restimulated every two days after initial activation for a total of 8 days (three additional stimulations). On day 8, cells from each condition were split in half, where half of the cells were used to isolate RNA and the other half for isolating nuclei for ATAC-seq. For RNA-seq, isolated total RNA was shipped to Genewiz (Azenta Life Sciences, Burlington, MA) for RNA-seq library preparation and sequencing. For ATAC-seq, nuclei were transposed using Illumina’s (San Diego, CA) Tn5, and the Omni-ATAC protocol from Corces. M., et al. (Nature Methods 2017, 14, 959-~962, incorporated herein by reference) was followed. The ATAC-seq libraries were then sequenced using 50M paired- end reads / sample.[000178] Computational analysis of RNA and ATAC-seq data. RNA-seq data was analyzed with DeSeq2 for differential gene enrichment analysis. Differential genes were then entered into EnrichR for biological pathway analysis and GSEA for individual dataset enrichment analysis. Differentially accessible chromatin regions were analyzed using DeSeq2, and these regions were annotated for nearest gene using ChipSeeker. Cumulative ATAC-signal was analyzed by comparing a set of CD8+ T cell dysfunctional peaks from Pritykin Y, et. al. (Molecular Cell, 2021 , 81, 2477-2493, incorporated herein by reference) to cumulative peak signal from bigwig files generated from the ATAC-seq pipeline and plotted using the plotHeatmap function in R.[000179] Chronic stimulation CRISPRa TF-ome screen & TOX tiling CRISPRi screens. CD8+ T cells from three human donors were thawed and initially stimulated with anti- CD3 / CD28 dynabeads. Cells were transduced 24 hours later with lentivirus libraries corresponding to either the CRISPRa or CRISPRi library, and cells were restimulated for anadditional three times every two days over the course of 6 days. Cells were then harvested on day 10, stained for TOX expression using Miltenyi Biotec (Germany) TOX antibody clone REA473, and sorted for top and bottom 10% TOX expressing cells. Genomic DNA was isolated from these sorted cells, gRNAs were amplified with Illumina (San Diego, CA) adapter sequences, and the libraries were sequenced with Illumina (San Diego, CA) Miseq instrument.[090180] TF-orne ORF chronic versus acute stimulation screens. CD8+ T ceils from three human donors were thawed, stimulated with anti-CD3 / CD28 dynabeads, and split into two conditions: acute versus chronic stimulation. Ceils were transduced 24 hours later with lentivirus libraries for each condition. After 3 days puromycin selection was used to select for transduced cells at 1 ug / mL. Acute conditions were expanded for an additional 6 days and harvested on day 9 post-transduction, Celis in chronic conditions were restimulated 2 and 4 days with anti-CD3 / CD28 dynabeads after puromycin selection and harvested on day 10 post-transduction. Cells were then sorted for top and bottom 10% TOX expressing cells with the aforementioned antibody. Genomic DNA was isolated from these sorted cells. The barcode sequences corresponding to each TF were amplified with Illumina adapter sequences, and the libraries were sequenced with Illumina (San Diego, CA) Miseq instrument.[000181] Screen computational analysis. All screens were aligned to respective custom bowtie indexes and analyzed for differential guide / ORF enrichment using DeSeq2.[000182] Individual TF ORF validation. For individual TF validations, the same protocol as the TF ORF chronic versus acute screens was followed, except cells were individually transduced with TF lentivirus and with a final harvest where conditions were split in half for RNA isolation and FACS staining. For FACS staining of TOX, Miltenyi Biotec (Germany) TOX antibody clone REA473 was used. For FACS exhaustion / memory marker staining, the following antibodies were used: anti-human TIGIT clone A15153G from Biolegend (San Diego, CA), anti-human LAG-3 clone 3DS223H from Invitrogen (Waltham, MA), anti-human PD-1 clone A17188B from Biolegend (San Diego, CA), anti-human IL7R clone HIL-7R-M21 from BD Horizon (Franklin Lakes, NJ), and anti-human CD8 clone SK1 from Biolegend (San Diego, CA). For TOX qPCR, total RNA was isolated, reverse transcription was conducted for complementary DNA generation, and qPCR was conducted using primers that flanked exon regions of the TOX locus to quantify relative TOX transcript expression.[000183] In vitro tumor killing assay. Two separate tumor killing assays were conducted with either peripheral blood CD8+ T cells or tumor-infiltrating lymphocytes from non-smallcell lung cancer patients from three different donors, respectively (six donors total: three from healthy PBMC patients, and three from lung cancer patients). For the peripheral blood T cells, CD8s were isolated using a StemCell Technologies (Vancouver, Canada) CD8+ negative isolation kit (catalog # 344710). The CD8+ T cells were activated with anti- CD3 / CD28 dynabeads, and individual viruses with the construct described in FIG. 12A were added to transduce the T cells 24 hours later. Three days after transduction, cells were restimulated with anti-CD3 / CD28 dynabeads. On day 10 post transduction, cells were stained with a CD8 antibody (anti-human CD8 clone SKI from Biolegend, San Diego, CA) and an anti-mouse Myc antibody (Cell Signaling Technologies, Danvers, MA, catalog #3739) to sort for CD8+ CAR+ T cells. Cells were then added at an effector to target cell ratio of 1 :4 and imaged using the Incucyte S5 live / imaging system. For tumor-infiltrating lymphocytes, the same experimental setup was followed, except there were no additional restimulations with anti-CD3 / CD28 antibodies.[000184] Smf / fe-ce / f SHARE-seq chronic-unique stimulation screen. The same experimental setup was followed for this screen as the bulk TOX TF-ome ORF screen, except that a lentiviral pool was generated from a subpool of the “chronic-unique” TFs and an additional 2-hour PMA / IO stimulation was conducted at the end of day 10 along with a vehicle control for each condition. Cells were then gently fixed with 0.2% formaldehyde for 5 minutes at room temperature. Fixation was quenched using a 2.5M Glycine, Tris-HCI, and 7.5% BSA solution. Cells were washed and frozen with Cryostor.[000185] THAP6 FACS exhaustion / memory validation and cytokine expression analysis. CD8+ T cells from three distinct donors were thawed, and the same experimental timeline as described above for “Single-cell SHARE-seq chronic-unique stimulation screen” was followed, except for a FACS analysis of exhaustion and memory markers with antibodies as listed above (CD8, LAG3, TIGIT, PD-1 , and IL7R). For cytokine analysis, 100k cells were seeded into wells of a 96-well flat-bottomed plate per condition and stimulated with either PMA / IO or vehicle control for 2 hours. Supernatant from each condition was then collected, and a bead-based cytokine analysis kit was used (Biolegend Legendplex CD8 / NK cell panel, San Diego, CA) to capture cytokine expression. Analysis of data was conducted using Biolegend’s Legendplex analysis software.Example 2The Immune Response to Cancer is Restrained by T Cell Exhaustion[000186] The immune system is reactive to cancer cells, being able to identify them via antigen recognition through the T cell receptor. Thanks to the robustness of the adaptive immune system, reactive T cells are able to quickly eliminate any cancerous ceils that are identified. Unfortunately, many cancers can escape this immune response by forming an immunosuppressive environment. One of the main ways that cancer survives T cell attack is by inducing the T cell exhaustion program through chronic stimulation of the T cell receptor. T cell exhaustion is loosely defined as a hypofunctional T cell state that is specific to chronic stimulation and / or immunosuppression by the tumor microenvironment. Exhausted T cells are defined by a transcriptional and epigenetic program that drives expression of immune checkpoint markers, and hampers both the effector function and metabolic state of T cells. This T ceil state leads to decreased cancer cell killing and reduced T cell proliferation. FIG.1 shows an example of exhausted T cells in the tumor microenvironment and the most successful immunotherapy strategies involving inhibition of the PD-1 / PD-L1 immune checkpoint receptors. Represented as two opposite ends of the spectrum, TCF1 is thought to be a master regulator of memory T cell formation (a highly functional T cell state partly responsible for immunological memory) while TOX is thought to be a master regulator driving the T cell exhaustion program, driving increased expression of exhaustion markers (e.g., immune checkpoint markers), decreasing T cell effector function, and increasing metabolic disfunction.Example 3Differential Gene Analysis Between Chronic and Acute Stimulation Conditions Revealed Upregulation of Many Exhaustion-Associated Genes and Downregulation of Memory-Associated Genes[000187] Using screening technologies, novel master regulators of the exhaustion program were identified that prevent exhaustion from occurring, work with TOX to drive exhaustion, and may reverse exhaustion. An in vitro model of T cell exhaustion was used to perform these studies (FIG. 2). To screen for regulators of exhaustion at a large scale, an in vitro restimulation assay was used to drive the exhaustion program in primary human CD8+ T ceils. T cells were cultured and then harvested on day 8 using CD3 / CD28 dynabeads. For half of the cells, RNA was isolated and sequenced to determine which genes were being expressed. For the other half of the cells, the cell nuclei were isolated, a transposase-accessible chromatin with sequencing (ATAC-seq) library was prepared to determine chromatin accessibility across the genome, and the resulting sequences were determined. Chronic stimulation of the T ceil receptor resulted in exhaustion phenotype in primary human CD8+ T cells.[000188] To ensure that this model successfully recapitulated terminal exhaustion versus regular effector differentiation, acutely stimulated T cells (one TCR stimulation) and chronically stimulated T cells (three additional stimulations) were submitted to bulk RNA and ATAC-seq. Since antibody and Dynabead repeat stimulated conditions were comparable, Dynabead repeat-stimmed conditions and acutely-stimmed conditions were compared in all future studies, FIG. 3A is an RNA-seq volcano plot of differentially expressed genes and accessible regions showing that gene pathways in primary human CD8+ T cells that are related to exhaustion were upregulated with repeat stimulations with anti-CD3 / CD28 Dynabeads in vitro (i.e., chronic stimulation; “Chronic”) as compared to acutely stimulated T cells (“Acute”). 5,705 differentially expressed genes were found. Upregulated exhaustion markers included TIGIT, PDCD1 , CLTA4, and LAG3. Downregulated memory markers included SELL, TCF7, and IL7R. Similarly, chromatin regions near exhaustion genes were differentially accessible, and regions near memory genes were differentially inaccessible. FIGS. 3B-3C show accessible / inaccessible chromatin in primary human CD8+ T cells that are related to exhaustion were upregulated with repeat stimulations with anti-CD3 / CD28 Dynabeads in vitro based on the bulk RNA-seq data. Chromatin regions near exhaustion genes were differentially accessible (LAG3, PD1 , CD39, TIM3, CTLA4, TIGIT, TOX) and regions near memory genes were differentially inaccessible (CCR7, IL7R, TCF7; FIG. 38).[000189] Gene set enrichment analysis (GSEA) was used to analyze exhaustion genes. The GSEA of chronic vs acute model compared to CD8 T cell exhaustion datasets used were GSE9650__Exhausted__vs__memory__cd8__tcell__up and GSE9650__Effector__vs__exhausted_cd8_tcell__dn. GSEA demonstrated enrichment for a T cell exhaustion gene signature (FIGS. 3D-3E). Global ATAC peak analysis was also conducted, and it revealed an increase in chromatin accessibility in regions associated with terminally exhausted T cells (FIG. 3F). Terminally dysfunctional chromatin regions were taken from Pritykin Y, et. al, Molecular Cell, 2021 , 81, 2477-2493, incorporated herein by reference; the unified human ATAC seq dataset for differentially accessible peaks in terminally dysfunctional versus progenitor t cells. There were several differentially accessible regions in the TOX locus as shown by an ATAC track of the TOX locus (FIG. 3G).[000190] With an exhausted T cell (TEX) model defined, screens to discover novel regulators of TOX and the exhausted T cell state were conducted. The aims of theseexperiments were to discover novel regulators of TOX expression, prevent T cells from reaching terminal exhaustion, and determine which TFs regulate TOX expression. A CRISPR activation (CRISPRa) TF-ome library was designed by selecting 1 ,639 TFs based on a list of ail human TFs as defined by Lambert et al., Ce / / . 2018;172(4): 650-665, incorporated herein by reference (FIG. 4). To understand what other TF regulators might be driving or preventing T cell exhaustion upstream of TOX, a T cell repeat stimulation screen using the CRISPRa library targeting all the TFs in the human genome with TOX protein expression as a reporter was developed. For library design, 6 gRNAs targeting each TF promoter were used. T cells from three distinct donors were transduced with a lentiviral pool of the gRNA library and chronically stimulated. Finally, high and low TOX expressing cells were sorted via fluorescence activated cell sorting (FACS), genomic DNA was isolated, gRNA sequences were amplified for next-generation sequencing, and each TOX high / low bin was analyzed for gRNA enrichment. For all TFs that did not have 6 gRNAs in Calabrese and were present in Horlbeck et al. (eLife. 2016; 5:e19760, incorporated herein by reference), the TF gRNAs were supplemented to 6 with top gRNAs from Horlbeck et al. The experimental timeline used for the CRISPRa TF-ome chronic stimulation TOX screen is shown in FIG, 5A. A CRISPR interference (CRISPRi) tiling screen was also conducted using the protocol shown in HG. SB. The TOX locus CRISPRi tiling screen library was designed to target the TOX gene locus ±100 kb upstream and downstream, which consisted of a total of 7,052 sgRNAs with ~5% of the library being non-targeting negative controls.The results of the TF-ome CRISPRa with TOX readout are shown in FIG. SC, and the results of the tiling screen are shown in FIG. SD. The results showed that regulatory elements that positively and negatively regulate TOX expression were successfully identified. For example, it was found that PRDM1 downregulates expression of TOX whereas BATF and PAX7 upregulate expression of TOX. The CRISPRa screen targeting all human TFs in genome confirmed previously characterized TFs controlling TOX expression and the CRISPRi tiling screen targeting the TOX locus uncovered regulatory elements that modulate TOX expression in T cell chronic stimulation model.Example 4TF Overexpression Screens Reveal Chronic-Unique TF Regulators of TOX[000191] TF overexpression screens with T cell exhaustion were performed as shown in FIGS. 6A-SB. To maximize the likelihood for a cell state conversion, an open-reading frame (ORF) library encoding all human transcription factors provided by Addgene (Watertown, MA; Joung et al., Cell. 2023; 186, 209-229, incorporated herein by reference) was used.The lentiviral library included all TF open-reading frames in the human genome constitutivelyexpressed by a EF-1a promoter. To understand what other TF regulators might be driving or preventing T cell exhaustion upstream of TOX, a T cell stimulation screen using the ORF library of all the TFs in the human genome with TOX protein expression as a reporter was developed. Comparing screen results in the context, of acute versus chronic stimulation facilitated identification of factors specific to the TEX state. Transduced CD3+ T cells from three distinct donors were used in either an acute stimulation setting (one TCR stimulation with anti-CD3 / CD28 beads) or a chronic stimulation setting (at least two additional stimulations). CD8+ T cells were thawed and activated. A lentiviral library as described herein was added. Cells from the same donor were split into either acute or chronic stimulation conditions. Cells were then stained for TOX and sorted based on high or low TOX expression. Genomic DNA was isolated and sorted bins were sequenced for differential TF enrichment analysis. The lentiviral construct used for the foregoing comprised a SV40 promoter driving expression of a puromycin selection cassette and an EF1 alpha promoter driving expression of the TF cDNA and barcode (FiG. 6C). The ORF screen of all human TFs in the human genome confirmed previously characterized TFs and discovered novel TFs that regulate T cell exhaustion.[000192] Differential analysis of the two sorted bins (high or low TOX expression) revealed many potential regulators of TOX (FIGS. 7A-7C and FiG. 8). Over 100 TFs were enriched in TOX-high and TOX-low bins, including both known and novel regulators of TOX. TOX emerged as a highly significant hit for both screens, serving as a positive control that the screens worked. Many TFs regulated TOX dependent on stimulation condition. Since the aim was to discover TFs that prevented exhaustion uniquely in the chronic stimulation setting (so TFs that don’t impair TOX function in normal effector differentiation), TFs in the “chronic only” bins were pursued (i.e., TFs that emerged as hits in the chronic stimulation setting but not the acute stimulation setting). Some TFs identified were RARA (binding is enriched in exhausted CD8 T ceils), XBP1 (promotes T cell exhaustion), ID2 and ID3 (control formation of memory CD8 T cells), Flit (represses optimal effector CD8+ T cells during exhaustion), FOXP1 (regulates T cell quiescence and is thought to drive exhaustion program), RUNX1 (plays an important role in CD8 T cell development), and KLF2 (associated with T cell quiescence maintenance).Example §Validations of Top Chronic-Uniqae Hits[000193] Initial validation efforts focused on the top 13 chronic-unique TFs ranked by p- value. The validation setup followed the screen timeline, with the key differences focused onindividually transducing TFs and measuring TOX protein levels (via FACS), exhaustion / memory markers staining (via FACS), TOX transcript levels (via RT-qPCR) with individual TF transductions, and transcriptomic changes (via bulk RNA-seq). The validation protocol is shown in FIG. 9A and FIG. 10A. Validations of initial protein and RNA-levels confirmed that there was modulation of the exhaustion program (FIGS. 9B-9C, FIGS. 10B- 10C, and FIGS, 11A-1 W). As expected, most TFs were validated in terms of TOX expression on the protein level via FACS. TFs that downregulated TOX were readily captured via FACS, but since baseline expression was high it was hard to capture upregulators. Via qPCR, nearly all TFs were validated and the effects of upregulators were effectively captured. These individual validations of the top enriched TFs unique to chronic stimulation confirmed modulation of TOX expression in the expected direction at the protein and transcript level.[000194] T cell exhaustion / memory marker expression was also measured via FACS (FIGS. 10D-10G). This FACS staining of exhaustion (LAG3, PD-1 , and TIGIT) and memory (IL7R) surface markers revealed significant differences in expression levels, suggesting potential phenotypic changes in T cell function. In terms of expression of exhaustion / memory surface markers, TOX upregulated PD-1 , as has been previously characterized , which served as a positive control that the overexpression was functionally working. POU2F2 significantly upregulated PD-1 expression (FIG. 10D). MEF2B significantly downregulated expression of LAG3 (FIG. WE). PAX2 significantly downregulated expression of TIGIT (FIG. 10F). And finally, MEF2B upregulated expression of the memory marker IL7R (FIG. 10G). As it can be seen, many TFs had varying effects on exhaustion / memory marker expression.[000195] Functional validation was also conducted using an in vitro CAR-T cell cancer coculture model. The protocol is shown in FIG. 12A. FIG. 12B is a graph showing cell counts of the SKBR3 human breast cancer cell line tagged with nuclear GFP overtime that were co-cultured with CD8+ chronically stimulated CAR T cells expressing various TFs. FIG. 12C is a graph showing cell counts of the SKBR3 human breast cancer cell line over time that were co-cultured with CD8+ CAR+ TILs expressing various TFs. As it can be seen in FIGS. 12B-12C, chronic-unique TFs impacted cancer killing in vitro.Example 6Single-Cell T ceil Stimulation Screening[000196] To understand what effect the TFs were having on a transcriptomic and epigenomic level along with the rest of the chronic-unique hits, a single-cell screen was performed to obtain a more global picture of these cell state changes (FIGS. 13A-13B). A chronic versus acute stimulation screen with a subpool of chronic-unique TFs was conducted, and SHARE-seq was used as a reporter (paired RNA+ATAC seq on top of TF capture). To add an additional layer of functional information, a final PMA / IO stimulation in both conditions was performed to dissect functional capacity of T cells with specific TFs being overexpressed. A 770k single-cell atlas was generated that contained matched RNA+ATAC+TF information with different stimulation conditions. As is represented by the UMAP, it was observed that the cells clustered based on stimulation condition (FIGS. 13A- 1348).[000197] Then, a gene module was generated for stimulation response and a chromatinbased exhaustion score to capture TFs that affect both stimulation response and exhaustion. FIG. 13C shows RNA stimulation scores based on pseudo-bulked single-cell RNA-seq data in exhausted stimulated cells (TEX). THAP6 and DMRT3 pushed TEX away from exhaustion and improved stimulation response. FIG. 13D shows chromatin exhaustion scores based on pseudo-bulked single-cell ATAC-seq data in exhausted unstimulated cells. FIG. 13E shows RNA stim scores in TEX. THAP6 improved TEX stimulation and pushed away from exhaustion. FIG. 13F is a graph showing chromatin exhaustion scores in exhausted unstimulated cells. The TFs that improved the stimulation response via an increase in the RNA module score and TFs that pushed away from an exhausted chromatin state were validated with the ATAC module score (FIGS. 13C-13F). There were many TFs that pushed T cells in both directions, but the initial validation efforts focused on THAP6 due to its undescribed role in T cell biology and preliminary validation results.[000198] To validate THAP6 phenotypically and functionally, surface staining was performed for the same exhaustion / memory markers and cytokine expression was measured via 13-plex CD8 / NK cytokine panel (FIG. 14A). Through FACS analysis of exhaustion markers, it was found that THAP6 reduces protein expression of LAG3 for all donors, and PD-1 and TIGIT for most donors (FIGS. 14B-14G). Through multiplexed cytokine expression analysis, it was found that there was an improvement in IFN-gamma production, a drastic improvement in Granzyme A production, and an improvement in granzyme B production after a final PMA / IO stimulation (FIG. 14H). In addition, THAP6improved secretion of T cell effector molecules (interferon gamma, sFasL, granzyme A and B, and perforin; FIGS. 15A-1SJ). Overall, it was found that THAP6 decreased exhaustion markers and improved T cell effector function.Example 7Cytotoxicity Analysis[000199] To analyze cytotoxicity, a co-culture killing assay was performed. The assay included: (1) co-transducing CD8+ T ceils with HERCAR-mCherry and TF lenti; (2) restimulating the cells two times; (3) co-culturing the cells with SKBR3s-GFP ceils; and (4) imaging with IncuCyte®. The assay results are shown in FIGS. 16A-16B.Example 8RNA-seq[000200] Bulk RNA-seq revealed widespread transcriptomic changes when overexpressing most of the top candidates, as illustrated by FIG. 17A. Direct TOX overexpression did not lead to widespread changes since T cells were already pushed to exhaustion. Looking at a condensed gene expression heatmap of the bulk RNA-seq data (FIG. 17B) revealed many things. As quality control, it was observed that the TF ORFs were being properly overexpressed. Generally speaking, direct TOX overexpression in the chronic stimulation setting drove upregulation of NR4A2 and PD-1 as has been previously described, but TOX overexpression was unable to further upregulate other exhaustion markers (this could be due to the already high levels of TOX expression in this model). HOXB9 drove upregulation of virtually all exhaustion markers while ZBTB18 had mixed results. Finally, PAX2 drove decreased expression of many exhaustion markers as expected, while the results with MEF2B were not as clear. In terms of T ceil memory related genes, TOX and HOXB9 drove downregulation of most genes as expected, while ZBTB18 had mixed results. MEF2B drove upregulation of memory markers, and PAX2 seemingly had no effect. While novel regulators of exhaustion were identified, it was also sought to understand the global transcriptomic and epigenomic effect of each TF for all TFs that regulated TOX expression, so a single-cell screen of all chronic-unique hits was performed.[000201] Overall, TFs that delay TEX formation and improve TEX function were found (THAP6 being one candidate), novel TFs that increase TOX in a stimulation-dependent manner were found, and a multimodal single-cell atlas with stimulation conditions was generated. THAP6 may be further characterized using in vitro and in vivo T cell killingassays. Additional TFs may be validated using the single-cell screen. TFs may be further examined using a pre-exhausted model to find TFs that potentially reverse exhaustion.Example 9CRiSPR Activation of TFs to Modulate T Ceils[000202] CRISPRa will be used to increase the expression of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST 1 , NKX6, and FEV. A dCas9 protein (such as dSpCas9) will be fused to an activation domain, such as at least one domain of VP64, p300, or p300 core. The resulting fusion protein may be VP64-dCas9- VP64 (SEQ ID NO: 43). T cells will be transduced with a lentiviral vector encoding the fusion protein such as VP64-dCas9-VP64 and a gRNA targeting a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV. Exemplary gRNAs are shown in TABLE 3. The transduced T cells will be assayed for TOX activity by measuring TOX transcript levels (via RT-qPCR), TOX protein levels (via FACS), exhaustion / memory markers staining (via FACS), and transcriptomic changes (via bulk RNA- seq), as described in Example 5. The transduced cells will show decreased TOX transcription, decreased TOX expression, decreased exhaustion markers, and improved T cell effector function.[000203] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.[000204] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.[000205] AH publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually indicated to be incorporated by reference for all purposes.[000206] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:[000207] Clause 1. An isolated polynucleotide encoding a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof.[000208] Clause 2. The isolated polynucleotide of clause 1 , wherein the isolated polynucleotide comprises a sequence selected from SEQ ID NOs: 75-84.[000209] Clause 3. The isolated polynucleotide of clause 1 or 2, wherein the transcription factor is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.[000210] Clause 4, A vector encoding a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof.[000211] Clause 5. The vector of clause 4, wherein the vector comprises a promoter operably linked to a polynucleotide sequence encoding the transcription factor.[000212] Clause 6. The vector of clause 5, wherein the promoter is non-endogenous to the transcription factor.[000213] Clause 7. The vector of clause 5 or 6, wherein the promoter is a constitutive promoter, or a ubiquitous promoter, or an inducible promoter, or a cell-specific promoter, or a tissue-specific promoter.[000214] Clause 8. The vector of any one of clauses 4-7, wherein the vector comprises an open reading frame (ORF) of the transcription factor.[000215] Clause 9, The vector of any one of clauses 4-8, wherein the vector comprises a sequence selected from SEQ ID NOs: 75-84 or encodes a polypeptide comprising a sequence selected from SEQ ID NOs: 85-94.[000216] Clause 10. The vector of any one of clauses 4-9, wherein the transcription factor is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.[000217] Clause 11. The vector of any one of clauses 4-10, wherein the vector is a viral vector.[000218] Clause 12. The vector of clause 11 , wherein the vector is a lentiviral vector.[000219] Clause 13. The vector of clause 11 , wherein the vector is an adeno-associated virus (AAV) vector.[000220] Clause 14. The vector of clause 13, wherein the AAV vector is selected from AAV1 , AAV2, AAV5, AAV6, AAV8, AAV9, and an engineered AAV vector.[000221] Clause 15. A method of modulating T cells, the method comprising administering to a T cell or a subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.[000222] Clause 16. The method of clause 15, wherein modulating T cells comprises increasing T cells, or increasing memory T cells, or preventing T cell exhaustions, or reversing T cell exhaustions, or a combination thereof,[000223] Clause 17. A method of increasing T cells, the method comprising administering to a T cell or a subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.[000224] Clause 18. A method of enhancing adoptive T cell therapy (ACT) in a subject, the method comprising administering to the subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.[000225] Clause 19. A method of treating cancer in a subject, the method comprising administering to the subject or to a T cell of the subject, an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.[000226] Clause 20. The method of any one of clauses 15-19, wherein the gene is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.[000227] Clause 21 . The method of any one of clauses 15-20, wherein the activator modulates T cells, and wherein modulating T cells comprises increasing T cells, or increasing memory T cells, or increasing the lifetime of a T cell, or preventing T cell exhaustions, or reversing T cell exhaustions, or reducing T cell exhaustion, or enhancing the therapeutic potential of T cells, or a combination thereof.[000228] Clause 22. The method of any one of clauses 15-21 , wherein the activator modulates gene expression within the T cell.[000229] Clause 23. The method of any one of clauses 15-22, wherein the activator decreases expression of TOX in the T cell.[000230] Clause 24. The method of any one of clauses 15-23, wherein the activator comprises a polypeptide, or a polynucleotide, or a small molecule, or a combination thereof.[000231] Clause 25. The method of any one of clauses 15-24, wherein the activator comprises a polynucleotide encoding the gene.[000232] Clause 26. The method of any one of clauses 15-25, wherein the activator comprises a polynucleotide comprising the open reading frame of the gene or a polynucleotide encoding a protein encoded by the gene.[000233] Clause 27. The method of any one of clauses 15-26, wherein the activator comprises a polynucleotide selected from SEQ ID NOs: 75-84 or a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 85-94.[000234] Clause 28. The method of any one of clauses 15-27, wherein the activator comprises a polypeptide comprising a protein encoded by the gene.[000235] Clause 29. The method of any one of clauses 15-24, wherein the activator comprises a polypeptide selected from SEQ ID NOs: 85-94.[000236] Clause 30. The method of any one of clauses 15-28, wherein the activator comprises the vector of any one of clauses 4-14.[000237] Clause 31 . The method of any one of clauses 15-30, wherein the activator or a polynucleotide encoding the activator is encapsulated within a lipid nanoparticle or polymeric carrier.[000238] Clause 32. The method of any one of clauses 15-31 , the method further comprising administering at least one cancer therapy or at least one antiviral therapy.[000239] Clause 33. A vector comprising the isolated polynucleotide of any one of clauses 1-3.[000240] Clause 34. A cell comprising the isolated polynucleotide of any one of clauses 1- 3, or the vector of any one of clauses 4-14, or the vector of clause 33.[000241] Clause 35. The cell of clause 34, wherein the cell is a CD8+ T ceil.[000242] Clause 36. A pharmaceutical composition comprising: the isolated polynucleotide of any one of clauses 1-3, or the vector of any one of clauses 4-14, or the vector of clause 33, or a combination thereof.[000243] Clause 37. The pharmaceutical composition of clause 36, further comprising at least one cancer therapy or at least one antiviral therapy.[000244] Clause 38. A composition for increasing T cells, the composition comprising an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof.[000245] Clause 39. The composition of clause 38, wherein the gene is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.[000246] Clause 40. The composition of clause 38 or 39, wherein the activator comprises a polynucleotide encoding the gene, or a polynucleotide encoding the open reading frame of the gene, or a polypeptide encoded by the gene, or a combination thereof.[000247] Clause 41 . The composition of clause 40, wherein the activator comprises a polynucleotide selected from SEQ ID NOs: 75-84 or a polypeptide selected from SEQ ID NOs: 85-94.[000248] Clause 42. The composition of any one of clauses 38-41 , further comprising at least one cancer therapy or at least one antiviral therapy,[000249] Clause 43. The composition of any one of clauses 38-42, wherein the activator comprises a DNA targeting composition, the DNA targeting composition comprising: (a) a Cas9 protein and at least one guide RNA (gRNA) that targets the Cas9 protein to the gene or a regulatory element thereof; or (b) a meganuclease, or (c) a fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, wherein the firstpolypeptide domain comprises a zinc finger protein or a TALE or a Cast 2 protein or a Cas13 protein or a Cas9 protein, and wherein the second polypeptide domain has an activity selected from transcription activation activity, transcription repression activity, nuclease activity, base editing activity, prime editing activity, transcription release factor activity, histone modification activity, nucleic acid association activity, methylase activity, and demethylase activity, wherein when the first polypeptide domain comprises a Cas9 protein the DNA targeting composition further comprises at least one guide RNA (gRNA) that targets the Cas9 protein to the gene or a regulatory element thereof.[000250] Clause 44. The composition of clause 43, wherein the second polypeptide domain comprises a meganuclease,[000251] Clause 45. A DNA targeting composition comprising: a Cas9 protein or a fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, wherein the first polypeptide domain comprises a zinc finger protein or a TALE or a Cas12 protein or a Cast 3 protein or a Cas9 protein, and wherein the second polypeptide domain has an activity selected from transcription activation activity, transcription repression activity, nuclease activity, base editing activity, prime editing activity, transcription release factor activity, histone modification activity, nucleic acid association activity, methylase activity, and demethylase activity; and at least one guide RNA (gRNA) that targets the Cas9 protein to a target gene or a regulatory element thereof when the DNA targeting composition comprises a Cas9 protein, wherein the target gene is selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV.[000252] Clause 46. The composition of clause 45, wherein the gene is THAP6, DMRT3, or MEF2B.[000253] Clause 47. The composition of any one of clauses 43-46, wherein the gRNA is encoded by a polynucleotide comprising a sequence selected from SEQ ID NOs: 95-114, or comprises a sequence selected from SEQ ID NOs: 115-134.[000254] Clause 48. The composition of any one of clauses 43-47, wherein the Cas protein comprises a Streptococcus pyogenes Cas9 protein, or a Staphylococcus aureus Cas9 protein, or any fragment thereof.[000255] Clause 49. The composition of any one of clauses 43-48, wherein the Cas9 protein comprises the amino acid sequence of one of SEQ ID NOs: 26-29, or any fragment thereof, and / or wherein the Cas9 protein is encoded by a polynucleotide comprising a sequence selected from SEQ ID NOs: 30-39, and / or wherein the Cas9 protein comprises anamino acid sequence having al feast 90% or greater identity to a sequence selected from SEQ ID NOs: 26-29, or any fragment thereof, and / or wherein the Cas9 protein is encoded by a polynucleotide comprising a sequence having at least 90% or greater identity to a sequence selected from SEQ ID NOs: 30-39, or any fragment thereof, and / or wherein the Cas9 protein comprises an amino acid sequence having one, two, three, four, five or more changes selected from amino acid substitutions, insertions, or deletions, relative to a sequence selected from SEQ ID NOs: 26-29, or any fragment thereof, and / or wherein the Cas9 protein is encoded by a polynucleotide comprising a sequence having one, two, three, four, five or more changes selected from nucleotide substitutions, insertions, or deletions, relative to a sequence selected from SEQ ID NOs: 30-39, or any fragment thereof.[000256] Clause 50. The composition of any one of clauses 43-49, wherein the fusion protein comprises more than one second polypeptide domain.[000257] Clause 51 . The composition of any one of clauses 43-50, wherein the second polypeptide domain has transcription activation activity.[000258] Clause 52. The composition of clause 51 , wherein the second polypeptide domain comprises a polypeptide selected from VP16, VP64, p65, TET1 , VPR, VPH, Rta, and p300, or a fragment thereof.[000259] Clause 53. The composition of clause 52, wherein the second polypeptide domain comprises VP64, p300, VPH, or VPR, or a fragment thereof.[000260] Clause 54. The composition of one of clauses 43-53, wherein the second polypeptide domain comprises the amino acid sequence of SEQ ID NO: 41 , 42, 53, or 55, or any fragment thereof, and / or wherein the second polypeptide domain is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 54 or 56, and / or wherein the second polypeptide domain comprises an amino acid sequence having at least 90% or greater identity to SEQ ID NO: 41 , 42, 53, or 55, or any fragment thereof, and / or wherein the second polypeptide domain is encoded by a polynucleotide comprising a sequence having at least 90% or greater identity to SEQ ID NO: 54 or 56, or any fragment thereof, and / or wherein the second polypeptide domain comprises an amino acid sequence having one, two, three, four, five or more changes selected from amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 41 , 42, 53, or 55, or any fragment thereof, and / or wherein the second polypeptide domain is encoded by a polynucleotide comprising a sequence having one, two, three, four, five or more changes selected from nucleotide substitutions, insertions, or deletions, relative to SEQ ID NO: 54 or 56, or any fragment thereof.[000261] Clause 55. The composition of any one of clauses 43-54, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 43, or any fragment thereof, and / or wherein the fusion protein is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 44, and / or wherein the fusion protein comprises an amino acid sequence having at least 90% or greater identity to SEQ ID NO: 43, or any fragment thereof, and / or wherein the fusion protein is encoded by a polynucleotide comprising a sequence having at least 90% or greater identity to SEQ ID NO: 44, or any fragment thereof, and / or wherein the fusion protein comprises an amino acid sequence having one, two, three, four, five or more changes selected from amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 43, or any fragment thereof, and / or wherein the fusion protein is encoded by a polynucleotide comprising a sequence having one, two, three, four, five or more changes selected from nucleotide substitutions, insertions, or deletions, relative to SEQ ID NO: 44.[009262] Clause 56. The composition of any one of clauses 43-55, further comprising at least one cancer therapy or at least one antiviral therapy.[000263] Clause 57. An isolated polynucleotide sequence encoding the composition of any one of clauses 43-56.[000264] Clause 58. A vector comprising the isolated polynucleotide sequence of clause 57.[000265] Clause 59. A cell comprising the composition of any one of clauses 43-56, or the isolated polynucleotide sequence of clause 57, or the vector of clause 58, or a combination thereof.[000266] Clause 60. The cell of clause 59, wherein the cell is a CD8+ T cell.[000267] Clause 61. A pharmaceutical composition comprising: the composition of any one of clauses 43-56, or the isolated polynucleotide sequence of clause 57, or the vector of clause 58, or a combination thereof.[000268] Clause 62. A method of modulating T cells, the method comprising administering to a cell or a subject the composition of any one of clauses 43-56, or the isolated polynucleotide sequence of clause 57, or the vector of clause 58, or the cell of clause 59 or 60, or the pharmaceutical composition of clause 61 , or a combination thereof.[000269] Clause 63. The method of clause 62, wherein modulating T cells comprises increasing T cells, or increasing memory T cells, or preventing T cell exhaustions, or reversing T cell exhaustions, or a combination thereof.[000270] Clause 64. The method of clause 62 or 63, wherein the composition or isolated polynucleotide sequence or vector is administered to a T cell, and wherein the T cell thereby decreases expression of TOX.[000271] Clause 65. A method of increasing T cells, the method comprising administering to a cell or a subject the composition of any one of clauses 43-56, or the isolated polynucleotide sequence of clause 57, or the vector of clause 58, or the cell of clause 59 or 60, or the pharmaceutical composition of clause 61 , or a combination thereof.[000272] Clause 66. A method of enhancing adoptive T cell therapy (ACT) in a subject, the method comprising administering to the subject the composition of any one of clauses 43-56, or the isolated polynucleotide sequence of clause 57, or the vector of clause 58, or the cell of clause 59 or 60, or the pharmaceutical composition of clause 61 , or a combination thereof.[0(80273] Clause 67. A method of treating cancer in a subject, the method comprising administering to the subject the composition of any one of clauses 43-56, or the isolated polynucleotide sequence of clause 57, or the vector of clause 58, or the ceil of clause 59 or 60, or the pharmaceutical composition of clause 61 , or a combination thereof.SEQUENCESSEQ ID NO: 1NRG (R = A or G; N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 2NGG (N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 3NAG (N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 4NGGNG (N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 5NNAGAAW (W = A or T; N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 6NAAR (R ~ A or G; N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 7NNGRR (R = A or G; N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 8NNGRRN (R = A or G; N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 9NNGRRT (R = A or G; N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 10NNGRRV (R = A or G; N can be any nucleotide residue, e.g., any of A, G, C, or T; V = A orC or G)SEQ ID NO: 11NNNNGATT (N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 12NNNNGNNN (N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 13NGA (N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 14NNNRRT (R = A or G; N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 15ATTCCTSEQ ID NO: 16NGAN (N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 17NGNG (N can be any nucleotide residue, e.g., any of A, G, C, or T)SEQ ID NO: 18DNA sequence of the gRNA constant region gtttaaga.gctatgctggaaacagcatagcaagtt .taaataaggctagt ccgttat caacttgaaaaa g t gg c a c c gag t c gg t gcSEQ ID NO: 19RNA sequence of the gRNA constant region guuuaagagcuaugcuggaaacagcauagcaaguuuaaauaaggcuaguccguuaucaacuugaaaaa guggca ccgagucggugcSEQ ID NO: 20SV40 NLS (Pro-Lys-Lys-Lys-Arg-Lys-Vai)SEQ ID NO: 21GS linker (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer between 0 and 10SEQ ID NO: 22Gly-Gly-Gly-Giy-GlySEQ ID NO: 23Gly-Gly-Ala-Gly-GlySEQ ID NO: 24Gly-Gly-Gly-Gly-Ser-Ser-SerSEQ ID NO: 25Gly-Gly-Gly-Gly-Ala-Ala-AlaSEQ iD NO: 26Streptococcus pyogenes Cas9MDKKYS IGLDIGTNSVGWAVITDEYKVPSKKFKVIJGNTDRHS IKKNLIGALDFDSGETAEATRLKRTA RRRYTORKHRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRilYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPOTKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDIEED IVLTETLFEDREMIEEREKTYAHLFDDKVMKQIJKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFEKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRESDYDVDHIVPQSFEKDDSIDNKVETRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREISnmiHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKL,IARKI<DWDPKKYGGFDSPTVAYSVIJv7VAKVEKGKSKK LKSVKEIJLGITIMERSSFEKNPIDFLEAKGYKEVKKDDI IKEPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVMFLYEASHYEKEKGSPEDNEQKQLFVEQHKHYEDEI IEQISEFSKRVILADANEDKVLS AYRKHRDKPIREQAEMT IHEFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATEIHQS ITGEYETRI DLSQLGGDSEQ !D NO: 27Staphylococcus aureus Cas9MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVK KLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKE QISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDL LETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDEN EKLEYYEKFQIIEWFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKE IIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEErEQISNLKGYTGTHNLSLKAINLILDELW HTNDNQ I Al FNRLKLVPKKVDLSQQKE I PTTLVDDFILS PWKRS F IQS I KV IM Al I KKYGDPND I I I ELAREKNSKDAQKMINEMQKRNRQTNERIEEI IRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLE DLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLA KGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATOGLMNLLRSYFRVNNLDVKVKSINGGF TSFLRRKWKFKORNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQ EYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKL KKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYG NKLNAHLD I TDD Y PNS RNKWKL S L KP YRFD VYLDNGVY KF VTVKNLD V I KKENYYE VNS KC YEE AKK LKKISNQAEFIASFYMMDLIKIMGELYRVIGVNNDLLMRIEVNMIDITYREYLENMNDKRPPRI IKTI AS KTQS I KKYSTD I LGMLYE VKS KKHPQ 11 KKGSEQ !D NO: 28Streptococcus pyogenes Cas9 (with D1 OA)MDKKYS IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALEFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQEPEKYKE I FFDQSKNGYAGYIDGGASQEEFYKF I KP I LEKMDGTEELLVKLNREDLERKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQWIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGDSEQ iD NO: 29Streptococcus pyogenes Cas9 (with D10A, H849A)MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKE I FFDQSKNGYAGYIDGGASQEEFYKF I KP I LEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQWIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI D L S QLGGDSEQ NO: 30Poiynucieotide sequence of D10A mutant of S. aureus Cas9 atgaaaagga actacattct ggggctggcc atcgggatta caagcgtggg gtatgggatt attgactatg aaacaaggga cgtgatcgac gcaggcgtca gactgttcaa ggaggccaac gtggaaaaca atgagggacg gagaagcaag aggggagcca ggcgcctgaa aegaeggaga aggcacagaa tccagagggt gaagaaactg ctgttcgatt acaacctgct gaccgaccat tctgagctga gtggaattaa tccttatgaa gccagggtga aaggcctgag t cagaagctg t cagaggaag agt r ttccgc agctctgctg cacctggcta agcgccgagg agtgcataac gtcaatgagg tggaagagga caccggcaac gagctgtcta caaaggaaca gatctcacgc aatagcaaag ctctggaaga gaagtatgtc gcagagctgc age tggaacg getgaagaaa gatggcgagg tgagagggtc aattaatagg ttcaagacaa gcgactacgt caaagaagcc aagcagctgc tgaaagtgca gaaggcttac caccagctgg atcagagctt catcgatact tatatcgacc tgctggagac tcggagaacc tactatgagg gaccaggaga agggagcccc ttcggatgga aagacatcaa ggaatggtac gagatgctga tgggacattg cacctatttt ccagaagagc tgagaagcgt caagtacgct tataacgcag at ctgtacaa cgccctgaat gacctgaaca acctggt cat caccagggat gaaaacgaga aactggaata ctatgagaag ttccagatca tcgaaaacgt gt ttaagcag aagaaaaagc ctacactgaa acagattgct aaggagatcc tggtcaacga agaggaca tc aagggctacc gggtgacaag cactggaaaaaaggagatce tggtcaacga agaggaca tc aagggctacc gggtgacaag esc tggaaaa ccagagttca ccaatctgaa agtgtatcac gatattaagg acatcacagc acggaaagaa atcattgaga acgccgaact gctggatcag a i- i_gc caaga tcctgactat ctaccagagc tccgaggaca tccaggaaga gctgact aac ctgaacagcg agctgaccca ggaagagatc gaacagatta gtaatctgaa gggg tacacc ggaacacaca acctgt ccct gaaagctatc aatetgatte tggatgagc ti gtggcataca aacgacaa tic agat tgcaat c tttaa ccgg etgaagetgg t c c c a a a a a a ggtggacctg agticagcaga aagagatccc aaccacac tig gtggacgatt tcattctgtc acccgtggtc aagcggagct tcatccagag catcaaagtg atcaacgcua tcatcaagaa gtacggcctg c c c a a t ga n a teat tat ega gctggc tiagg gagaagaaca gcaaggacgc acagaagatg atcaatgaga tgcagaaacg aaaccggcag accaatgaac gcattgaaga gattatccga actaccggga aagagaaege aaagtacctg attgaaaaaa tcaagctgca cgat atgcag gagggaaagt gtctgtat tc tctggaggcc atccccctgg aggacctgct gaacaatcca ttcaactacg aggtegat ea tatt atcccc agaagegtgt ccttcgacaa ttcctttaac aacaaggtgc tggteaagea ggaagaggcc tc taaaaagg gaaataggac tcctttccag tacctgtcta gttcagat tc caagatctct taegaaacc t ttaaaaagca cattctgaat c tgq c c aaag gaaagggeeg c a ti c ag c a ag accaaaaagg agtacctgct ggaagagegg g a c a t c a a c a gattctccgt ccagaaggat tttattaacc ggaatctggt ggacacaaga tacgctactc geggeetgat gaatctgctg cgatcctatt tccgggtgaa caatctggat gtgaaagtca agtccatcaa cggcgggttc acatct tttc tgaggcgcaa atggaagttt aaaaaggagc gcaacaaagg gtacaagcac catgccgaag atgctctga t tatcgcaaat gccgac t tic a tot t taagga gtggaaaaag ctggacaaag ccaagaaag t: gatggagaac cagatgt teg aagagaagca ggccgaa tict atgcccgaaa tcgagacaga acaggagtac aaggaga ti ti ti tea toactcc ticac c a gate a age a Lav ua aggatttcaa ggac tacaag tiactctca cc gggtggataa aaagcccaac agagagetga tcaatgacac cctgtatagt acaagaaaag aegataaggg gaataccctg attgtgaaca atctgaacgg actgtacgac aaagataatg acaagctgaa aaagetgate aacaaaagtc ccgagaagct gctgatgtac caccatgatc ctcagacata teagaaaetg aagetgatta t ggagcagta aagaacccac tgtataagta ctatgaagag ac tgggaac t acctgaccaa g tiatagcaaa a ag g a ti a a t g gccccgtgat caagaagatc aagtactatg ggaacaagct gaatgccca ti ctgga catca cagacga t ta ccc tiaacagt cgcaacaagg tggtcaagct gteaetgaag ccatacagat tegatgteta tctggacaac ggcgtgtata aatttgtgac ti g ii c a a g a a n ctggatgtca t uaaaaag ga g a a c c a c t a ti gaagtgaata gcaagtgcta egaagagget a a a a a Q C t g a aaaagat tag c a a c c agg c a gagttcatcg cctcctttta caacaacgac c i_gat caaga teaatggega aetgtatagg gtcatcgggg tgaacaatga tctgctgaac cgcattgaag tgaatatgat tgacatcact taccgagagt at ctggaaaa catgaat gat aagcgccccc ctcgaattat caaaacaatt gactctaaga ctcagagta t aaaaaagtac ticaaccga ca ttc tgggaaa oetgtatgag gtgaagagca aaaagcaccc tcaga ttatc a a a a a g g g cSEQ ID NO: 32cagattgggg atcagtacgc egatetettt ttggcagcaa agaacctgtc cgacgccatc ctgttgagcg atatcttgag agtgaacacc gaaatrac LS aagcacccct tagcgcatct atgatcaagc ggtacgacga gcatcatcag gatctgaccc tgetgaagge tettgtgagg caacagct cc ccgaaaaata caaggaaatc t t ctttgacc agagcaaaaa cggctacgct ggetatatag atggtggggc cagt caggag gaattctata aatt catcaa gcccatt ctc gagaaaatgg acggcacaga ggagt tgctg g t c a aa c 11 a acagggagga cctgctgcgg aagcagcgga cc tttgacaa eggg tetate ccccaccaga ttca tctggg cgaactgcac gcaatcctga ggaggcagga ggatttttat ccttttc tta aagataaeeg C g a g 333 a a. a gaaaag ac t c ttacattcag gatcccgtac tacgtgggac ctc tcgcccg gggcaa ttca cggtttgcct ggatgacaag gaagteagag gagactatta C 3 C C TL IL CJCJ 33 ettegaagaa gtggtggaca agggtgcatc tgcccagtct ttcatcgagc ggatgacaaa ttttgacaag aacctcccta atgagaaggt gctgcccaaa cattct ctgc t etaegagta cttt accgtc tacaatgaac tgactaaagt caagtacgt c accgagggaa tgaggaagee ggcattcctt ag tggagaac agaagaaggc ga ttgtagac ctgt tgttca agaccaacag gaaggtgact gtgaagcaac ttaaagaaga c taetttaag aagatcgaat gttttgacag tgtggaaatt tcaggggttg aagaccgctt caatgcgtca ttggggactt accatga tet tctcaagatc ataaaggaca aagacttcct ggacaacgaa gaaaa tgagg atattetega agacatcgtc ctcaccctga ccctgttcga agacagggaa atgatagaag agegettgaa aacctatgcc cacctcttcg acgataaagt tatgaagcag ctgaagcgca ggaga tacac aggatgggga agattgtcaa ggaagctgat caatggaatt agggataaac agagtggcaa gaccatactg gatttcctea aa tctgatgg ettcgccaat aggaact tea tgeaaetgat teaega tgac tctcttacct tcaaggagga c a 11 e a a a aq gctcagg tga gcgggcaggg agactccctt c a t q a a c a c a tcgcgaat t t ggcaggttcc co eget at ta aaaagggcat ccttcaaact gtcaaggtgg tggatgaa t is ggtcaaggta atgggcagac a t a a g c c a g a aaatattgtg ategagatgg cccgcgaaaa C C 3.Q 3 C C H C 3 cagaagggcc aqaaaaat aq tagagagegg atgaagagga tcgaggaggg catcaaagag ctgggatctc agattctcaa agaacacccc gtagaaaaca cacagctgca gaaegaaaaa ttgtacttgt actatctgca gaacggcaga gacatgtacg t cgaccaaga act tgatat t aatagactgt ccgactatga cgtagaccat atcgtgcccc agtcct tcct gaaggacga c tcca t tgata a eaaagt e tt gacaagaagc gacaagaaca ggggtaaaag tgataatg tg cctagcgagg aggtggtgaa aaaa atgaag aactactggc gacagctgct taatgeaaag ct ca t tacac a acggaagtt eg a taatetg acgaaagcag agagaggtgg ettgtetgag t tgg a o aagg cagggtt tat taagcggcag ctggtggaaa ctaggcagat cacaaagcac g t gg c g c a g a ttttggacag ccggatgaac acaaaatacg acgaaaatga taaaetgata egagaggt ca aagttatcac getgaaaage aagctggtgt ccgatttt cg gaaagaette cagttctaca aagt t egega gattaataac taccat catg ct cacgatgc gtacctgaac gctgttgt cg ggaccgcctt gataaagaag tacccaaagc tggaatccga gttcgtatac ggggat taca aagtgtacga tgtgaggaaa atgatagcea ag tccgagca ggaga ttgga aaggccacag etaagtaett cttttat tet aacatcatga atttttttaa gaeggaaatt accctggcca aeggagagat cagaaagcgg ccccttatag 3. Cj H C J H H 3 U Q Q tgaaacaggt gaaategtet gggataaggg cagggatttc gctactgtga ggaaggtgct gagtatgcca caggtaaata t cgtgaaaaa aaccgaagta cagaccggag gattttccaa ggaaagcatt ttgcctaaaa gaaactcaga caagctcatc gcccgcaaga aagattggga ccctaagaaa tacgggggat t tgactcacc caccgtagcc tat t ctgtgc t ggtggtagc taaggtggaa aaaggaaagt etaagaaget gaagteegtg aaggaactc t tigggaa ticac ta tcatggaa aga tea tcct t tgaaaagaa ccc tat eg at ttcctggagg ctaagggt ta caaggagg tc aagaaagacc teat cat taa actgccaaaa tactctctc t tcgagctigga aaatggcagg aagaqaatg u tggccagcgc eggagagetg caaaagggaa acgagcctgc tctgccctcc aaatatgtta attttctcta tctcgcttcc caeca tga aa agctgaaagg gtctcccgaa gataaegage agaagcagct gttcgtcgaa cagcacaagc actatctgga tgaaataatc gaacaaataa gcgagttcag caaaagggtt atcctggcgg atgctaat t t ggacaaagta etgtetgett ataacaagca ccgggataag cctattaggg aacaagccga gaatataatt cacctctt ta cactcacgaa tctcggagcc cccgccgcct tcaaatac t: t tgata cgact a tcgaccgga aacggtatac cagtaccaaa gaggtcctcg atgceaccct catccaccag teaattaetg gee tgtaega aacacggatc gacctc tctc aactgggcgg cgac tagSEQ ID NO: 33 codon optimized nucleic acid sequences encoding S. aureus Cas9 atgaaaagga actacattct ggggctggac atcgggatta caagcgtggg gtatgggatt attgactatg aaacaaggga cgtgatcgac gcaggcgtca gactgttcaa ggaggccaac gtggaaaaca atgagggacg gagaagcaag aggggagcca ggcgcctgaa acgacggaga aggcacagaa tccagagggt gaagaaactg ctgttcgatt acaacctgct gaccgaccat t ctgagctga gtggaattaa tccttatgaa gccagggtga aaggcctgag tcagaagctg t cagaggaag agtr ttccgc agctctgctg cacctggcta agcgccgagg agtgcataac gtcaatgagg tggaagagga caccggcaac gagctgtcta caaaggaaca gatctcacgc aatagcaaag ctctggaaga gaag tatgtc gcagagctgc agctggaacg gctgaagaaa gatggcgagg tgagaggg tc aattaatagg ttcaagacaa gcgactacgt caaagaagcc aagcagctgc tgaaagtgca gaaggcttac caccagctgg atcagagctt catcgatact tatatcgacc tgctggagac tcggagaacc tactatgagg gaccaggaga agggagcccc ttcggatgga aagacatcaa ggaatggtac gagatgctga tgggacattg cacctatttt ccagaagagc tgagaagcgt caagtacgct tataacgcag ar ctgtacaa cgccctgaat gacctgaaca acctggtcat caccagggat gaaaacgaga aactggaata ctatgagaag ttccagatca tcgaaaacgt gtttaagcag aagaaaaagc ctacactgaa acagattgct aaggagatcc tggtcaacga agaggaca tc aagggctacc gggtgacaag cactggaaaa ccagagttca ccaatctgaa agtgtatcac gata ttaagg acatcacagc acggaaagaa atcattgaga acgccgaact gctggatcag attgctaaga tcctgactat ctaccagagc tccgaggaca tccaggaaga gctgactaac ctgaacagcg agctgaccca ggaagagatc gaacagatta gtaatctgaa ggggtacacc ggaacacaca acctgtccct gaaagctatc aatctgat tc tggatgagct gtggcataca aacgacaatc agattgcaat cr ttaaccgg ctgaagctgg tcccaaaaaa ggtggacctg agt cagcaga aagagatccc aaccacactg gtggacgatt tcattctgtc acccgtggtc aagcggagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaatgata tca ttatcga gctggc tagg gagaagaaca gcaaggacgc acagaagatg atcaatgaga tgcagaaacg aaaccggcag accaatgaac gcattgaaga gattatccga actaccggga aagagaacgc aaagtacctg attgaaaaaa tcaagctgca cgatatgcag gagggaaagt gtctgtattc tctggaggcc tccccctgg aggacctgct gaacaatcca ttcaactacg aggtcgatca tattatcccc agaagcgtgt ccttcgacaa ttcctttaac aacaaggtgc tggtcaagca ggaagagaac tctaaaaagg gcaataggac tccr ttccag tacctgtcta gr tcagat t c caagatctct tacgaaacct ttaaaaagca cattctgaat ctggccaaag gaaagggccg catcagcaag accaaaaagg agtacctgct ggaagagcgg gacatcaaca gattctccgt ccagaaggat tttattaacc ggaatctggt ggacacaaga tacgctactc gcggcctgat gaatctgctg cgatcatatt tccgggtgaa caatctggat gtgaaagtca agtccatcaa cggcgggttc acatcttttc tgaggcgcaa atggaagttt aaaaaggagc gcaacaaagg gtacaagcac catgccgaag atgctctgat tatcgcaaat gccgacttca tctttaagga gtggaaaaag ctggacaaag ccaagaaagt gatggagaac cagatgttcg aagagaagca ggccgaatct atgcccgaaa tcgagacaga acaggagtac aaggagattt tcatcactcc tcaccagat c aagcatatca aggatttcaa ggac tacaag tactctcacc gggtggataa aaagcccaac agagagctga tcaatgacac cctg tatagt acaagaaaag acgataaggg gaataccctg attgtgaaca atctgaacgg actgtacgac aaagataatg acaagctgaa aaagctgatc aacaaaagtc ccgagaagct gctgatgtac caccatgatc ctcagacata tcagaaactg aagctgatta tggagcagta cggcgacgag aagaacccac tgtataagta ctatgaagag actgggaact acctgaccaa gtatagcaaa aaggataatg gccccgtgat caagaagatc aagtactatg ggaacaagct gaat gcccat ctggacatca cagacgat ta ccctaacagt cgcaacaagg tggtcaagct gtcactgaag ccatacagat tcgatgtcta tctggacaac ggcg t g t a t a aatttgtg a c t g t c a ag a at c tggatgtca t c a a a a a gga g a a c t a c t a t gaagtgaata gcaagtgcta cgaagaggct aaaaagctga aaaagattag caaccaggca gagttcatcg cctcctttta caacaacgac ctgattaaga tcaatggcga actgtatagg gtcatcgggg tgaacaatga. tctgctgaac cgcattgaag tgaatatgat tgacatcact taccgagagt atctggaaaa catgaatgat aagcgccccc ctcgaattat caaaacaattgcctctaaga ctcagagtat caaaaagtac tcaaccgaca ttctgggaaa cctgtatgag gtgaagagca aaaagcaccc tcagattatc aaaaagggcSEQ !D NO: 34 codon optimized nucleic acid sequences encoding S. aureus Cas9 atgaagcgga actacatcct gggcctggac atcggcatca ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag agaggcgcca gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact acaacctgct gaccgaccac agcgagctga gcggcatcaa cccc tacgag gccagagtga agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg gccgaactgc agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg accagagctt catcgacacc tacat cgacc tgctggaaac ccggcggacc tactatgagg gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga tgggccactg caccracttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt aagggctaca gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg agctgaccca ggaagagat c gagcagat ct ctaatctgaa gggctatacc ggcacccaca acctgagcct gaaggccat c aacctgatcc tggacgagct gtggcacacc aacgacaacc agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct tca tccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg accaccggca aagagaacgc caagtacctg atcgagaaga t caagctgca cgacatgcag gaaggcaagt gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc tcgtgaagca ggaagaaaac agcaagaagg gcaaccggac cccattccag tacctgagca gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga agtccatcaa tggcggcttc accagctt tc tgcggcggaa gtggaagttt aagaaagagc ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgatttca tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgt tcg aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagaga tct tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc gggtggacaa gaagcctaat agagagctga ttaacgacac cctg tactcc acccggaagg acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc cccagaccta ccagaaactg aagcrgatta tggaacagta cggcgacgag aagaatcccc tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctacagat tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct aagaagctga agaagatcag caaccaggcc gagtttatcg cctccttcta caacaacgat ctgatcaaga tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac cggatcgaag tgaacatgat cgacatcacctaccgcgag t acctggaaaa catgaacgac aagaggcccc c cagg a L c a t taagacaatc gcctccaaga cccagagcat taagaagtac agcacagaca ttctgggcaa cctguatgaa gtgaaatcta agaagcaccc tcagatcatc aaaaagggcSEQ !D NO: 35 codon optimized nucieic acid sequence encoding S. aureus Cas9 atgaagcgca actacatcct cggactggac atcggcatta cctccgtggg atacggcatc atcgattacg aaactaggga tgtgatcgac gctggagtca ggctgt tcaa agaggcgaac gtggagaaca acgaggggcg gcgctcaaag aggggggccc gccggctgaa gcgccgccgc agacatagaa tccagcgcgt gaagaagctg ctgttcgact acaaccttct gaccgaccac tccgaacttt ccggcatcaa ccca tatgag gctagagtga agggattgtc ccaaaagctg tccgaggaag agttctccgc cgcgttgctc cacctcgcca agcgcagggg agtgcacaat gtgaacgaag tggaagaaga taccggaaac gagctgtcca ccaaggagca gatcagccgg aactccaagg ccctggaaga gaaatacgtg gcggaactgc aactggagcg gctgaagaaa gacggagaag tgcgcggctc gatcaaccgc ttcaagacct cggactacgt gaaggaggcc aagcagctcc tgaaagtgca aaaggcctat caccaacttg accagtcct t tatcgatacc tacat cgatc tgctcgagac tcggcggact tactacgagg gt ccagggga gggct cccca tttggttgga aggatattaa ggagtggtac gaaatgctga tgggacactg cacatacttc cctgaggagc tgcggagcgt gaaatacgca tacaaogcag acctgtacaa cgcgctgaac gacctgaaca atctcgtgat cacccgggac gagaacgaaa agctcgagta ttacgaaaag ttccagatta ttgagaacgt gttcaaacag aagaagaagc cgacactgaa gcagattgcc aaggaaatcc tcgtgaacga agaggacatc aagggctatc gagtgacctc aacgggaaag ccggagttca ccaatctgaa ggtctaccac gacatcaaag acattaccgc ccggaaggag atcattgaga acgcggagct gttggaccag attgcgaaga ttctgaccat ctaccaatcc t ccgaggata ttcaggaaga actcaccaac ctcaacagcg aactgaccca ggaggagata gagcaaatct ccaacctgaa gggc tacacc ggaactcata acctgagcct gaaggccatc aacttgatcc tggacgagct gtggcacacc aacgataacc agatcgctat tttcaatcgg ctgaagctgg tccccaagaa agtggacctc tcacaacaaa aggagatccc tactaccctt gtggacgatt tcattctg tc ccccgtggtc aagagaagct tcatacagtc aatcaaagtg atcaatgcca ttatcaagaa atacggtctg cccaacgaca ttatcattga gctcgcccgc gagaagaact cgaaggacgc ccagaagatg attaacgaaa tgcagaagag gaaccgacag actaacgaac ggatcgaaga aatcatccgg accaccggga aggaaaacgc gaagtacctg atcgaaaaga tcaagctcca tgacatgcag gaaggaaagt gr ctgtact c gctggaggcc attccgctgg aggacttgct gaacaaccct tttaactacg aagtggatca tatcattccg aggagcgtg t; cattcgacaa t tccttcaac aacaaggtcc tcgtgaagca ggaggaaaac tcgaagaagg gaaaccgcac gccgttccag tacctgagca gcagcgactc caagatttcc tacgaaacct tcaagaagca catcctcaac ctggcaaagg ggaagggtcg catctccaag accaagaagg aatatctgct ggaagaaaga gacatcaaca gattctccgt gcaaaaggac ttcatcaacc gcaacctcgt ggatactaga tacgctactc ggggtctgat gaacctcctg agaagctact ttagagtgaa caatctggac gtgaaggtca agtcgattaa cggaggttt c acctcctt cc tgcggcgcaa gtggaagttc aagaaggaac ggaacaaggg ctacaagcac cacgccgagg acgccctgat cattgccaac gccgact tca tct Lcaaaga atggaagaaa cttgacaagg ctaagaaggt catggaaaac cagatgt tcg aagaaaagca ggccgagtct atgcctgaaa tcgagactga acaggagtac aaggaaatct tta ttacgcc acaccagatc aaacacatca aggatttcaa ggat tacaag tactcacatc gcgtggacaa aaagccgaac agggaactga tcaacgacac cctctactcc acccggaagg atgacaaagg gaataccctc atcgtcaaca accttaacgg cctgtacgac aaggacaacg ataagctgaa gaagctcatt aacaagtcgc ccgaaaagtt gctgatgtac caccacgacc ct cagact ta ccagaagctc aagctgatca tggagcagta tggggacgag aaaaacccgt tgtacaagta ctacgaagaa actgggaatt atctgactaa gtactccaag aaagataacg gccccgtgat taagaagatt aagtactacg gcaacaagct gaacgcccat ctggacatca ccgatgacta ccctaattcc cgcaacaagg tcgtcaagct gagcctcaag ccctaccggt ttgatgtgta ccttgacaat ggagtgta.ca agttcgtgac tgtgaagaac cttgacgtga tcaagaagga gaactactac gaagtcaact ccaagtgcta cgaggaagca aagaagttga agaagatctc gaaccaggcc gagttcat tg cctccttcta taacaacgac ctgattaaga tcaacggcga actgtaccgcgtcattggog tgaacaacga tctcctgaac egea togaag tgaacatgat cgacatcact taccgggaat acctggagaa tatgaacgac aagcgcccgc cecggatcat taagactatc gccteaaaga cccagtcgat caagaagtac agcaccgaca tcctgggcaa cctgtacgag gtcaaatcga agaagcaccc ccagat catc aagaagggaSEQ !D NO: 36 codon optimized nucleic acid sequence encoding S. aureus Cas9 atggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagccaagcggaactacatcct gggcctggacatcggcatcaccagcgtgggctacggcatcat cgactacgagacacgggacgtgatcg atgccggegtgcggctgttcaaagaggceaacg tggaaaacaaagagggeaggcggagcaagagaggc gccagaaggctgaagcggcggaggcggoatagaa tccagagagtgaagaagctgctgttcgactacaa cctgctgaccgaccacagcgagctgagcggcatcaacccctacgaggccagagtgaagggcctgagcc agaagctgagcgaggaagagttctctgccgccctgctgcacctggccaagagaagaggcgtgcacaac gtgaacgaggtggaagaggacaccggcaacgagctgtccaccagagagcagatcagccggaacagcaa ggccctggaagagaaatacgtggccgaactgcagctggaacggctgaagaaagacggcgaagtgcggg gcagcatcaacagattcaagaccagcgactacgtgaaagaagccaaacagctgctgaaggtgcagaag gcctaccaccagctggaccagagct t catcgacacctacatcgacctgctggaaacccggcggaccta eta tgagggaoctggcgagggcagocccttoggctggaaggaca teaaagaa tggtacgagatgc tga tgggccactgcacctacttccccgaggaactgcggagcgtgaag tacgcctacaacgccgacctg tac aaogcectgaacgacctgaacaa totcgtgateaccagggacgagaacgagaagctggaatattaoga gaagtt ccagat eat cgagaacgtgttcaagoagaagaagaagcocaccctgaagcagategccaaag aaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccggcaagcccgagttcacc aacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagattattgagaacgccgagct gctggatcagattgccaagatcctgaccatctaccagagcagcgaggacatccaggaagaactgacca atctgaactccgagctgacccaggaagagatcgagcagatctctaatctgaagggctataccggcacc eacaacotgagcc tgaaggooatcaaootgatcotggacgagctgtggoacaccaacgacaaoeagat egctatcttcaaccggctgaagctggtgcccaagaaggtggacctgtcceagcagaaagaga teccca ccaccctggtggaogacttoatcctgagccccgtogtgaagagaagcttcatccagagcatoaaagtg atcaacgccatca tcaagaagtacggootgcccaacgaca toattatogagctggocegcgagaagaa ctccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagaccaacgagcggatcg aggaaatcatccggaccaccggcaaagagaacgccaagtacctgatcgagaagatcaagctgcacgac atgcaggaaggcaagtgcctgtacagcctggaagccatccctctggaagatctgctgaacaacccctt caactatgaggtggaccacatcatccccagaagcgtgtccttcgacaacagcttcaacaacaaggtgc tcgtgaagcaggaagaaaacagcaagaagggcaaccggacccca t tccagtaectgagcagcagcgac agcaagatcagetacgaaaccttcaagaagcacatcc tgaatctggccaagggcaagggeagaatoag caagaccaagaaagagtatctgctggaagaacgggacatcaacaggttctccgtgcagaaagacttca tcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacctgctgcggagctacttc agagtgaacaacctggacgtgaaagtgaagtccatcaatggcggcttcaccagctttctgcggcggaa gtggaagtttaagaaagagcggaacaaggggtacaagcaccacgccgaggacgccctgatcattgcca acgccgatttcatcttcaaagagtggaagaaactggacaaggccaaaaaagtgatggaaaaccagatg ttcgaggaaaggcaggccgagagcatgcccgagatcgaaaccgagcaggagtacaaagagatcttcat caccccocaccagatcaagoaeattaaggacttoaaggac taeaagtaoagccaoogggtggaeaaga agcctaatagagagetgat taacgacacectgtactccacccggaaggacgacaagggcaacaecctg atcgtgaacaatctgaacggcctgtacgacaaggacaatgacaagctgaaaaagctgatcaacaagag ccccgaaaagctgotgatg taccaccaogacccooagacctaccagaaactgaagotgattatggaac agtacggcgacgagaagaatcccctgtacaagtactacgaggaaaccgggaactacctgaccaagtac tccaaaaaggacaacggccccgtgatcaagaagattaagtattacggcaacaaactgaacgcccatct ggacatcaccgacgactaccccaacagcagaaacaaggtcgtgaagctgtccctgaagccctacagat tcgacgtgtaoctggaoaatggcgtgtacaagttcgtgaccgtgaagaatc tggatgtgatcaaaaaa gaaaactac taegaag tgaatagcaagtgc tatgaggaagetaagaagetgaagaaga teagcaacea ggoogagtttatcgcctocttctaoaacaaogatctgatcaaga tcaacggogagctgtatagagtga tcggcgtgaaoaacgaootgctgaaccgga togaagtgaacatgatcgaca tcacctaocgcgagtac ctggaaaacatgaacgacaagaggccccccaggatcattaagacaatcgcctccaagacccagagcat taagaagtacagcacagacattctgggcaacctgtatgaagtgaaatctaagaagcaccctcagatca tcaaaaagggcaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagSEQ ID NO: 37 codon optimized nucleic acid sequence encoding S. aureus Cas9 accggtgcca ccatgtaccc a tacgatgtt ccagattacg cttcgccgaa Q 33333Gf Cq O aaggtcgaag cgtccatgaa aaggaactac attctggggc tggacatcgg ga t. L 3i*J3.3.q C gtggggtatg ggattattga ctatgaaaca agggacgtga tcgacgcagg cgtcagactg ttcaaggagg ccaacgtgga aaacaatgag ggacggagaa gcaagagggg agccaggcgc ctgaaacgac ggagaaggca cagaatccag agggtgaaga aactgctgtt cgattacaac ctgctgaccg accattctga gctgagt gga at t aatcct t atgaagccag ggtgaaaggc ctgagtcaga agctgtcaga ggaagagttt tccgcagctc tgctgcacct ggctaagogc cgaggagtgc ataaagtcaa tgaggtggaa gaggacaccg gcaacgagct gtatacaaag gaacaqacut cacgcaataQ aaaagccutg qaagagaaqc a ug togcaga gctgcagctg gaacggctga agaaagatgg cgaggtgaga gggtcaa tta ataggttcaa Q H C H 3 Cj C Q H C tacgtcaaag aagccaagca gctgctgaaa gtgcagaagg cttaccacca gctggatcag agcttcatcg atacttatat cgacctgctg gagactcgga gaacctacta tgagggacca ggagaaggga gcccct tcgg atggaaagac atcaaggaat ggtacgagat gctgatggga cat tgcacct attttccaga agagctgaga agcgt caagt acgcttat aa cgcagatct tacaaagccc tgaatgacct gaacaacc tg gtca tcacca gggatgaaaa cgagaaactg g a at a c t a t g a g a a g 11 c c a ga t: c a t c ga a a a eg t g 111 a a g c ag a a g a a aaagcctaca ctgaaacaga ttgetaagga gatcctggtc aacgaagagg acatcaaggg ctaccgggtg acaagcactg gaaaaccaga gttcaccaat etgaaagtgt atcacga tat taaggacatc acagcacgga aagaaatcat tgagaaegee gaaetgetgg atcagattgc taagatcctg actatctacc agagctccga ggacatccag gaagagetga ctaacctgaa cagcgagctg acccaggaag agat cgaaca gattagt aat ctgaaggggt acaccggaac acacaacct g t ccctgaaag ctatcaatct gatt etggat gagctgtggc atacaaacga caatcagat t gaaate ttta accggctgaa gctggtccca aaaaaggtgg acc tgagtca gcagaaagag a 1: e c c a a c c a c a c t gg t g ga c ga 111 c a 11 c t g t c a c c eg t gg t c a age g gagcttcata cagagcatca aagtgatcaa cgccatcatc aagaagtacg gcctgcccaa tgatatcatt ategagetgg ctagggagaa gaacagcaag gacgcacaga aga tgatcaa tgagatgcag aaacgaaacc ggcagaccaa tgaacgcatt gaagagatta t ccgaactac cgggaaagag aacgcaaagt acctgattga aaaaatcaag etgeaegata tgcaggaggg aaagtgtctg tat tetetgg aggccatccc cctggaggac ctgctgaaca ar ccattcaa ctacgaggtc gat catatta t ccccagaag cgtgtcctt c gacaattcct t t aacaacaa ggtgctggtc aagcaggaag agaactctaa aaagggcaa t: aggactcctt tccagtacct gtctagttca gattccaaga tetettaega aacctttaaa aagcacattc tgaatctgge caaaggaaag ggccgcatca gcaagaccaa aaaggagtac etgetggaag agcgggacat caacagattc tccgtccaga aggattttat taaccggaat ctggtggaca caagatacgc tactcgcggc etgatgaate tgetgegate ctatttccgg gtgaacaatc tggatgtgaa agtcaagtcc atcaacggcg ggttcacatc ttttctgagg egeaaatgga agtttaaaaa ggagcgcaac aaagggtaca agcaccatgc cgaagat gct etgattat eg caaatgccga ct tcatctt t. aaggagtgga aaaagctgga caaagccaag aaagtgatgg agaaccagat gt tcgaagag aagcaggaag aa tetatgee egaaategag acagaacagg agtaeaagga gattttcata actcctcacc agataaagca tatcaaggat ttcaaggact acaagtactc tcaccgggtg gataaaaagc ccaacagaga gctgatcaat gacaccctgt atagtacaag aaaagacgat aaggggaata cectgattgt gaacaatctg aacggactgt acgacaaaga taatgacaag etgaaaaage tgatcaacaa aagtcccgag aagctgctga tgtaccacca tgat cctcag acatatcaga aaetgaaget gattatggag cagtacggcg aegagaagaa cccactgtat aagtactatg aagagact gg gaactacctg accaagtata gcaaaaagga taatggcccc gtgatcaaga agatcaagta c tatgggaac aagc tgaatg ccaatctgga catcacagac ga ttacccta aaagtcgcaa caaggtgg to aagc tgtcac tgaagcca ta caga ttcgat gtetatctgg acaacggcgt gtataaattt gtgaetgtca agaatetgga tqtcatcaaa aaggagaact actatgaagt gaatagcaag tgetaegaag aggetaaaaa gctgaaaaag attagcaacc aggcagagtt catcgcctcc ttttacaaca acgacctgat taagatcaat ggcgaactgt atagggteat cggggtgaac aatgatctgc tgaaccgcat tgaagtgaat atgattgaca tcacttaccg agagtat ctg gaaaacatga atgataagcg cccccctcgacaattgcctc taagactcag agtatcaaaa agtactcaac cgacattctg atgaggtgaa gagcaaaaag caccctcaga ttatcaaaaa gggctaagaaSEQ ID NO: 38 codon optimized nucleic acid sequences encoding S. aureus Cas9 atggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagccaagcggaactacatcct gggcctggacatcggcatcaccagcgtgggctacggcatcat cgactacgagacacgggacgtgatcg atgccggcgtgcggctgttcaaagaggccaacgtggaaaacaacgagggcaggcggagcaagagaggc gccagaaggctgaagcggcggaggcggcatagaa tccagagagtgaagaagctgctgttcgactacaa cctgctgaccgaccacagcgagctgagcggcatcaacccctacgaggccagagtgaagggcctgagcc agaagctgagcgaggaagagttctctgccgccctgctgcacctggccaagagaagaggcgtgcacaac gtgaacgaggtggaagaggacaccggcaacgagctgtccaccaaagagcagatcagccggaacagcaa ggccctggaagagaaatacgtggccgaactgcagctggaacggctgaagaaagacggcgaagtgcggg gcagcatcaacagattcaagaccagcgactacgtgaaagaagccaaacagctgctgaaggtgcagaag gcctaccaccagctggaccagagct r catcgacacctacatcgacctgctggaaacccggcggaccta ctatgagggacctggcgagggcagccccttcggctggaaggacatcaaagaatggtacgagatgctga tgggccactgcacctacttccccgaggaactgcggagcgtgaag tacgcctacaacgccgacctg tac aacgccctgaacgacctgaacaa tctcgtga tcaccagggacgagaacgagaagctggaatattacga gaagttccagatcatcgagaacgtgttcaagcagaagaagaagcccaccctgaagcagatcgccaaag aaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccggcaagcccgagttcacc aacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagattattgagaacgccgagct gctggatcagattgccaagatcctgaccatctaccagagcagcgaggacatccaggaagaactgacca atctgaactccgagctgacccaggaagagatcgagcagatct ctaatctgaagggctataccggcacc cacaacctgagcctgaaggccatcaacctgatcctggacgagctgtggcacaccaacgacaaccagat cgctatcttcaaccggctgaagctggtgcccaagaaggtggacctgtcccagcagaaagaga tcccca ccaccc tggtggacgacttcatcctgagccccg tcgtgaagagaagct tcatccagagcatcaaagtg atcaacgccatca tcaagaagtacggcctgcccaacgaca tcattatcgagctggcccgcgagaagaa ctccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagaccaacgagcggatcg aggaaatcatccggaccaccggcaaagagaacgccaagtacctgatcgagaagatcaagctgcacgac atgcaggaaggcaagtgcctgtacagcctggaagccatccctctggaagatctgctgaacaacccctt caacratgaggtggaccacatcatccccagaagcgtgtccttcgacaacagct tcaacaacaaggtgc tcgtgaagcaggaagaaaacagcaagaagggcaaccggaccccat rccagtacctgagcagcagcgac agcaagatcagctacgaaaccttcaagaagcacatcctgaatctggccaagggcaagggcagaatcag caagaccaagaaagag t:a tctgc t;ggaagaacgggaca tcaacaggttctccgtgcagaaagact tea tcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacctgctgcggagctacttc agagtgaacaacctggacgtgaaagtgaagtccatcaatggcggcttcaccagctttctgcggcggaa gtggaagtttaagaaagagcggaacaaggggtacaagcaccacgccgaggacgccctgatcattgcca acgccgatttcatcttcaaagagtggaagaaactggacaaggccaaaaaagtgatggaaaaccagatg ttcgaggaaaagcaggccgagagcatgcccgagatcgaaaccgagcaggagtacaaagagatcttcat caccccccaccagatcaagcacattaaggacttcaaggactacaagtacagccaccgggtggacaaga agcctaatagagagctgat taacgacaccctgtactccacccggaaggacgacaagggcaacaccctg atcgtgaacaatc tgaacggcctgtacgacaaggacaatgacaagctgaaaaagctgatcaacaagag ccccgaaaagctgctgatg taccaccacgacccccagacctaccagaaactgaagctgattatggaac agtacggcgacgagaagaa tcccctg tacaagtactacgaggaaaccgggaactacctgaccaagtac tccaaaaaggacaacggccccgtgatcaagaagattaagtattacggcaacaaactgaacgcccatct ggacatcaccgacgactaccccaacagcagaaacaaggtcgtgaagctgtccctgaagccctacagat tcgacgtgtacctggacaatggcgtgtacaagttcgtgaccgtgaagaatctggatgtgatcaaaaaa gaaaactac taegaag tgaatagcaagtgc tatgaggaagetaagaagetgaagaaga tcagcaacca ggccgagtt tatcgcctccttctacaacaacgatctga tcaaga teaaegg egage tgtatagag tga tcggcgtgaacaacgacctgctgaaccgga tcgaagtgaacatgatcgaca tcacctaccgcgagtac ctggaaaacatgaacgacaagaggccccccaggatca ttaagacaatcgcctccaagacccagagcat taagaagtacagcacagacattctgggcaacctgtatgaagtgaaatctaagaagcaccctcagatca tcaaaaagggcaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagSEQ ID NO: 39 codon optimized nucleic acid sequences encoding S. aureus Cas9 aagcggaactacatcctgggcctggacatcggcatcaccagcgtgggctacggcatca tcgactacga gacacgggacgtgatcgatgccggcgtgcggctgttcaaagaggccaacgtggaaaacaacgagggca ggcggagcaagagaggcgccagaaggctgaagcggcggaggcggcatagaatccagagagtgaagaag ctgctgttcgactacaacctgctgaccgaccacagcgagctgagcggcatcaacccctacgaggccag a.gtgaagggcctgagccagaagctgagcgaggaaga.gttct ctgccgccctgctgca.cc tggccaaga gaagaggcgtgcacaacgtgaacgaggtggaagaggacaccggcaacgagctgtccaccaaagagcag atcagccggaacagcaaggccctggaagagaaa tacgtggccgaactgcagctggaacggctgaagaa agacggcgaagtgcggggcagcatcaacagattcaagaccagcgactacgtgaaagaagccaaacagc tgctgaaggtgcagaaggcctaccaccagctggaccagagcttcatcgacacctacatcgacctgctg gaaacccggcggacctactatgagggacctggcgagggcagccccttcggctggaaggaca tcaaaga atggtacgagatgctgatgggccactgcacctacttccccgaggaactgcggagcgtgaagtacgcct acaacgccgacctgtacaacgccctgaacgacctgaacaatctcgtgatcaccagggacgagaacgag aagctggaatattacgagaagttccagatcatcgagaacgtgttcaagcagaagaagaagcccaccct gaagcagatcgccaaagaaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccg gcaagcccgagttcaccaacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagatt at tgagaacgccgagctgctgga tcagattgccaaga tcctgaccatctaccagagcagcgaggacat ccaggaagaactgaccaatctgaactccgagctgacccaggaagagatcgagcagatctctaatctga agggctataccggcacccacaacctgagcctgaaggccatcaacctgatcctggacgagctgtggcac accaacgacaaccagatcgctatcttcaaccggctgaagctggtgcccaagaaggtggacctgtccca gcagaaagagatccccaccaccctggtggacgact teat cctgagccccgtcgtgaagagaagct tea tccagagcatcaaagtgatcaacgccatcatcaagaagtacggcctgcccaacgacatcattatcgag ctggcccgcgagaagaactccaaggacgcccagaaaatga tcaacgagatgcagaagcggaaccggca gaccaacgagcggatcgaggaaatca tccggaccaccggcaaagagaacgccaagtacctga tcgaga agatcaagctgcacgacatgcaggaaggcaagtgcctgtacagcctggaagcca tccctctggaagat ctgctgaacaaccccttcaactatgaggtggaccacatcatccccagaagcgtgtccttcgacaacag cttcaacaacaaggtgctcgtgaagcaggaagaaaacagcaagaagggcaaccggaccccattccagt acctgagcagcagcgacagcaagatcagctacgaaaccttcaagaagcacatcctgaatctggccaag ggcaagggcagaatcagcaagaccaagaaagagtatctgctggaagaacgggacatcaacaggttctc cgtgcagaaagacttcatcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacc tgctgcggagctacttcagagtgaacaacc tggacgtgaaagtgaagtcca tcaatggcggcttcacc agctttctgcggcggaagtggaagtttaagaaagagcggaacaaggggtacaagcaccacgccgagga cgccctgatcattgccaacgccgatttcatcttcaaagagtggaagaaactggacaaggccaaaaaag tgatggaaaaccagatgttcgaggaaaagcaggccgagagcatgcccgaga tcgaaaccgagcaggag tacaaagagatcttcatcaccccccaccagatcaagcacattaaggacttcaaggactacaagtacag ccaccgggtggacaagaagcctaatagagagctgattaacgacaccctgtactccacccggaaggacg acaagggcaacaccctgatcgt.gaacaat.ctgaacggcctgtacgacaaggacaatgacaagct.gaaa aagctgatcaacaagagccccgaaaagctgctgatgtaccaccacgacccccagacctaccagaaact gaagctgattatggaacag tacggcgacgagaagaatcccctgtacaagtactacgaggaaaccggga actacc tgaccaagtactccaaaaaggacaacggccccgtgatcaagaagattaagtattacggcaac aaactgaacgcccatctggacatcaccgacgactaccccaacagcagaaacaaggtcgtgaagctgtc cctgaagccctacagattcgacgtgtacctggacaatggcgtgtacaagttcgtgaccgtgaagaatc tggatgtgatcaaaaaagaaaactactacgaagtgaatagcaagtgctatgaggaagctaagaagctg aagaagatcagcaaccaggccgagtttatcgcctccttctacaacaacgatctgatcaagatcaacgg cgagctgtatagagtgatcggcgtgaacaacgacctgctgaaccggatcgaagtgaacatgatcgaca tcacctaccgcgagtacct ggaaaacatgaacgacaagaggccccccaggat cattaagacaatcgcc tccaagacccagagca t taagaagtacagcacagaca t tctgggcaacctg tatgaag tgaaatc taa g a agcaccctcag a t c a t c a a a a agggcSEQ ID NO: 40Vector (pDO242) encoding codon optimized nucleic acid sequence encoding S. aureusCas9 ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcatttt tta accaataggccgaaatcggcaaaa tccctta taaatcaaaagaa tagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgt ctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgta aagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtg gcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgct gcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccatt cgccatt caggc tgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagc tggcgaaaggggga tgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgt tgtaaaacgaeggc cagtgagcgcgcgtaatacgactcac tatagggcgaattgggtacCtttaattctagtactatgcaTg cgttgacattgattattgactagtta ttaatagtaatcaa ttacggggtcattagttcatagcccata tatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcc cattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgg gtggagtatttacggtaaactgcccacttggcagtacat caagtgtatcatatgccaagtacgccccc tattgacgtcaatgacggtaaatggcccgcct ggcattatgcccagtacatgaccttatgggactt tc ctacttggcagtacatctacgtat tagtea t eget at taccatggtgatgcggttttggcagtacatc aa tgggcgtggatagcggtttgactcacggggatttccaagtctccacccca ttgacgtcaatgggag tttgttttggcaccaaaatcaacgggactttccaaaa tgtcgtaacaactccgcccca ttgacgcaaa tgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactaccggtgccacc ATGAAAAGGAACTACATTCTGGGGCTGGACATCGGGATTACAAGCGTGGGGTATGGGATTATTGACTA TGJVtACAAGGGACGTGA'I'CGACGCAGGCG'j?CAGACTG'r'I'C'-\AGGAGGCC?iACGTGG?iAJGAC'-\ATGAGG GACGGAGAAGCAAGAGGGGAGCCAGGCGCCTGAAACGACGGAGAAGGCACAGAATCCAGAGGGTGAAG AAACTGCTGTTCGATTACAACCTGCTGACCGACCATTCTGAGCTGAGTGGAATTAATCCTTATGAAGC CAGGGTGAAAGGCCTGAGTCAGAAGCTGTCAGAGGAAGAGTTTTCCGCAGCTCTGCTGCACCTGGCTA AGCGCCGAGGAGTGCATAACGTCAATGAGGTGGAAGAGGACACCGGCAACGAGCTGTCTACAAAGGAA CAGATCTCACGCAATAGCAAAGCTCTGGAAGAGAAGTATGTCGCAGAGCTGCAGCTGGAACGGCTGAA GAAAGATGGCGAGGTGAGAGGGTCAATTAATAGGTTCAAGACAAGCGACTACGTCAAAGAAGCCAAGC AGCTGCTGAAAGTGCAGAAGGCTTACCACCAGCTGGATCAGAGCTTCATCGATACTTATATCGACCTG CTGGAGACTCGGAGAACCTACTATGAGGGACCAGGAGAAGGGAGCCCCTTCGGATGGAAAGACATCAA GGAATGGTACGAGATGCTGATGGGACATTGCACCTATTTTCCAGAAGAGCTGAGAAGCGTCAAGTACG CTTATAACGCAGATCTGTACAACGCCCTGAATGACCTGAACAACCTGGTCATCACCAGGGATGAAAAC GAGAAACTGGAATACTATGAGAAGTTCCAGATCATCGAAAACGTGTTTAAGCAGAAGAAAAAGCCTAC ACTGAAACAGATTGCTAAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTACCGGGTGACAAGCA CTGGAAAACCAGAGTTCACCAATCTGAAAGTGTATCACGATATTAAGGACATCACAGCACGGAAAGAzA ATCATTGAGAACGCCGAACTGCTGGATCAGATTGCTAAGATCCTGACTATCTACCAGAGCTCCGAGGA CATCCAGGAAGAGCTGACTAACCTGAACAGCGAGCTGACCCAGGAAGAGATCGAACAGATTAGTAATC TGAAGGGGTACACCGGAACACACAACCTGTCCCTGAAAGCTATCAATCTGATTCTGGATGAGCTGTGG CATACAAACGACAATCAGATTGCAATCTTTAACCGGCTGAAGCTGGTCCCAAAAAAGGTGGACCTGAG TCAGCAGAAAGAGATCCCAACCACACTGGTGGACGATTTCATTCTGTCACCCGTGGTCAAGCGGAGCT TCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAATGATATCATTATC GAGCTGGCTAGGGAGAAGAACAGCAAGGACGCACAGAAGATGATCAATGAGATGCAGAAACGAAACCG GCAGACCAATGAACGCATTGAAGAGATTATCCGAACTACCGGGAAAGAGAACGCAAAGTACCTGATTG AAAAAATCAAGCTGCACGATATGCAGGAGGGAAAGTGTCTGTATTCTCTGGAGGCCATCCCCCTGGAG GACCTGCTGAACAATCCATTCAACTACGAGGTCGATCATATTATCCCCAGAAGCGTGTCCTTCGACAA TTCCTTTAACAACAAGGTGCTGGTCAAGCAGGAAGAGAACTCTAAAAAGGGCAATAGGACTCCTTTCC AGTACCTGTCTAGTTCAGATTCCAAGATCTCTTACGAAACCTTTAAAAAGCACATTCTGAATCTGGCC AAAGGAAAGGGCCGCATCAGCAAGACCAAAAAGGAGTACCTGCTGGAAGAGCGGGACATCAACAGATT CTCCGTCC^GAAGGATTTTATTAACCGGAATCTGGTGGACACAAGATACGCTACTCGCGGCCTGATGA ATCTGCTGCGATCCTATTTCCGGGTGAACAATCTGGATGTGAAAGTCAAGTCCATCAACGGCGGGTTC ACATCTTTTCTGAGGCGCAAATGGAAGTTTAAAAAGGAGCGCAACAAAGGGTACAAGCACCATGCCGA AGATGCTCTGATTATCGCAAATGCCGACTTCATCTTTAAGGAGTGGAAAAAGCTGGACAAAGCCAAGA AAGTGATGGAGAACCAGATGTTCGAJiGAGAAGCAGGCCGAaVrCTATGCCCGAAATCGAGACAGAACAG GAGTACAAGGAGATTTTCATCACTCCTCACCAGATCAAGCATATCAAGGATTTCAAGGACTACAAGTA CTCTCACCGGGTGGATAAAAAGCCCAACAGAGAGCTGATCAATGACACCCTGTATAGTACAAGAAAAG ACGATAAGGGGAATACCCTGATTGTGAACAATCTGAACGGACTGTACGACAAAGATAATGACAAGCTG AAAAAGCTGATCAACAAAAGTCCCGAGAAGCTGCTGATGTACCACCATGATCCTCAGACATATCAGAA ACTGAAGCTGATTATGGAGCAGTACGGCGACGAGAAGAAGCCACTGTATAAGTACTATGAAGAGACTG GGAACTACCTGACCAAGTATAGCAAAAAGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTATGGGHDCPVCLPLKNAGDKRNQQPILTGAPVGLGNPSSLGVGQQSAPNLSTVSQIDPSSIERAYAALGLPYQ VNQMPTQPQVQAKNQQNQQPGQSPQGMRPMSNMSASPMGVNGGVGVQTPSLLSDSMLHSAINSQNPMM SENASVPSMGPMPTAAQPSTTGIRKQWHEDITQDLRNHLVHKLVQAIFPTPDPAALKDRRMENLVAYA RKVEGDMYESANNRAEYYHLLAEKIYKIQKELEEKRRTRLQKQNMLPNAAGMVPVSMNPGPNMGQPQp GMTSNGPLPDPSMIRGSVPNQMMPRITPQSGLNQFGQMSMAQPPIVPRQTPPLQHHGQLAQPGALNPP MGYGPRMQQPSNQGQFLPQTQFPSQGMNVTNIPLAPSSGQAPVSQAQMSSSSCPVNSPIMPPGSQGSH IHCPQLPQPALHQNSPSPVPSRTPTPHHTPPSIGAQQPPATTIPAPVPTPPAMPPGPQSQALHPPPRQ TPTPPTTQLPQQVQPSLPAAPSADQPQQQPRSQQSTAASVPTPTAPLLPPQPATPLSQPAVSIEGQVS NPPSTSSTEVNSQAIAEKQPSQEVKMEAKMEVDQPEPADTQPEDISESKVEDCKMESTETEERSTELK TEIKEEEDQPSTSATQSSPAPGQSKKKIFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPD YFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPV MQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQT TINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEI IWPAGFVCDGCLKKSARTRKENKFSAKR LPSTRLGTFLENRVNDFLRRQNHPESGEVTVRWHASDKTVEVKPGMKARFVDSGEMAESFPYRTKAL FAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKL GYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERrVHDYKDrFKQATEDRLT SAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLS RGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLT IARDKHLEFSSLRRAQWSTMCMLVELHTQSQDRFVYTCNECKHHVETRWHCTVCEDYDLCITCYNTKN HDHKMEKLGLGLDDESNNQQAAATQSPGDSRRLSIQRCIQSLVHACQCRNANCSLPSCQKMKRWQHT KGCKRKTNGGCPICKQLIALCCYHAKHCQENKCPVPFCLNIl«2KLRQQQLQHRLQQAQMLRRRMASMQ RTGWGQQQGLPSPTPATPTTPTGQQPTTPQTPQPTSQPQPTPPNSMPPYLPRTQAAGPVSQGKAAGQ VTPPTPPQTAQPPLPGPPPAAVEMAMQIQRAAETQRQMAHVQIFQRPIQHQMPPMTPMAPMGMNPPPM TRGPSGHLEPGMGPTGMQQQPPWSQGGLPQPQQLQSGMPRPAMMSVAQHGQPLNMAPQPGLGQVGISP LKPGTVSQQALQNLLRTLRSPSSPLQQQQVLSrLHANPQLLAAFIKQRAAKYANSNPQPIPGQPGMPQ GQPGLQPPTMPGQQGVHSNPAMQNMNPMQAGVQRAGLPQQQPQQQLQPPMGGMSPQAQQMNMNHNTMP SQFRDILRRQQMMQQQQQQGAGPGIGPGMANHNQFQQPQGVGYPPQQQQRMQHHMQQMQQGNMGQIGQ LPQALGAEAGASLQAYQQRLLQQQMGSPVQPNPMSPQQHMLPNQAQSPHLQGQQIPNSLSNQVRSPQP VPSPRPQSQPPHSSPSPRMQPQPSPHHVSPQTSSPHPGLVAAQANPMEQGHFASPDQNSMLSQLASNP GMANLHGASATDLGLSTDNSDLNSNLSQSTLDIHSEQ ID NO: 42Human p300 Core Effector protein (aa 1048-1664 of SEQ ID NO: 41)IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPW QYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLC TIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECG RKM-IQICVLHHEI IWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESG EVTVRVVHASDKTVEVKPGMKARFVPSGEMAESFPYRTKALFAFEEIDGVRLCFFGMHVQEYGSRCPP PNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQ KIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQE EEERKREENTSNESTPVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKH KEVFFVIRLIAGPAANSLPPIVEPDPLIPCDLMDGREAFLTLARDKHLEFSSLRRAQWSTMCI'4LVELH TQSQE)SEQ ID NO: 43VP64-dCas9-VP64 proteinRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMVNPKKKRKVGRGMDKKY SIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT RRKNRrCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK LVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAK AILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQ IHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV VrjKGASAQSFIERMTNFDKNLPNEKVLPK.HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHOLLKI IKOKDFLDNEENEDILE DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGESLHEHIANLAGSPAIKKGILQTVKWEELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRD MYVDQELDINRLSDYEVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNA KLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQE IGKATAKYFFYSNIMNFFKTE ITLANGE I RKRPL IETNGETGE I VWDKGREFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVOTLYI^HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGDSRADPKKKRKVASRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML ISEQ ID NO: 44VP64-dCas9-VP64 DNA cgggctgacgcattggacgattt tgatctggatatgctgggaagtgacgccotcgatga ttttgacct t ga c a t g c 11 gg 11 cgga t gc c c 11 g a t g a c 111 g a c c t c ga catgctcgg c a g t g a c g c c c 11 g a t g atttcgacctggacatggttaaccccaagaagaagaggaaggtgggccgcggaatggacaagaagtac tccattgggctcgcca tcggcacaaacagcgtcggctgggccgtcattacggacgagtacaaggtgcc gagcaaaaaattcaaagttctgggcaataccgatcgccacagcataaagaagaacctcattggcgccc tcctgttcgactccggggaaaccgccgaagccacgcggctcaaaagaacagcacggcgcagatatacc cgcagaaagaatcggatctgctacctgcaggagat ctttagtaatgagatggctaaggtggatgactc r ttcttccataggctggaggagtccttt .ttggtggaggaggataaaaagcacgagcgccacccaatct tit. gg c a a t a t eg t gga c g a gg t ggcg t a c c a t g a a a ag t a c c c a a c c at a t a t c a t c t g a g ga a g a a g cttgtagacagtactgataaggctgacttgcggt tgateta tctcgcgctggcgcatatgatcaaatt tcggggacacttcctcatcgagggggacctgaacccagacaacagcgatgtcgacaaactctttatcc aactggttcagacttacaa teagett ttcgaagagaacccgatcaacgcatccggagttgacgccaaa gcaatcctgagcgctaggctgtccaaatcccggcggctcgaaaacctcatcgcacagctccctgggga gaagaagaacggcctgtttggtaatcttatcgccctgtcactcgggctgacccccaactttaaatcta aett cgacctggccgaagatgccaagcttcaactgagcaaagacacctacgatgatgat ctcgacaat ctgctggcccagatcggcgaccagtacgcagaccttt t tttggcggcaaagaacctgtcagacgccat t c t gc t g a gtgatattctg c g a g t ga a c a c gg aga t cac c a a a g c t c c g c t g a g c g c t ag t a t g a t c a agegetatga tgagcaccaccaagacttgactttgctgaaggcccttgtcagacagcaactgcctgag aagtacaaggaaattttcttcga tcagtctaaaaatggctacgccggatacattgacggcggagcaag ccaggaggaattttacaaattta ttaagcccatcttggaaaaaa tggacggcaccgaggagctgctgg taaagcttaacagagaagatctgttgcgcaaacagcgcactttcgacaatggaagcatcccccaccag attcacctgggcgaactgcacgctatcctcaggcggcaagaggatttctacccctttttgaaagataa cagggaaaagattgagaaaat cctcacat ttcggataccctactatgtaggccccct cgcccggggaa attccagattcgcgtggatgactcgcaaatcagaagagaccatcactccctggaacttcgaggaagtc gtggataagggggcctctgcccagtccttcatcgaaaggatgactaactttgataaaaatctgcctaa c ga a a a gg t g c 11 c c t a a a c a c t c t c t g c t g t a egag t a c 11 c a c a g 111 a t a a egag c t cac c a ag g tcaaatacgtcacagaagggatgagaaagccagcattcctgtctggagagcagaagaaagctatcgtg gacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaagaagactatttcaaaaagat tgaatgtttcgactctgttgaaatcagcggagtggaggatcgcttcaacgcatccctgggaacgtatc acgat ctcctgaaaatcat taaagacaaggacttcctggacaatgaggagaacgaggacattcttgag gacat tgtcct cacccttacgttgt rtgaagatagggagatgattgaagaacgcttgaaaacttacgc tea tctcttcgacgacaaagtca tgaaacagctcaagaggcgccgatatacaggatgggggcggc tgt caagaaaac tgatcaa tgggatcogagacaagcagag tggaaagacaatcc tggattt tettaag tec gatggatttgccaaccggaacttcatgcagttgatccatgatgactctctcacctttaaggaggacat ccagaaagcacaagtttctggccagggggacagtcttcacgagcacatcgctaatcttgcaggtagcc cagctatcaaaaagggaatactgcagaccgttaaggtcgtggatgaactcgtcaaagtaatgggaagg cataagcccgagaatatcgttatcgagatggcccgagagaaccaaactacccagaagggacagaagaa cagtagggaaaggatgaagaggattgaagagggtataaaagaactggggtcccaaatccttaaggaacacccagttgaaaacacccagcttcagaatgagaagctctacctgtactacctgcagaacggcagggac atgtacgtggatcaggaactggacatcaatcggctctccgactacgacgtggatgccatcgtgcccca gtcttttctcaaagatgattctattgataataaagtgttgacaagatccgataaaaatagagggaaga gtgataacgtccccr cagaagaagttgt caagaaaatgaaaaattattggcggcagctgctgaacgcc aaactgat cacacaacggaagt tcgataatctgactaaggctgaacgaggtggcctgtctgagt tgga taaagccggcttcatcaaaaggcagc t: tgttgagacacgccagatcaccaagcacgtggcccaaattc tcgattcacgcatgaacaccaagtacgatgaaaa tgacaaactgattcgagagg tgaaagt tat tact ctgaagtctaagctggtctcagatttcagaaaggactttcagtttta taaggtgagagaga tcaacaa ttaccaccatgcgcatgatgcctacc tgaatgcagtggtaggcactgcacttatcaaaaaa tatccca agcttgaatctgaatttgtttacggagactataaagtgtacgatgttaggaaaatgatcgcaaagtct gagcaggaaataggcaaggccaccgctaagtacttcttttacagcaatattatgaattttttcaagac cgagattacacrggccaar ggagagattcggaagcgaccacttat cgaaacaaacggagaaacaggag aaat cgtgtgggacaagggtagggar ttcgcgacagtccggaaggr cctgtccatgccgcaggtgaac atcgttaaaaagaccgaagtacagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacag cgacaagctgatcgcacgcaaaaaagattgggaccccaagaaatacggcgga ttcgat tctcctaoag tcgcttacagtgtactggttgtggccaaagtggagaaagggaagtctaaaaaactcaaaagcgtcaag gaactgctgggcatcacaatcatggagcga tcaagcttcgaaaaaaaccccatcgactttctcgaggc gaaaggatataaagaggtcaaaaaagacctcatcattaagcttcccaagtactctctctttgagcttg aaaacggccggaaacgaatgctcgctagtgcgggcgagctgcagaaaggtaacgagctggcactgccc r ctaaatacgttaat ttcttgtatctggccagccactatgaaaagctcaaagggtct cccgaagataa tgagcagaagcagctgttcgtggaacaacacaaacactaccttgatgagatcatcgagcaaa taagcg a a 11 c t c caaaagagtgatcctcgc c gacgctaacctcgataaggtgctttctgcttacaataagca c agggataagccca tcagggagcaggcagaaaaca ttatccacttgtt tactctgaccaact tgggcgc gcctgcagccttcaagtac ttcgacaccaccatagacagaaagcggtacacctctacaaaggaggtcc tggacgccacactgattca tcagtcaattacggggctcta tgaaacaagaatcgacctctctcagctc ggtggagaGagcagggctgaccGcaagaagaagaggaaggtggGtagccgGgccgacgcgctggacga tttcgatctcgacatgctgggttctgatgccctcgatgactttgacctggatatgttgggaagcgacg cattggatgact ttgatct ggacatgctcggctccgatgctctggacgattt cgatctcgatatgt ta ateSEQ ID NO: 45Polypeptide sequence of KRAB proteinRTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVSEQ ID NO: 46Polynucleotide sequence for KRAB cggacactggtgaccttcaaggatgtgtttgtggacttcaccagggaggagtggaagctgct ggacactgctcagcagatcctgtacagaaatgtgatgctggagaactataagaacctggttt ccttgggttatcagcttactaagccagatgtgatcctccggttggagaagggagaagagccc tggctggtgSEQ ID NO: 47Polypeptide sequence of Sfreptococctis pyogenes dCas9-KRAB proteinMDYKDHIX3DYKDHDIDYKDDDDKMAPKKKRKVGRGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNFJ4AKVDDSFFHRL BESFLVEBDKKHERHPIFGNIVDEVAYHBKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFL IEGDLNPDNSDVQKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLI.AQLPGEKKNGL FGNLIAESLGLTPNFKSNFDEAIEDAKLQLSKDTYDDPLDNLEAIQIGQQYAIPLFLAAKJMLSQAILLSDILRVNTEITKAPLSASMIKRYDEHHQELTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY KFIKPIEEKMDGTEELLVKLNREDLLRKQRTFQNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSBETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNBKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKBDYFKKIBCFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRBMIEERLKTYAHLFDD KVMKQLKRRRYTGWGRLSRKLINGIRDl«2SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSIJHEHIANIJAGSPAIKKGILQTVKVVDEIJVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVEN'j?QLQNEKLYLYYLQNGRQMYVRQELDINRLSQYDVRAIVPQSFLiCDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENGKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKEajI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIGLSQLGGGSRAEPKKKRK?7ASGAKSLTAWSRTLVTFKEVFVQFTREEWKLLDTAQQILYRIYVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQ ETHPDSETAFEIKSSVPKKKRKVSEQ ID NO: 48Polynucleotide sequence encoding Streptococcus pyogenes dCas9-KRAB atggactacaaagaccatgacggtgattataaagatcat gacatcgattacaaggatgacgatgacaa gatggcccccaagaagaagaggaaggtgggccgcggaat .ggacaagaagtactccattgggctcgcca t cggc a c a a a c ag eg t egg c t g g g c c g t c a t; t a c gg a c g ag t a c a a gg t g c c gage a a a a a a 11 c a a a gt tctgggcaataccga tcgccacagcataaagaagaacctcat tggcgccctcctgt tcgactccgg ggaaaccgccgaagccacgcggctcaaaagaacagcacggcgcagatatacccgcagaaagaatcgga tctgctacctgcaggagatctttagtaatgagatggctaaggtggatgactctttcttccataggctg gaggagtcctttttggtggaggaggataaaaagcacgagcgccacccaatctttggcaatatcgtgga cgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaagaagcttgtagacagtactg ataaggctgacttgcggttgatctatct cgcgctggcgcatatgatcaaatttcggggacact tcctc atcgagggggacctgaacccagacaacagcgatgt cgacaaactctttatccaactggttcagactta caatcagcttttcgaagagaacecga teaaegea tccggagttgacgccaaagcaatcctgagcgcta ggctgtccaaatcccggcggctcgaaaacctca tcgcac age tecctggggagaagaagaa cggc etg tttggtaatcttatcgccctgtcactcgggctgacccccaactttaaatctaacttcgacctggccga agatgccaagcttcaactgagcaaagacacctacgatgatgatctcgacaatctgctggcccagatcg gcgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattctgctgagtgatatt ctgcgagtgaacacggagatcaccaaagctccgctgagcgctagtatgatcaagcgctatgatgagca ccaccaagact rgactttgctgaaggcccttgtcagacagcaactgcctgagaagtacaaggaaat r t to t; tcgatcagtctaaaaatggctacgccggatacat tgacggcggagcaagccaggaggaattt tac aaa tttattaagcccatcttggaaaaaatggacggcaccgaggagctgctggtaaagct taacagaga a ga t c t g 11 g c g c a a a c ag c g c a c 111 eg a c a a t gg a ag c a t c c c c c a c c a g a 11 c a c o t ggg c g a a c tgeaegeta tcctcaggcggcaagaggatttctacccctttttgaaagataacagggaaaagattgag aaaatcctcacatttcggataccctactatgtaggccccctcgcccggggaaattccagattcgcgtg gatgactcgcaaatcagaagagaccatcactccctggaacttcgaggaagtcgtggataagggggcct ctgcccagtccttcatcgaaaggatgactaactttgataaaaatctgcctaacgaaaaggtgcttcct aaacact ctctgctgtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacaga agggatgagaaagccagcatt cctgt ct ggagagcagaagaaagc tat cgtggacet ee t ct tcaaga cgaaccggaaagt taccgtgaaacagctcaaagaagacta t ttcaaaaagattgaatgtttcgaetct gttgaaatcagcggagtggaggatcgcttcaacgcatccc tgggaacgtatcacgatctcc tgaaaat cattaaagacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattgtcctcaccc ttacgttgtttgaagatagggagatgattgaagaacgcttgaaaacttacgctcatctcttcgacgac aaagtcatgaaacagctcaagaggcgccgatatacaggatgggggcggctgtcaagaaaactgatcaa tgggatccgagacaagcagagtggaaagacaatcctggattttct taagteegatggat ttgccaacc ggaacttcatgcagttgatccatgatgactct ctcacct ttaaggaggacat ccagaaagcacaagtt tc tggccagggggacagtcttcacgagcaca tegetaa tcttgcaggtagcccagcta tcaaaaaggg aa tactgcagaccgttaaggtcg tggatgaactcgtcaaagtaa tgggaaggcataagcccgagaata tcgttatcgagatggcccgagagaaccaaactacccagaagggacagaagaacagtagggaaaggatg aagaggattgaagagggtataaaagaactggggtcccaaatccttaaggaacacccagttgaaaacac ccagcttcagaatgagaagctctacctgtactacctgcagaacggcagggacatgtacgtggatcagg aactggacatcaatcggctctccgactacgacgtggatgccatcgtgccccagtcttttctcaaagat gattctattgataataaagtgttgacaagatccgataaaaatagagggaagagtgataacgtcccetcag aagaag L Lg ■_ c aag aaaa tga aaaa t i.a L tggegg cage tg c Lgaacg c c aaac i.g a ■_ cac a c aac ggaagttcgataatctgactaaggctgaacgaggtggcctgtctgagttggataaagccggcttcatc aaaaggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctcgattcacgcatgaa caccaagracgatgaaaatgacaaactgattcgagaggtgaaagttattactctgaagtctaagctgg tctcagatttcagaaaggact ttcagtt ttataaggtgagagagatcaacaattaccaccatgcgcat gatgcctacctgaatgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaatctgaatt tgtttacggagac tataaagtgtaega tgttaggaaaatga tcgcaaagtctgagcaggaaa taggca aggccaccgctaagtacttcttttacagcaata ttatgaa ttttttcaagaccgagattacactggcc aatggagagattcggaagcgaccacttat.cgaaacaaacggagaaacaggagaaatcgtgtgggacaa gggtagggatttcgcgacagtccggaaggtcctgtccatgccgcaggtgaacatcgttaaaaagaccg aagtacagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacagcgacaagctgatcgca cgcaaaaaagattgggaccccaagaaatacggcggattcgattct cctacagtcgcttacagtgtact ggttgtggccaaagtggagaaagggaagtctaaaaaact caaaagcgtcaaggaactgctgggcat ca caa tcatggagcgatcaagcttcgaaaaaaaccccatcgactttctcgaggcgaaagga tataaagag g t c a a a a a a ga c c t c a t c a 11 a a g c 11 c c c a a g t a c t c t c t c 111 gage 11 g a a a a c g g c c gg a a a c g aatgctcgctagtgcgggcgagctgcagaaaggtaacgagctggcactgccctctaaatacgttaatt tcttgtatctggccagccactatgaaaagctcaaagggtctcccgaagataatgagcagaagcagctg ttcgtggaacaacacaaacactaccttgatgagatcatcgagcaaataagcgaattctccaaaagagt gatcctcgccgacgctaacctcgataaggtgctttctgcttacaataagcacagggataagcccatca gggagcaggcagaaaacattatccacttgtttact ctgaccaacttgggcgcgcctgcagcct tcaag tacttcgacaccaccatagacagaaagcggtacacctct acaaaggaggt.cc tggacgccacactgat tcatcagtcaattacggggctctatgaaacaagaatcgacctctctcagctcggtggagacagcaggg ctgaccccaagaagaagaggaaggtggctagcga tgctaagtcactgactgcctggtcccggacactg gtgaccttcaaggatgtgt ttgtggacttcaccagggaggagtggaagctgctggacactgctcagca gatcctgtacagaaatgtgatgctggagaactataagaacctggtttccttgggttatcagcttacta agccagatgtgatcctccggttggagaagggagaagagccctggctggtggagagagaaattcaccaa gagacc cat cctgattcagagact goat ttgaaatcaaat cat cagttccgaaaaagaaacgcaaagt ttgaSEQ ID NO: 49Polypeptide sequence of Staphylococcus aureus dCas9-KRAB proteinMAP KKKRKVG I HGVPAAKRNY I LGL A I G I TS VGYG 11 D Y ETRD V I D AGVRL F KE ANVENNEGRRS KRG ARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY NALINDLNNLVITRDENETKLEYYEKFQI IENVFKQKKKPTLKQIAKEIIJVNEEDIKGYRVTSTGKPEFT NLKVYHDIKDITARKEIIENAELLDQIAKrLTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGT HNLSLKAINLILQELWHTNDNQIAIFNRLKLVPKKVELSQQKEIPTTLVDEFILSPVVKRSFIQS IKV INAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHD MQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSD SKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYF RVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQM FEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL IVNNLNGLYDKENDKLKKLINKSPEKLLMYHHEPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKY SKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKWKESLKPYRFEVYLENGVYKFVTVKNLDVIKK ENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNQLLNRIEVNMIDITYREY LENMNDKRPPRI IKTIASKTQSIKKYSTDIEGNEYEVKSKKHPQI IKKGKRPAATKKAGQAKKKKGSD AKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGE EPWLVERE IHQETHPDSETAFE I KSS VPKKKRKVSEQ ID NO: 50Polynucleotide sequence of Staphylococcus aureus dCas9-KRAB protein atggccccaaagaagaagcggaaggtcggtat ccacggagtcccagcagccaagcggaactacatcct gggcctggccatcggca tcaccagcgtgggctacggca tcatcgactacgagacacgggacgtga teg atgeeggeg tgcggctgttcaaagaggccaacgtggaaaacaacgagggcaggcggagcaagagaggcgccagaaggctgaagcggcggaggcggcatagaatccagagagtgaagaagctgctgttcgactacaa cctgctgaccgaccacagcgagctgagcggcatcaacccctacgaggccagagtgaagggcctgagcc agaagctgagcgaggaagagttctctgccgccctgctgcacctggccaagagaagaggcgtgcacaac gtgaacgaggtggaagaggacaccggcaacgagctgtccaccaaagagcagatcagccggaacagcaa ggccctggaagagaaatacgtggccgaactgcagctggaacggctgaagaaagacggcgaagtgcggg gaagca tcaacagattcaagaccagcgactacg tgaaagaagccaaacagctgctgaaggtgcagaag gcctaccaccagc tggaccagagcttcatcgacacctaca tcgacctgctggaaacccggcggaccta ctatgagggacctggcgagggcagccccttcggctggaaggacatcaaagaatggtacgagatgctga tgggccactgcacctacttccccgaggaactgcggagcgtgaagtacgcctacaacgccgacctgtac aacgccctgaacgacctgaacaatctcgtgatcaccagggacgagaacgagaagctggaatattacga gaagttccagatcatcgagaacgtgttcaagcagaagaagaagcccaccctgaagcagatcgccaaag aaat cctcgtgaacgaagaggatat r aagggctacagagtgaccagcaccggcaagcccgagttcacc aaccrgaaggtgtaccacgacatcaaggacat taccgcccggaaagagattar tgagaacgccgagct gctagatcagattgccaagatcctgaccatctaccagagcagcgaggacatccaggaagaactgacca atctgaactccgagctgacccaggaagaga tcgagcagatctctaatctgaagggcta taccggcacc cacaacctgagcctgaaggccatcaacctgatcctggacgagctgtggcacaccaacgacaaccagat cgctatcttcaaccggctgaagctggtgcccaagaaggtggacctgtcccagcagaaagagatcccca ccaccctggtggacgacttcatcctgagccccgtcgtgaagagaagcttcatccagagcatcaaagtg atcaacgccatcatcaagaagtacggcctgcccaacgacatcattatcgagctggcccgcgagaagaa ctccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagaccaacgagcggatcg aggaaa tcatccggaccaccggcaaagagaacgccaagtacctgatcgagaaga tcaagctgcacgac atgcaggaaggcaagtgcc tgtacagcctggaagccatccctctggaagatctgctgaacaacccctt caacta tgaggtggaccaca tcatccccagaagcgtgtcct tcgacaacagcttcaacaacaaggtgc tcgtgaagcaggaagaagccagcaagaagggcaaccggaccccattccagtacctgagcagcagcgac agcaagatcagctacgaaaccttcaagaagcacatcctgaatctggccaagggcaagggcagaatcag caagaccaagaaagagtatctgctggaagaacgggacatcaacaggttctccgtgcagaaagacttca tcaaccggaacctggtggataccagatacgccaccagaggcctgargaacctgctgcggagctact t c agagrgaacaacctggacgtgaaagr gaagtccatcaat ggcggcr tcaccagctttctgcggcggaa gtggaagtt taagaaagagcggaacaaggggtacaagcaccacgccgaggacgccctga tcattgcca acgccgatt tcatcttcaaagag tggaagaaactggacaaggccaaaaaag tgatggaaaaccagatg ttcgaggaaaagcaggccgagagcatgcccgagatcgaaaccgagcaggag tacaaagagatcttcat caccccccaccagatcaagcaca ttaaggacttcaaggactacaagtacagccaccgggtggacaaga agcctaatagagagctgattaacgacaccctgtactccacccggaaggacgacaagggcaacaccctg atcgtgaacaatctgaacggcctgtacgacaaggacaatgacaagctgaaaaagctgatcaacaagag ccccgaaaagctgcrgatgtaccaccacgacccccagacctaccagaaactgaagctgattatggaac agtacggcgacgagaagaatcccctgtacaagtacracgaggaaaccgggaactacctgaccaagtac tccaaaaaggacaacggccccgtgatcaagaaga ttaagta ttacggcaacaaactgaacgcccatct ggacatcaccgacgactaccccaacagcagaaacaaggtcgtgaagctgtccctgaagccctacagat tcgacgtgtacctggacaa tggcgtg tacaagttcgtgaccgtgaagaatctggatgtgatcaaaaaa gaaaactactacgaagtgaatagcaagtgctatgaggaagctaagaagctgaagaagatcagcaacca ggccgagtttatcgcctccttctacaacaacgatctgatcaagatcaacggcgagctgtatagagtga tcggcgtgaacaacgacctgctgaaccggatcgaagtgaacatgatcgacatcacctaccgcgagtac ctggaaaacatgaacgacaagaggccccccaggatcatt aagacaatcgcct ccaagacccagagcat taagaagtacagcacagacattctgggcaacctgtatgaagtgaaatctaagaagcaccctcagat ca tcaaaaagggcaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatccgat gctaagtcactgactgcctggtcccggacactggtgaccttcaaggatgtgt ttgtggacttcaccag ggaggagtggaagctgctggacactgctcagcagatcctgtacagaaatgtgatgctggagaactata agaacctggtttccttgggttatcagcttactaagccagatgtgatcctccggttggagaagggagaa gagccctggctggtggagagagaaattcaccaagagacccatcctgattcagagactgcatttgaaat caaatcatcagttccgaaaaagaaacgcaaagttSEQ ID NO: 51Polypeptide sequence of TetI CDLPTCSCLDRVIQKDKGPYYTHLGAGPSVAAVREIMENRYGQKGNAIRIEIWYTGKEGKSSHGCPIAKWVLRRSSDEEKVLCLVRQRTGHHCPTAVMVVLIMVWDGIPLPMADRLYTELTENLKSYNGHPTDRRCTLNENRTCTCQGIDPETCGASFSFGCSWSMYFNGCKFGRSPSPRRFRIDPSSPLHEKNLEDNLQSLATRLAPIYKQYAPVAYQNQVEYENVARECREGSKEGRPFSGVTACLRFCAHPHRDIHNMNNGSTVVCTLTREDNRSLGVIPQDEQLHVLPLYKLSDTDEFGSKEGMEAKIKSGAIEVLAPRRKKRTCFTQPVPRSGKKRAAMMTEVLAHKIRAVEKKPIPRIKRKNNSTTTNNSKPSSLPTLGSNTETVQPEVKSETEPHFILKSSDNTKTYSLMPSAPHPVKEASPGFSWSPKTASATPAPLKNDATASCGFSERSSTPHCTMPSGRLSGANAAAADGPGISQLGEVAPLPTLSAPVMEPLINSEPSTGVTEPLTPHQPNHQPSFLTSPQDLASSPMEEDBQHSEADEPPSDEPLSDDPLSPAEEKLPHIDEYWSDSEHIFLDANIGGVAIAPAHGSVLIECARRELHATTPVEHPNRNHPTRLSLVFYQHKNLNKPQHGFELNKIKFEAKEAKNKKMKASEQKDQAANEGPEQSSEVNELNQYPSHKALTLTHDNVVTVSPY'ALiTHVAGPYNHWVSEQ ID NO: 52Polynucleotide sequence of TetI CDCTGCCCACCTGCAGCTGTCTTGATCGAGTTATACAA / AAAGACAzAAGGCCCATATTATACACACCTTGG GGCAGGACCAAGTGTTGCTGCTGTCAGGGAAATCATGGAGAATAGGTATGGTCAAAAAGGAAACGCAA TAAGGATAGAAATAGTAGTGTACACCGGTAAAGAAGGGAAAAGCTCTCATGGGTGTCCAATTGCTAAG TGGGTTTTAAGAAGAAGCAGTGATGAAGAAAAAGTTCTTTGTTTGGTCCGGCAGCGTACAGGCCACCA CTGTCCAACTGCTGTGATGGTGGTGCTCATCATGGTGTGGGATGGCATCCCTCTTCCAATGGCCGACC GGCTATACACAGAGCTCACAGAGAATCTAAAGTCATACAATGGGCACCCTACCGACAGAAGATGCACC CTCAATGAAAATCGTACCTGTACATGTCAAGGAATTGATCCAGAGACTTGTGGAGCTTCATTCTCTTT TGGCTGTTCATGGAGTATGTACTTTAATGGCTGTAAGTTTGGTAGA / AGCCCAAGCCCCAGAAGATTTA GAATTGATCCAAGCTCTCCCTTACATGAAAAAAACCTTGAAGATAACTTACAGAGTTTGGCTACACGA TTAGCTCCAATTTATAAGCAGTA1X3CTCCAGTAGCTTACCAAAATCAGGTGGAATATGAAAATGTTGC CCGAGAATGTCGGCTTGGCAGCAAGGAAGGTCGACCCTTCTCTGGGGTCACTGCTTGCCTGGACTTCT GTGCTCATCCCCACAGGGACATTCACAACATGAATAATGGAAGCACTGTGGTTTGTACCTTAACTCGA GAAGATAACCGCTCTTTGGGTGTTATTCCTCAAGATGAGCAGCTCCATGTGCTACCTCTTTATAAGCT TTCAGACACAGATGAGTTTGGCTCCAAGGAAGGAATGGAAGCCAAGATCAAATCTGGGGCCATCGAGG TCCTGGCACCCCGCCGCAAAAAAAGAACGTGTTTCACTCAGCCTGTTCCCCGTTCTGGAAAGAAGAGG GCTGCGATGATGACAGAGGTTCTTGCACATAAGATAzAGGGCAGTGGAAAAGAAACCTATTCCCCGAAT CAAGCGGA / AGAATAACTCAACAACAACAAACAACAGTAAGCCTTCGTCACTGCCAACCTTAGGGAGTA ACACTGAGACCGTGCAACCTGAAGTAAAAAGTGAAACCGAACCCCATTTTATCTTAAAAAGTTCAGAC AACACTAAAACTTATTCGCTGATGCCATCCGCTCCTCACCCAGTGAAAGAGGCATCTCCAGGCTTCTC CTGGTCCCCGAAGACTGCTTCAGCCACACCAGCTCCACTGAAGAATGACGCAACAGCCTCATGCGGGT TTTCAGAAAGAAGCAGCACTCCCCACTGTACGATGCCTTCGGGAAGACTCAGTGGTGCCAATGCTGCA GCTGCTGATGGCCCTGGCATTTCACAGCTTGGCGAAGTGGCTCCTCTCCCCACCCTGTCTGCTCCTGT GATGGAGCCCCTCATTAATTCTGAGCCTTCCACTGGTGTGACTGAGCCGCTAACGCCTCATCAGCCAA ACCACCAGCCCTCCTTCCTCACCTCTCCTCAAGACCTTGCCTCTTCTCCAATGGAAGAAGATGAGCAG CATTCTGAAGCAGATGAGCCTCCATCAGACGAACCCCTATCTGATGACCCCCTGTCACCTGCTGAGGA GAAATTGCCCCACATTGATGAGTATTGGTCAGACAGTGAGCACATCTTTTTGGATGCAAATATTGGTG GGGTGGCCATCGCACCTGCTCACGGCTCGGTTTTGATTGAGTGTGCCCGGCGAGAGCTGCACGCTACC ACTCCTGTTGAGCACCCCAACCGTAATCATCCAACCCGCCTCTCCCTTGTCTTTTACCAGCACAAAAA CCTAAATAAGCCCCAACATGGTTTTGAACTAAACAAGATTAAGTTTGAGGCTAAAGAAGCTAAGAATA AGAAAATGAAGGCCTCAGAGCAAAAAGACCAGGCAGCTAATGAAGGTCCAGAACAGTCCTCTGAAGTA AATGAATTGAACOUU^TTCCTTCTCATAAAGCATTAACATTAACCCATGACAATGTTGTCACCGTGTC CCCTTATGCTCTCACACACGTTGCGGGGCCCTATAACCATTGGGTCSEQ ID NO: 53Protein sequence for VPHDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSLPSASVEFEGSGGPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVL FELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVSSEQ iD NO: 54DNA sequence for VPHGatgctttagacgattttgacttagatatgcttggttcagacgcgttagacgacttcgacctagacat gttaggctcagatgcattggacgacttcgatttagatatgttgggctccgatgccctagatgactttg atctagatatgctagggtcactacccagcgccagcgtcgagttcgaaggcagcggcgggccttcaggg cagatcagcaaccaggccctggctctggcccctagctccgctccagtgctggcccagactatggtgcc ct.ctagtgctatggtgcctctggcccagccacctgctccagcccctgtgctgaccccaggaccacccc agtcactgagcgccccagtgcccaagtctacacaggccggcgaggggact ctgagtgaagctctgctg cacctgcagttcgacgctga tgaggacctgggagctctgctggggaaca.gcaccga.tcccgga.gtgtt cacaga tctggcc tccgtggacaactctgagtt tcagcagctgctgaa tcagggcgtgtcca tgtctc atagtacagccgaaccaatgctgatggagtaccccgaagccattacccggctggtgaccggcagccag cggccccccgaccccgctccaactcccctgggaaccagcggcctgcctaatgggctgtccggagatga agacttctcaagcatcgctgatatggactttagtgccctgctgtcacagatttcctctagtgggcagg gaggaggtggaagcggcttcagcgtggacaccagtgccctgctggacctgttcagcccctcggtgacc gtgcccgacatgagcctgcctgacct tgacagcagcctggccagtatccaagagctcctgtctcccca ggagccccccaggcctcccgaggcagagaacagcagcccggattcagggaagcagctggtgcactaca cagcgcagccgctgttcctgctggaccccggctccgtggacaccgggagcaacgacctgccggtgctg tt tgagctgggagagggctcctacttctccgaaggggacggcttcgccgaggaccccaccatctccct gctgacaggctcggagcctcccaaagccaaggaccccactgtctccSEQ iD NO: 55Protein sequence for VPRDAWDFDWMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSPKKKRKVGSQYLPDTDERHRIEEKRKRTYETFKSIMKKSPFSGPTPPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSESTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSAEAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISSGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLFSEQ iD NO: 56DNA sequence for VPR gatgct ttagacgattttgacttagatatgcttggttcagacgcgttagacgact tcgacctagacat gttaggctcagatgcattggacgacttcgatttagatatgttgggctccgatgccctagatgactttg at ctagatatgctaggtag tcccaaaaagaagaggaaagtgggatcccagtatctgcccga cacaga t gatagacaccgaatcgaagagaaacgcaagcgaacgtatgaaaccttcaaatcgatcatgaagaaatc gcccttctcgggtccgaccgatcccaggcccccaccgagaaggattgcggtcccgtcccgctcgtcgg ccagcgtgccgaagcctgcgccgcagccctaccccttcacgtcgagcctgagcacaatcaattatgac gagt t cccgacgatggtgt tcccct cgggacaaatctcacaagcct cggcgct cgcaccagcgcct cc ccaagtccttccgc a a g c g c c t g c c c c ag c g c c t g c a c c ggc a atggtgtccg c c c t eg c a c agg c c c c t g c g c c c g t c c c eg t g c t c g c g c c t gg a c c g c c c c a gg c gg t c g c t c c a c c ggc t c c g a age c g a c g caggccggagagggaacactctccgaagcacttcttcaactccagtttgatgacgaggatcttggagc actccttggaaactcgacagaccctgcggtgtttaccgacctcgcgtcagtagataactccgaatttc agcagcttttgaaccagggtatcccggtcgcgccacatacaacggagcccatgttgatggaatacccc gaagcaatcacgagacttgtgacgggagcgcagcggcctcccgatcccgcacccgcacctttgggggc acctggcct.ccctaacggact tt.tgagcggcgacgaggattt ct.cctccat.cgccgat.atggatt.tct cagccttgctgtcacagattt ccagcggctctggcagcggcagccgggat tccagggaagggatgttt ttgccgaagcctgaggccggctccgc tattagtgaegtgt t tgagggccgcgaggtgtgccagccaaa a c ga a t c c gg c c a 111 c a t c c t c c ag ga a g t c c a t ggg c c a a c c g c c c a c t c c c c g c c a g c c t c g c a c caacaccaaccggtccagtacatgagccagtcgggtcactgaccccggcaccagtccctcagccactg gatccagcgcccgcagtgactcccgaggccagtcacctgttggagga tcccgatgaagagacgagcca ggctgtcaaagcccttcgggagatggccgatactgtgattccccagaaggaagaggctgcaatctgtg gccaaatggacctttcccatccgcccccaaggggccatctggatgagctgacaaccacacttgagtcc atgaccgaggat ctgaacctggact cacccct gaccccggaattgaacgagat tctggataccttcctgaacgacgagtgcctcttgcatgccatgcatatcagcacaggactgtccatcttcgacacatctctgt ttSEQ ID NO: 57ABE ecTadA wild-type, proteinSEVEFSHEYNMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGIL ADECAALLSDFFRMRRQEIKAQKKAQSSTDSEQ ID NO: 58ABE ecTadA*7.9, proteinSEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGIL ADECNALLCYFFRMPRQVFNAQKKAQSSTDSEQ ID NO: 59ABE ecTadA*7.10, proteinSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGIL ADECAALLCYFFRMPRQVFNAQKKAQSSTDSEQ ID NO: 60ABE ecTadA*8e, proteinSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGIL ADECAALLCDFYRMPRQVFNAQKKAQSSINSEQ ID NO: 61ABE ecTadA*8.8, proteinSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVrGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGIL AQECAALLCRFFRMPRRVFNAQKKAQSSTQSEQ ID NO: 62ABE ecTadA*8.13, proteinSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGIL ADECAALLCRFFRMPRRVFNAQKKAQSSTDSEQ ID NO: 63ABE ecTadA*8.17, proteinSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRWFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGIL ADECAALLCYFFRMPRRVFNAQKKAQSSTDSEQ ID NO: 64ABE ecTadA*8.20, protein ecTadA*8.20SEVEFSHEYWRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQmrRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGIL ADECAALLCRFFRMPRRVFNAQKKAQSSTDSEQ ID NO: 65ABE ecTadA wild-type, DNA tctgaagtcgagtttagccacgagtattggatgaggcacgcactgaccctggcaaagcgagcatggga tgaaagagaagtccccgtgggcgccgtgctggtgcacaacaatagagtgatcggagaggga tggaaca ggccaatcggccgccacgaccctaccgcacacgcagagatcatggcactgaggcagggaggcctggtc atgcagaattaccgcctgatcgatgccaccctgtatgtgacactggagccatgcgtgatgtgcgcagg agcaatgatccacagcaggatcggaagagtggtgttcggagcacgggacgccaagaccggcgcagcag gctccctgatggatgtgctgcaccaccccggcatgaaccaccgggtggagatcacagagggaatcctg gcagacgagtgcgccgccc tgctgagcgatttc t ttagaa tgcggagacaggaga tcaaggcccagaa gaagg c a c agag c t c c a c c ga cSEQ !D NO: 66ABE ecTadA*7,9, DNA tccgaagtcgagt tttccca tgagtactggatgagacacgcattgactctcgcaaagagggctctcga tgaacgcgaggtgcccgtgggggcag tact eg tact caacaatcgcgtaatcggcgaaggttggaata gggcaa tcggactccacgaccccactgcacatgcggaaatcatggcccttcgacagggagggcttgtg atgcagaattatcgacttatcgatgcgacgctgtacgtcacgtttgaaccttgcgtaatgtgcgcggg agctatgattcactcccgcattggacgagttgtattcggtgttcgcaacgccaagacgggtgccgcag gttcactgatggacgtgctgcattacccaggcatgaaccaccgggtagaaat cacagaaggcatat tg gcggacgaatgtaacgcgctgttgtgttacttttttcgcatgcccaggcaggtctttaacgcccagaa a a a age: a c a a t c c t c t a c t ga cSEQ ID NO: 67ABE ecTadA*7.10, DNA tccgaagtcgagt tttcccatgagtactgga tgagacacgcattgactctcgcaaagagggctcgaga tgaacgcgaggtgcccgtgggggcagtactcgtgctcaacaatcgcgtaatcggcgaaggttggaata gggcaa tcggactccacgaccccactgcacatgcggaaatcatggcccttcgacagggagggcttgtg atgcagaattatcgacttatcgatgcgacgctgtacgtcacgtttgaaccttgcgtaatgtgcgcggg agctatgattcactcccgcattggacgagttgtattcggtgttcgcaacgccaagacgggtgccgcag gttcactgatggacgtgctgcattacccaggcatgaaccaccgggtagaaat cacagaaggcatattg gcggacgaatgtgcggcgctgttgtgttacttttttcgcatgcccaggcaggtctttaacgcccagaa aaaagcacaatcctctactgacSEQ !D NO: 68ABE ecTadA*8e, DNA tctgaggtggagttttcccacgagtactggatgagacatgccctgaccctggccaagagggcacggga tgagagggaggtgcctgtgggagccg tgctggtgctgaacaatagag tgatcggcgagggc tggaaca gagcca tcggcctgcacgacccaacagcccatgccgaaat tatggccctgagacagggcggcctggtc atgcagaactacagactga ttgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccgg cgccatgatccactctaggatcggccgcgtggtgtttggcgtgaggaactcaaaaagaggcgccgcag gctccctgatgaacgtgctgaactaccccggcatgaatcaccgcgtcgaaattaccgagggaatcctg gcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacaggtgttcaatgctcagaa gaaggcccagagctccatcaacSEQ ID NO: 69ABE ecTadA*8.8, DNA tccgaagtcgagttttcccatgagtactggatgagacacgcattgactctcgcaaagagggctcgaga tgaacgcgaggtgcccgtgggggcagtactcgtgctcaacaatcgcgtaatcggcgaaggttggaata gggcaa tcggactccacgaccccactgcaca tgcggaaatcatggcccttcgacagggagggcttgtg a t g c a g a a 11 a t c g a ettategatge ga c g c t g t a cgtcacgtttgaaccttg c g t a a t g t geg c ggg agctatgattcactcccgcattggacgagttgtattcggtgttcgcaacgccaagacgggtgccgcag gttcactgatggacgtgctgcatcatccaggcatgaaccaccgggtagaaat cacagaaggcatattg gcggacgaatgtgcggcgctgttgtgtcgtttttttcgcatgcccaggcgggtctttaacgcccagaa aaaagcacaatcctctactgactctggtggttcttctggtggttctagcggcagcgagactcccggga cctcagagtccgccacacccgaaagttctggtggttcttctggtggttctSEQ ID NO: 70ABE ecTadA*8.13, DNA tccgaagtcgagttttcccatgagtactggatgagacacgcattgactctcgcaaagagggctcgaga tgaacgcgaggtgcccgtgggggcagtactcgtgctcaacaatcgcgtaatcggcgaaggttggaata gggcaatcggactccacgaccccactgcacatgcggaaatcatggcccttcgacagggagggcttgtg atgcagaattatcgactttatgatgcgacgctgtacgtcacgtttgaaccttgcgtaatgtgcgcggg age tat gat tcact cccgcat tggacgagttgt at tcggtgttcgcaacgccaagacgggtgccgcag gttcactgatggacgtgctgcat cat ccaggcatgaaccaccgggtagaaat cacagaaggcatattg g c gg a cgaatgtgcggcgctgttgtgtcgtttttttcgcatgc c c a ggcggg t c 111 a a c g c c c a g a a a a a ag c a c a a t c c t c t a c t g a cSEQ ID NO: 71ABE ecTadA*8,17, DNA tccgaagtcgagt tttccca tgagtactggatgagacacgcattgactctcgcaaagagggctcgaga tgaacgcgaggtgcccgtgggggcag tactcgtgctcaacaatcgcgt...

Claims

CLAIMS1 . An isolated polynucleotide encoding a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof.

2. The isolated polynucleotide of claim 1 , wherein the isolated polynucleotide comprises a sequence selected from SEQ ID NOs: 75-84.

3. The isolated polynucleotide of claim 1 or 2, wherein the transcription factor is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.

4. A vector encoding a transcription factor selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof.

5. The vector of claim 4, wherein the vector comprises a promoter operably linked to a polynucleotide sequence encoding the transcription factor.

6. The vector of claim 5, wherein the promoter is non-endogenous to the transcription factor.

7. The vector of claim 5 or 6, wherein the promoter is a constitutive promoter, or a ubiquitous promoter, or an inducible promoter, or a cell-specific promoter, or a tissue-specific promoter,8. The vector of any one of claims 4-7, wherein the vector comprises an open reading frame (ORF) of the transcription factor.

9. The vector of any one of claims 4-8, wherein the vector comprises a sequence selected from SEQ ID NOs: 75-84 or encodes a polypeptide comprising a sequence selected from SEQ ID NOs: 85-94.

10. The vector of any one of claims 4-9, wherein the transcription factor is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.11 . The vector of any one of claims 4-10, wherein the vector is a viral vector.

12. The vector of claim 11 , wherein the vector is a lentiviral vector.

13. The vector of claim 11 , wherein the vector is an adeno-associated virus (AAV) vector,14. The vector of claim 13, wherein the AAV vector is selected from AAV1 , AAV2, AAV5,AAV6, AAV8, AAV9, and an engineered AAV vector.

15. A method of modulating T cells, the method comprising administering to a T cell or a subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 . NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.

16. The method of claim 15, wherein modulating T cells comprises increasing T cells, or increasing memory T cells, or preventing T ceil exhaustions, or reversing T cell exhaustions, or a combination thereof.

17. A method of increasing T cells, the method comprising administering to a T cell or a subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.

18. A method of enhancing adoptive T cell therapy (ACT) in a subject, the method comprising administering to the subject an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KL.F2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.

19. A method of treating cancer in a subject, the method comprising administering to the subject or to a T cell of the subject, an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof, wherein the activator increases the expression of the gene or increases the level of a protein encoded by the gene.

20. The method of any one of claims 15-19, wherein the gene is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.21 . The method of any one of claims 15-20, wherein the activator modulates T cells, and wherein modulating T cells comprises increasing T cells, or increasing memory T cells, or increasing the lifetime of a T cell, or preventing T cell exhaustions, or reversing T cell exhaustions, or reducing T cell exhaustion, or enhancing the therapeutic potential of T cells, or a combination thereof.

22. The method of any one of claims 15-21 , wherein the activator modulates gene expression within the T cell.

23. The method of any one of claims 15-22, wherein the activator decreases expression of TOX in the T cell.

24. The method of any one of claims 15-23, wherein the activator comprises a polypeptide, or a polynucleotide, or a small molecule, or a combination thereof.

25. The method of any one of claims 15-24, wherein the activator comprises a polynucleotide encoding the gene.

26. The method of any one of claims 15-25, wherein the activator comprises a polynucleotide comprising the open reading frame of the gene or a polynucleotide encoding a protein encoded by the gene.

27. The method of any one of claims 15-26, wherein the activator comprises a polynucleotide selected from SEQ ID NOs: 75-84 or a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 85-94.

28. The method of any one of claims 15-27, wherein the activator comprises a polypeptide comprising a protein encoded by the gene.

29. The method of any one of claims 15-24, wherein the activator comprises a polypeptide selected from SEQ ID NOs: 85-94.

30. The method of any one of claims 15-28, wherein the activator comprises the vector of any one of claims 4-14.31 . The method of any one of claims 15-30, wherein the activator or a polynucleotide encoding the activator is encapsulated within a lipid nanoparticle or polymeric carrier.

32. The method of any one of claims 15-31 , the method further comprising administering at least one cancer therapy or at least one antiviral therapy.

33. A vector comprising the isolated polynucleotide of any one of claims 1-3,34. A cell comprising the isolated polynucleotide of any one of claims 1 -3, or the vector of any one of claims 4-14, or the vector of claim 33.

35. The cell of claim 34, wherein the cell is a CD8+ T cell.

36. A pharmaceutical composition comprising: the isolated polynucleotide of any one of claims 1-3, or the vector of any one of claims 4-14, or the vector of claim 33, or a combination thereof.

37. The pharmaceutical composition of claim 36, further comprising at least one cancer therapy or at least one antiviral therapy.

38. A composition for increasing T cells, the composition comprising an activator of a gene selected from THAP6, DMRT3, MEF2B, PAX2, ATOH7, KLF2, KLF1 , TWIST1 , NKX6, and FEV, or a combination thereof.

39. The composition of claim 38, wherein the gene is selected from THAP6, DMRT3, and MEF2B, or a combination thereof.

40. The composition of claim 38 or 39, wherein the activator comprises a polynucleotide encoding the gene, or a polynucleotide encoding the open reading frame of the gene, or a polypeptide encoded by the gene, or a combination thereof.41 . The composition of claim 40, wherein the activator comprises a polynucleotide selected from SEQ ID NOs: 75-84 or a polypeptide selected from SEQ ID NOs: 85-94.

42. The composition of any one of claims 38-41 , further comprising at least one cancer therapy or at least one antiviral therapy.

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