Compositions, systems, and methods for modulating T cell function

Abstract

Provided herein is an epigenetic modification DNA targeting system, such as CRISPR-Cas / guide RNA (gRNA) system, which binds to or targets the target site in gene or its regulatory element in T cell.In some aspects, provided epigenetic modification DNA targeting system regulates T cell function, such as T cell phenotype or activity.In some aspects, provided herein is also a method and use related to provided composition, for example, in regulating T cell (including those related to adoptive T cell therapy methods). TIFF2025531268000128.tif86166

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Application No. 63 / 408,081, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR MODULATING T CELL FUNCTION," filed September 19, 2023; U.S. Provisional Application No. 63 / 459,969, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR MODULATING T CELL FUNCTION," filed April 17, 2023; U.S. Provisional Application No. 63 / 466,678, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR MODULATING T CELL FUNCTION," filed May 15, 2023; and U.S. Provisional Application No. 63 / 466,678, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR MODULATING T CELL FUNCTION," filed July 31, 2023. This application claims priority to U.S. Provisional Application No. 63 / 530,030, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR MODULATING T CELL FUNCTION," filed August 11, 2023; U.S. Provisional Application No. 63 / 532,345, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR MODULATING T CELL FUNCTION," filed September 11, 2023; and U.S. Provisional Application No. 63 / 581,949, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR MODULATING T CELL FUNCTION," filed September 11, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application is filed with an electronic Sequence Listing, which is provided as a file named 224742002240.xml, created on September 18, 2023, and is 734,197 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field In some aspects, the present disclosure relates to an epigenetic modification DNA targeting system, such as a CRISPR-Cas / guide RNA (gRNA) system, that binds to or targets a target site in a gene or its regulatory element in a T cell. In some aspects, the provided epigenetic modification DNA targeting system of the present disclosure regulates T cell function, such as T cell phenotype or activity. In some aspects, the present disclosure is directed to methods and uses related to the provided compositions, such as in regulating T cells (including those related to adoptive T cell therapy methods). [Background technology]

[0004] background The administration of T cells targeting specific antigens, also known as adoptive cell therapy (ACT), is a promising approach for treating diseases such as cancer. However, current ACT treatments face challenges, such as suboptimal T cell function, expansion, and persistence. Therefore, new and improved methods to overcome these challenges are needed. The present disclosure addresses these and other needs. Summary of the Invention

[0005] overview Provided herein is an epigenetic modification DNA targeting system comprising at least one DNA targeting module for suppressing transcription of one or more genes in a T cell, wherein each of the at least one DNA targeting module comprises a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site for one of the one or more genes selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2; and (b) at least one transcriptional repression effector domain for suppressing transcription of the one or more genes in a T cell.

[0006] In some aspects, provided herein is an epigenetic modification DNA targeting system comprising at least one DNA targeting module for suppressing transcription of one or more genes in a T cell, wherein each of the at least one DNA targeting module comprises a fusion protein comprising: (a) a DNA binding domain capable of being targeted to a target site in one of the one or more genes in a T cell selected from the group consisting of CD5, KDM1A, CBLB, DGKZ, MYB, RASA2, ELOB, GATA3, CISH, PRDM1, MED12, and CCNC, or a regulatory DNA element thereof; and (b) at least one transcriptional repression effector domain for suppressing transcription of the one or more genes in a T cell.

[0007] Also provided herein is an epigenetic modification DNA targeting system comprising at least one DNA targeting module for increasing transcription of one or more genes in a T cell, wherein each of the at least one DNA targeting module comprises a fusion protein comprising: (a) a DNA binding domain capable of being targeted to a target site for one of the one or more genes selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1; and (b) at least one transcriptional activation effector domain for increasing transcription of the one or more genes in a T cell.

[0008] In some aspects, provided herein is an epigenetic modification DNA targeting system comprising at least one DNA targeting module for increasing transcription of one or more genes in a T cell, wherein each of the at least one DNA targeting module comprises a fusion protein comprising: (a) a DNA binding domain capable of being targeted to a target site in one of the one or more genes in a T cell selected from the group consisting of VAV1, IL2, and IL2RB, or a regulatory DNA element thereof; and (b) at least one transcriptional activation effector domain for increasing transcription of the one or more genes in a T cell.

[0009] In some of the provided embodiments, transient delivery of the epigenetic modification DNA targeting system to T cells promotes increased T cell effector function upon T cell stimulation compared to T cell effector function in the absence of T cell stimulation. In some of the provided embodiments, the T cell effector function is characterized by an activity selected from the group consisting of IL-2 production, IFN-gamma production, TNF-alpha production, T cell proliferation, or any combination thereof. In some of the provided embodiments, the T cell effector function is characterized by IL-2 production. In some of the provided embodiments, the T cell effector function is characterized by IFN-gamma production. In some of the provided embodiments, the T cell effector function is characterized by IL-2 production and IFN-gamma production. In some of the provided embodiments, the T cell effector function is characterized by multifunctional production of IL-2, IFN-gamma, and TNF-alpha. In some of the provided embodiments, the T cell effector function is characterized by an activity further comprising T cell proliferation. In any of the embodiments herein, the T cell effector function is characterized by an activity that further comprises target cell killing. In any of the embodiments herein, the T cell effector function is characterized by an activity that further comprises T cell persistence.

[0010] In some of any of the provided embodiments of the epigenetic modification DNA targeting system, an increase in T cell effector function is observed 48 hours or more after transient delivery of the epigenetic modification DNA targeting system to the T cell. In some of the provided embodiments, an increase in T cell effector function is observed by 6 days, 9 days, 12 days, 15 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 71 days, or more after transient delivery of the epigenetic modification DNA targeting system to the T cell.

[0011] In some of any of the provided embodiments of the epigenetic modification DNA targeting system, the T cell stimulation is with anti-CD3 and anti-CD28 activating reagents.

[0012] In any of the embodiments herein, the T cells express an engineered antigen receptor, optionally a chimeric antigen receptor (CAR) or T cell receptor (eTCR). In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR) against an antigen, and the T cell stimulation is antigen-specific stimulation of the CAR or eTCR, optionally, the T cell stimulation is by antigen-expressing target cells. In some of the provided embodiments of the epigenetic modification DNA targeting system, the T cells express a chimeric antigen receptor (CAR) against an antigen, and the T cell stimulation is antigen-specific stimulation of the CAR, optionally, the T cell stimulation is by antigen-expressing target cells.

[0013] In some of any of the provided embodiments of the epigenetic modification DNA targeting system, the T cell stimulation is restimulation of the T cells after at least one prior T cell stimulation.

[0014] In some of any of the provided embodiments of the epigenetic modification DNA targeting system, the DNA targeting system does not introduce gene disruption or DNA breaks.

[0015] In some of the provided embodiments of the epigenetic modification DNA targeting system, the fusion protein of each DNA targeting module comprises a DNA-binding domain selected from a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) protein or variant thereof, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or an I-SceI enzyme or variant thereof. In some of any such embodiments, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing.

[0016] In any of the aspects herein, the at least one DNA targeting module is a single DNA targeting module that targets a target site for one of the one or more genes.

[0017] In some of the provided embodiments of the epigenetic modification DNA targeting system, the at least one DNA targeting module is a plurality of DNA targeting modules for targeting a plurality of target sites of one or more of the one or more genes or their regulatory elements.In some of the embodiments herein, the at least one DNA targeting module is a plurality of DNA targeting modules for suppressing the transcription of one or more genes in T cells, each DNA targeting module targets a target site for one of the one or more genes.In some of the embodiments herein, the at least one DNA targeting module is a plurality of DNA targeting modules for increasing the transcription of one or more genes in T cells, each DNA targeting module targets a target site for one of the one or more genes.In some of the provided embodiments, the plurality of DNA targeting modules is 2, 3, 4, 5 or 6 DNA targeting modules.

[0018] In any of the embodiments herein, the multiple DNA targeting modules for suppressing transcription of one or more genes in a T cell target at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some of the embodiments provided, the multiple DNA targeting modules target at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of CD5, KDM1A, CBLB, DGKZ, MYB, RASA2, ELOB, GATA3, CISH, PRDM1, MED12, and CCNC. In any of the embodiments herein, the multiple DNA targeting modules for suppressing transcription of one or more genes in a T cell target at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of CBLB, CISH, MED12, MYB, PRDM1, and RASA2. In some embodiments, the multiple DNA targeting modules for suppressing transcription of one or more genes in a T cell target at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some of any of the provided embodiments, the multiple DNA targeting modules target at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of CD5, KDM1A, CBLB, DGKZ, MYB, RASA2, ELOB, GATA3, CISH, PRDM1, MED12, and CCNC.In some embodiments, the multiple DNA targeting modules for suppressing transcription of one or more genes in a T cell target at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of CBLB, CISH, MED12, MYB, PRDM1, and RASA2.

[0019] In any of the embodiments herein, the multiple DNA targeting modules are selected from the group consisting of CBLB and CCNC; CBLB and CD5; CBLB and CISH; CBLB and DGKZ; CBLB and ELOB; CBLB and FAS; CBLB and Fli1; CBLB and GATA3; CBLB and KDM1A; CBLB and MED12; CBLB and MYB; CBLB and PRDM1; CBLB and RASA2; CD5 and CISH; CD5 and MYB; CISH and DGKZ; CISH and MYB; CISH and RASA2; GATA3 and CD5; GATA3 and CISH; GATA3 and MYB; MED12 and CBLB. ;MED12 and CD5;MED12 and CISH;MED12 and DGKZ;MED12 and ELOB;MED12 and GATA3;MED12 and MYB;MED12 and PRDM1;MED12 and RASA2;MYB and RASA2;PRDM1 and CISH;PRDM1 and GATA3;PRDM1 and MYB;PRDM1 and RASA2;CD5, CISH, and MYB;GATA3, CBLB, and MYB;GATA3, CD5, and MYB;PRDM1, GATA3, and CISH, TGFBR2 and MED12; and TGFBR2, MED12, and CISH are targeted. In some of the provided embodiments, the multiple DNA targeting modules target CBLB and MYB; CD5 and CISH; CD5, CISH and MYB; CD5 and MYB; CISH and DGKZ; CISH and MYB; CISH and RASA2; GATA3, CBLB and MYB; GATA3 and CD5; GATA3, CD5 and MYB; GATA3 and CISH; GATA3 and MYB; MYB and RASA2; PRDM1 and CISH; PRDM1 and GATA3; PRDM1, GATA3 and CISH; PRDM1 and MYB; PRDM1 and RASA2; CBLB and CCNC; and CBLB and MED12.

[0020] In any of the embodiments herein, the multiple DNA targeting modules target a combination of genes selected from MED12 and CBLB; MED12 and CISH; CBLB and MYB; and CBLB and RASA2. In some embodiments, the first and second genes are CBLB and MYB. In some embodiments, the first and second genes are CBLB and MED12. In some embodiments, the first and second genes are CBLB and CCNC.

[0021] In any of the embodiments herein, the multiple DNA targeting modules for increasing transcription of one or more genes in T cells target at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some of the embodiments provided, the multiple DNA targeting modules for increasing transcription of one or more genes in T cells target at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of VAV1, IL-2, and IL2RB. In any of the embodiments herein, the multiple DNA targeting modules for increasing transcription of one or more genes in T cells target at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of EOMES, IL-2, LCP2, and TBX21. In any of the embodiments herein, the multiple DNA targeting modules for increasing transcription of one or more genes in a T cell target at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In any of the embodiments herein, the multiple DNA targeting modules for increasing transcription of one or more genes in a T cell target a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of EOMES, IL-2, LCP2, and TBX21.

[0022] In any of the embodiments herein, the multiple DNA targeting modules target a combination of genes selected from BATF and IL-2; BATF and VAV1; CD28 and BATF; CD28 and EOMES; CD28 and IL-2; CD28 and LCP2; CD28 and TBX21; CD28 and VAV1; EOMES and BATF; EOMES and LCP2; EOMES and TBX21; EOMES and VAV1; EOMES and IL-2; LCP2 and BATF; LCP2 and IL-2; LCP2 and TBX21; LCP2 and VAV1; TBX21 and BATF; TBX21 and IL-2; TBX21 and TBX21; TBX21 and VAV1; and VAV1 and IL-2. In some of the embodiments provided, the first and second genes are IL2RB and VAV1. In some of any of the provided embodiments, the first and second genes are IL2 and VAV1. In some embodiments, the first and second genes are IL2 and LCP2. In some embodiments, the first and second genes are IL2 and TBX21. In some embodiments, the first and second genes are IL2 and EOMES.

[0023] In any of the embodiments herein, the target site for the gene or the target site for each of the one or more genes is in the gene and / or its regulatory DNA element.In some of the embodiments provided for the epigenetic modification DNA targeting system, the target site for the gene or each of the one or more genes is in its regulatory DNA element.In some of the embodiments provided for, the regulatory DNA element is an enhancer or a promoter.

[0024] In any of the embodiments herein, the target site is within 1000 base pairs (bp) of the transcription start site of the gene. In some of the provided embodiments of the epigenetic modification DNA targeting system, the target site is within 500 base pairs (bp) of the transcription start site of the gene.

[0025] In some of the provided embodiments of the epigenetic modification DNA targeting system, the target site is selected from the group consisting of: (a) a target site for CD5 having the sequence set forth in any one of SEQ ID NOs: 1-3, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for KDM1A having the sequence set forth in any one of SEQ ID NOs: 4-6, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for CBLB having the sequence set forth in any one of SEQ ID NOs: 10-12, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for DGKZ having the sequence set forth in any one of SEQ ID NOs: 13-15, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for RASA2, having a sequence set forth in any one of SEQ ID NOS: 19-21, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for ELOB, having a sequence set forth in any one of SEQ ID NOS: 22-24, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for GATA3, having a sequence set forth in any one of SEQ ID NOS: 25-27, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for CISH, having a sequence set forth in any one of SEQ ID NOS: 28-30, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(j) a target site for PRDM1 having a sequence set forth in any one of SEQ ID NOs: 31 to 33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (k) a target site for MED12 having a sequence set forth in any one of SEQ ID NOs: 80 to 90, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (l) a target site for CCNC having a sequence set forth in any one of SEQ ID NOs: 102 to 112, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (m) a target site for FAS having a sequence set forth in any one of SEQ ID NOs: 200 to 205 and 292 to 295, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (o) a target site for Fli1 having the sequence set forth in any one of SEQ ID NOS: 206-211, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (o) a target site for TGFBR2 having the sequence set forth in any one of SEQ ID NOS: 300-302 and 306-308, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;

[0026] In some of the provided embodiments of the epigenetic modification DNA targeting system, the target site is selected from the group consisting of: (a) a target site for CD5 having the sequence set forth in any one of SEQ ID NOs: 1-3, or a complementary sequence thereof; (b) a target site for KDM1A having the sequence set forth in any one of SEQ ID NOs: 4-6, or a complementary sequence thereof; (c) a target site for CBLB having the sequence set forth in any one of SEQ ID NOs: 10-12, or a complementary sequence thereof; (d) a target site for DGKZ having the sequence set forth in any one of SEQ ID NOs: 13-15, or a complementary sequence thereof; (e) a target site for MYB1 having the sequence set forth in any one of SEQ ID NOs: 16-18, or a complementary sequence thereof; (f) a target site for RASA2 having the sequence set forth in any one of SEQ ID NOs: 19-21, or a complementary sequence thereof; (h) a target site for GATA3 having the sequence set forth in any one of SEQ ID NOS: 25-27 or the complementary sequence of any of the foregoing; (i) a target site for CISH having the sequence set forth in any one of SEQ ID NOS: 28-30 or the complementary sequence of any of the foregoing; (j) a target site for PRDM1 having the sequence set forth in any one of SEQ ID NOS: 31-33 or the complementary sequence of any of the foregoing; (k) a target site for MED12 having the sequence set forth in any one of SEQ ID NOS: 80-90 or the complementary sequence of any of the foregoing; and (l) a target site for CCNC having the sequence set forth in any one of SEQ ID NOS: 102-112 or the complementary sequence of any of the foregoing; (m) a target site for GATA3 having the sequence set forth in any one of SEQ ID NOS: 29-310 or the complementary sequence of any of the foregoing; a target site for FAS having a sequence set forth in any one of SEQ ID NOs: 200-205 and 292-295, or a complementary sequence of any of the foregoing;(n) a target site for Fli1 having the sequence set forth in any one of SEQ ID NOS: 206-211, or a complementary sequence thereof; and (o) a target site for TGFBR2 having the sequence set forth in any one of SEQ ID NOS: 300-302 and 306-308, or a complementary sequence thereof;

[0027] In any of the embodiments herein, (a) the target site is selected from a target site for CBLB having the sequence set forth in SEQ ID NO:11 or its complementary sequence; (b) the target site is selected from a target site for MYB having the sequence set forth in SEQ ID NO:18 or its complementary sequence; (c) the target site is selected from a target site for RASA2 having the sequence set forth in SEQ ID NO:19 or its complementary sequence; (d) the target site is selected from a target site for CISH having the sequence set forth in SEQ ID NO:28 or its complementary sequence; (e) the target site is selected from a target site for PRDM1 having the sequence set forth in SEQ ID NO:33 or its complementary sequence; and (f) the target site is selected from a target site for MED12 having the sequence set forth in SEQ ID NO:81 or its complementary sequence.

[0028] In any of the embodiments herein, the target site is selected from the group consisting of: (a) a target site for VAV1 having the sequence set forth in any one of SEQ ID NOs: 7-9, 156, and 170, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for IL2 having the sequence set forth in SEQ ID NO: 78, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for BATF having the sequence set forth in any one of SEQ ID NOs: 172-174, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for CD28 having the sequence set forth in any one of SEQ ID NOs: 144-146 and 189-191, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for EOMES having the sequence set forth in any one of SEQ ID NOS: 175 to 177, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for LAT having the sequence set forth in any one of SEQ ID NOS: 184 to 186, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LCP2 having the sequence set forth in any one of SEQ ID NOS: 150 to 152 and 187 to 188, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (i) a target site for IRF4 having the sequence set forth in any one of SEQ ID NOS: 175 to 177, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. The target site for TBX21 is selected from the group consisting of a sequence set forth in any one of NOS: 153 to 155, a continuous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0029] In any of the embodiments herein, the target site is selected from the group consisting of: (a) a target site for VAV1 having the sequence set forth in any one of SEQ ID NOs: 7-9, 156, and 170, or a complementary sequence of any of the foregoing; (b) a target site for IL2 having the sequence set forth in SEQ ID NO: 78, or a complementary sequence of any of the foregoing; (c) a target site for BATF having the sequence set forth in any one of SEQ ID NOs: 172-174, or a complementary sequence of any of the foregoing; (d) a target site for CD28 having the sequence set forth in any one of SEQ ID NOs: 144-146 and 189-191, or a complementary sequence of any of the foregoing; (e) a target site for EOMES having the sequence set forth in any one of SEQ ID NOs: 147-149, or a complementary sequence of any of the foregoing; (g) a target site for IRF4 having the sequence set forth in any one of SEQ ID NOS: 175 to 177, or a complementary sequence of any of the foregoing; (g) a target site for LAT having the sequence set forth in any one of SEQ ID NOS: 184 to 186, or a complementary sequence of any of the foregoing; (h) a target site for LCP2 having the sequence set forth in any one of SEQ ID NOS: 150 to 152 and 187 to 188, or a complementary sequence of any of the foregoing; and (i) a target site for TBX21 having the sequence set forth in any one of SEQ ID NOS: 153 to 155, or a complementary sequence of any of the foregoing.

[0030] In any of the embodiments herein, the target site is selected from: (a) a target site for IL-2 having the sequence set forth in SEQ ID NO:78, or a complementary sequence of any of the foregoing; (b) a target site for EOMES having the sequence set forth in SEQ ID NO:149, or a complementary sequence of any of the foregoing; (c) a target site for LCP2 having the sequence set forth in SEQ ID NO:151, or a complementary sequence of any of the foregoing; and (d) a target site for TBX21 having the sequence set forth in SEQ ID NO:155, or a complementary sequence of any of the foregoing.

[0031] In any of the aspects herein, the DNA-binding domain of each of the at least one DNA-targeting module is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) protein or a variant thereof, and each of the at least one DNA-targeting module further comprises at least one gRNA for targeting the DNA-binding domain to a target site in the one or more genes.

[0032] In any of the embodiments herein, the Cas protein or variant thereof is an inactivated (dCas) protein. In some embodiments, the dCas protein lacks nuclease activity. In some embodiments, the dCas protein is a dCas9 protein. In some embodiments, the dCas protein is a dCasl2 protein. In some embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, based on the numbering of positions in SEQ ID NO: 124. In some embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 125, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, dSaCas9 is set forth in SEQ ID NO:125. In some embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, based on the numbering of positions in SEQ ID NO:126. In some embodiments, dSpCas9 comprises the sequence set forth in SEQ ID NO:127, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, dSpCas9 is set forth in SEQ ID NO:127.

[0033] In any of the embodiments herein, the gRNA comprises a gRNA spacer that is complementary to the target site of the gene.

[0034] In any of the embodiments herein, the DNA targeting module is a module for suppressing transcription of the one or more genes, and the gRNA is selected from the group consisting of: (a) a gRNA that targets a target site for CD5 and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 35 to 37, or a continuous portion thereof that is at least 14 nt; (b) a gRNA that targets a target site for KDM1A and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 38 to 40, or a continuous portion thereof that is at least 14 nt; (c) a gRNA that targets a target site for CBLB and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 44 to 46, or a continuous portion thereof that is at least 14 nt; and (d) a gRNA that targets a target site for DGKZ and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 45 to 46. (e) a gRNA targeting a target site for MYB and comprising a gRNA spacer sequence comprising a sequence as set forth in any one of SEQ ID NOs: 50 to 52 or a continuous portion thereof that is at least 14 nt; (f) a gRNA targeting a target site for RASA2 and comprising a gRNA spacer sequence comprising a sequence as set forth in any one of SEQ ID NOs: 53 to 55 or a continuous portion thereof that is at least 14 nt; (g) a gRNA targeting a target site for ELOB and comprising a gRNA spacer sequence comprising a sequence as set forth in any one of SEQ ID NOs: 56 to 58 or a continuous portion thereof that is at least 14 nt; (h) a gRNA targeting a target site for GATA3 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 57 to 59; (i) a gRNA that targets a target site for CISH and comprises a gRNA spacer sequence that comprises a sequence set forth in any one of SEQ ID NOs: 59 to 61 or a continuous portion thereof that is at least 14 nt; (ii) a gRNA that targets a target site for CISH and comprises a gRNA spacer sequence that comprises a sequence set forth in any one of SEQ ID NOs: 62 to 64 or a continuous portion thereof that is at least 14 nt;(j) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 65 to 67 or a continuous portion thereof that is at least 14 nt; (k) a gRNA that targets a target site for MED12 and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 91 to 101 or a continuous portion thereof that is at least 14 nt; (l) a gRNA that targets a target site for CCNC and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 113 to 123 or a continuous portion thereof that is at least 14 nt; (m) a gRNA that targets a target site for FAS and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 212 to 217 and 296 to 299 or a continuous portion thereof that is at least 14 nt; (n) a gRNA that targets a target site for Fli1 and comprises a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 212 to 217 and 296 to 299 or a continuous portion thereof that is at least 14 nt. (o) a gRNA that targets a target site for TGFBR2 and comprises a gRNA spacer sequence that includes a sequence set forth in any one of SEQ ID NOs: 218-223 or a continuous portion thereof that is at least 14 nt; and (o) a gRNA that targets a target site for TGFBR2 and comprises a gRNA spacer sequence that includes a sequence set forth in any one of SEQ ID NOs: 303-305 and 309-311 or a continuous portion thereof that is at least 14 nt;

[0035] In any of the embodiments herein, the DNA targeting module is a module for suppressing transcription of the one or more genes, and the gRNA is selected from the group consisting of: (a) a gRNA that targets a target site for CD5 and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 35 to 37; (b) a gRNA that targets a target site for KDM1A and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 38 to 40; (c) a gRNA that targets a target site for CBLB and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 44 to 46; (d) a gRNA that targets a target site for DGKZ and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 47 to 49; (e) a gRNA that targets a target site for MYB and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 50 to 52; and (f) a gRNA that targets a target site for RASA2 and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 53 to 54. (g) a gRNA targeting a target site for ELOB and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 56-58; (h) a gRNA targeting a target site for GATA3 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 59-61; (i) a gRNA targeting a target site for CISH and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 62-64; (j) a gRNA targeting a target site for PRDM1 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 65-67; ​​(k) a gRNA targeting a target site for MED12 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 91-101; (l) a gRNA targeting a target site for CCNC and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: a gRNA comprising a gRNA spacer sequence shown in any one of NOs: 113 to 123;(m) a gRNA that targets a target site for FAS and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOS: 212-217 and 296-299; (n) a gRNA that targets a target site for Fli1 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOS: 218-223; and (o) a gRNA that targets a target site for TGFBR2 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOS: 303-305 and 309-311.

[0036] In any of the embodiments herein, the DNA targeting module is a module for suppressing transcription of the one or more genes, and the gRNA is selected from (a) a gRNA that targets a target site for CBLB and comprises a gRNA spacer sequence set forth in SEQ ID NO: 45; (b) a gRNA that targets a target site for MYB and comprises a gRNA spacer sequence set forth in SEQ ID NO: 52; (c) a gRNA that targets a target site for RASA2 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 53; (d) a gRNA that targets a target site for CISH and comprises a gRNA spacer sequence set forth in SEQ ID NO: 62; (e) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 67; and (f) a gRNA that targets a target site for MED12 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 92.

[0037] In any of the embodiments herein, the DNA targeting module is a module for increasing transcription of the one or more genes, and the gRNA is selected from the group consisting of: (a) a gRNA that targets a target site for VAV1 and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 41 to 43, 169, and 171, or a continuous portion thereof that is at least 14 nt; (b) a gRNA that targets a target site for IL2 and comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 79, or a continuous portion thereof that is at least 14 nt; (c) a gRNA that targets a target site for BATF and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 178 to 180, or a continuous portion thereof that is at least 14 nt; and (d) a gRNA that targets a target site for CD28 and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 179 to 180. (e) a gRNA targeting a target site for EOMES and comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 160-162 or a continuous portion thereof which is at least 14 nt; (f) a gRNA targeting a target site for IRF4 and comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 181-183 or a continuous portion thereof which is at least 14 nt; (g) a gRNA targeting a target site for LAT and comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 192-194 or a continuous portion thereof which is at least 14 nt; (h) a gRNA targeting a target site for LCP2 and comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 193-194 or a continuous portion thereof which is at least 14 nt. a gRNA comprising a gRNA spacer sequence comprising a sequence set forth in any one of NOs: 163-165 and 195-196 or a continuous portion thereof that is at least 14 nt;and (i) a gRNA that targets a target site for TBX21 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 166 to 168, or a contiguous portion thereof that is at least 14 nt;

[0038] In any of the embodiments herein, the DNA targeting module is a module for increasing transcription of the one or more genes, and the gRNA is selected from the group consisting of: (a) a gRNA targeting a target site for VAV1 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 41-43, 169, and 171; (b) a gRNA targeting a target site for IL2 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 79; (c) a gRNA targeting a target site for BATF and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 178-180; (d) a gRNA targeting a target site for CD28 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 157-159 and 197-199; and (e) a gRNA targeting a target site for EOMES and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 158-159. (f) a gRNA that targets a target site for IRF4 and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 181 to 183; (g) a gRNA that targets a target site for LAT and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 192 to 194; (h) a gRNA that targets a target site for LCP2 and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 163 to 165 and 195 to 196; and (i) a gRNA that targets a target site for TBX21 and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 166 to 168.

[0039] In any of the embodiments herein, the DNA targeting module is a module for increasing transcription of the one or more genes, and the gRNA is selected from (a) a gRNA that targets a target site for IL-2 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 79; (a) a gRNA that targets a target site for EOMES and comprises a gRNA spacer sequence set forth in SEQ ID NO: 162; (a) a gRNA that targets a target site for LCP2 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 164; and (a) a gRNA that targets a target site for TBX21 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 168.

[0040] In any of the embodiments herein, the gRNA comprises a spacer sequence that is 14 nt to 24 nt, or 16 nt to 22 nt in length, or 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0041] In any of the embodiments herein, the gRNA further comprises a scaffold sequence as shown in SEQ ID NO:69.

[0042] In any of the aspects herein, the at least one transcriptional repression effector domain is capable of reducing transcription of the one or more genes.

[0043] In any of the embodiments herein, the transcriptional repression effector domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3A-DNMT3L fusion protein domain, an ERF repression domain, an Mxi1 repression domain, a SID4X repression domain, a Mad-SID repression domain, an LSD1 repression domain, an EZH2 repression domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing.

[0044] In any of the embodiments herein, the transcriptional repression effector domain is a KRAB domain, a DNMT3A domain, or a DNMT3L domain, or any combination of the foregoing.

[0045] In any of the embodiments herein, the at least one transcriptional repression effector domain comprises a KRAB domain or a variant or portion thereof that exhibits transcriptional repressor activity. In any of the embodiments herein, the at least one transcriptional repression effector domain comprises a sequence set forth in any one of SEQ ID NOs:70, 235, and 355-358, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0046] In any of the embodiments herein, the at least one transcriptional repression effector domain comprises a DNMT3A domain or a variant or portion thereof that exhibits transcriptional repressor activity. In any of the embodiments herein, the at least one transcriptional repression domain comprises the sequence set forth in SEQ ID NO: 131 or 238, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0047] In any of the embodiments herein, the at least one transcriptional repression domain comprises a DNMT3L domain or a variant or portion thereof that exhibits transcriptional repressor activity. In any of the embodiments herein, the at least one transcriptional repression domain comprises a sequence set forth in any one of SEQ ID NOs: 133 and 240-242, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0048] In any of the embodiments herein, the at least one transcriptional repression domain is a DNMT3A-DNMT3L fusion protein domain, a DNMT3B-DNMT3L fusion protein domain, or a variant thereof that exhibits transcriptional repressor activity. In any of the embodiments herein, the at least one transcriptional repression domain comprises a sequence set forth in any one of SEQ ID NOs: 135, 137, or 363, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In any of the embodiments herein, the fusion protein comprises a sequence set forth in any one of SEQ ID NOs: 138-141, 332-351, and 365-384, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0049] In any of the embodiments herein, the at least one transcriptional activation effector domain is capable of increasing transcription of the one or more genes.

[0050] In any of the embodiments herein, the at least one transcription activation effector domain is selected from the group consisting of a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain (optionally the TET protein is TET1), a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing.

[0051] In any of the embodiments herein, the at least one transcription activation effector domain comprises at least one VP16 domain and / or VP16 tetramer ("VP64") or a variant thereof. In any of the embodiments herein, the at least one transcription activation effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcription activation activity. In any of the embodiments herein, the at least one transcription activation effector domain is VP64. In any of the embodiments herein, the at least one transcription activation effector domain comprises the sequence set forth in SEQ ID NO:142, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In any of the embodiments herein, the fusion protein comprises the sequence set forth in SEQ ID NO:77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0052] Also provided herein is a combination of epigenetic modification DNA targeting systems comprising at least two of the DNA targeting systems described herein, wherein each DNA targeting system suppresses transcription of a different gene among the one or more genes.

[0053] Also provided herein is a combination of epigenetic modification DNA targeting systems comprising at least two of the DNA targeting systems described herein, wherein each DNA targeting system increases transcription of a different gene among the one or more genes.

[0054] Also provided herein are guide RNAs (gRNAs) that target target sites for genes selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the target site for a gene is within the gene or in a regulatory DNA element thereof. In some embodiments, the regulatory DNA element is an enhancer or promoter. In some embodiments, the target site is within 1000 base pairs (bp) of the transcription start site of the gene. In some embodiments, the target site is within 500 base pairs (bp) of the transcription start site of the gene. In any of the embodiments herein, the target site is selected from the group consisting of: (a) a target site for CD5 having the sequence set forth in any one of SEQ ID NOs: 1-3, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for KDM1A having the sequence set forth in any one of SEQ ID NOs: 4-6, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for CBLB having the sequence set forth in any one of SEQ ID NOs: 10-12, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for DGKZ having the sequence set forth in any one of SEQ ID NOs: 13-15, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for MYB having a sequence set forth in any one of SEQ ID NOs: 16 to 18, a contiguous portion thereof being at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for RASA2 having a sequence set forth in any one of SEQ ID NOs: 19 to 21, a contiguous portion thereof being at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(g) a target site for ELOB having a sequence set forth in any one of SEQ ID NOs: 22 to 24, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for GATA3 having a sequence set forth in any one of SEQ ID NOs: 25 to 27, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for CISH having a sequence set forth in any one of SEQ ID NOs: 28 to 30, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (j) a target site for PRDM1 having a sequence set forth in any one of SEQ ID NOs: 31 to 33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (k) a target site for GATA3 having a sequence set forth in any one of SEQ ID NOs: 26 to 28, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (l) a target site for CCNC having the sequence set forth in any one of SEQ ID NOS: 102 to 112, a contiguous portion thereof which is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (m) a target site for FAS having the sequence set forth in any one of SEQ ID NOS: 200 to 205 and 292 to 295, a contiguous portion thereof which is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (n) a target site for Fli1 having the sequence set forth in any one of SEQ ID NOS: 206 to 211, a contiguous portion thereof which is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (o) a target site for FAS having the sequence set forth in any one of SEQ ID NOS: 206 to 211, a contiguous portion thereof which is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. The target site for TGFBR2 is selected from the group consisting of a sequence set forth in any one of SEQ ID NOs: 300-302 and 306-308, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;

[0055] In any of the embodiments herein, the target site is selected from the group consisting of: (a) a target site for CD5 having the sequence set forth in any one of SEQ ID NOs: 1-3, or a complementary sequence thereof; (b) a target site for KDM1A having the sequence set forth in any one of SEQ ID NOs: 4-6, or a complementary sequence thereof; (c) a target site for CBLB having the sequence set forth in any one of SEQ ID NOs: 10-12, or a complementary sequence thereof; (d) a target site for DGKZ having the sequence set forth in any one of SEQ ID NOs: 13-15, or a complementary sequence thereof; (e) a target site for MYB having the sequence set forth in any one of SEQ ID NOs: 16-18, or a complementary sequence thereof; (f) a target site for RASA2 having the sequence set forth in any one of SEQ ID NOs: 19-21, or a complementary sequence thereof; (h) a target site for GATA3 having the sequence set forth in any one of SEQ ID NOS: 25 to 27 or the complementary sequence of any of the foregoing; (i) a target site for CISH having the sequence set forth in any one of SEQ ID NOS: 28 to 30 or the complementary sequence of any of the foregoing; (j) a target site for PRDM1 having the sequence set forth in any one of SEQ ID NOS: 31 to 33 or the complementary sequence of any of the foregoing; (k) a target site for MED12 having the sequence set forth in any one of SEQ ID NOS: 80 to 90 or the complementary sequence of any of the foregoing; (l) a target site for CCNC having the sequence set forth in any one of SEQ ID NOS: 102 to 112 or the complementary sequence of any of the foregoing; (n) a target site for FAS having the sequence set forth in any one of SEQ ID NOS: 200-205 and 292-295, or a complementary sequence of any of the foregoing; (n) a target site for Fli1 having the sequence set forth in any one of SEQ ID NOS: 206-211, or a complementary sequence of any of the foregoing;and (o) a target site for TGFBR2 having a sequence set forth in any one of SEQ ID NOs: 300-302 and 306-308, or a complementary sequence of any of the foregoing;

[0056] In any of the embodiments herein, the target site is selected from (a) a target site for CBLB having the sequence set forth in SEQ ID NO:11 or a complementary sequence of any of the foregoing; (b) a target site for MYB having the sequence set forth in SEQ ID NO:18 or a complementary sequence of any of the foregoing; (c) a target site for RASA2 having the sequence set forth in SEQ ID NO:19 or a complementary sequence of any of the foregoing; (d) a target site for CISH having the sequence set forth in SEQ ID NO:28 or a complementary sequence of any of the foregoing; (e) a target site for PRDM1 having the sequence set forth in SEQ ID NO:33 or a complementary sequence of any of the foregoing; and (f) a target site for MED12 having the sequence set forth in SEQ ID NO:81 or a complementary sequence of any of the foregoing. In any of the embodiments herein, the gRNA is selected from the group consisting of: (a) a gRNA that targets a target site for CD5 and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 35-37, or a continuous portion thereof that is at least 14 nt; (b) a gRNA that targets a target site for KDM1A and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 38-40, or a continuous portion thereof that is at least 14 nt; (c) a gRNA that targets a target site for CBLB and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 44-46, or a continuous portion thereof that is at least 14 nt; (d) a gRNA that targets a target site for DGKZ and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 47-49, or a continuous portion thereof that is at least 14 nt; and (e) a gRNA that targets a target site for MYB and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 48-49. a gRNA comprising a gRNA spacer sequence comprising a sequence set forth in any one of NOs: 50 to 52 or a continuous portion thereof of at least 14 nt;(f) a gRNA targeting a target site for RASA2 and comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 53 to 55 or a continuous portion thereof that is at least 14 nt; (g) a gRNA targeting a target site for ELOB and comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 56 to 58 or a continuous portion thereof that is at least 14 nt; (h) a gRNA targeting a target site for GATA3 and comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 59 to 61 or a continuous portion thereof that is at least 14 nt; (i) a gRNA targeting a target site for CISH and comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 62 to 64 or a continuous portion thereof that is at least 14 nt; (j) a gRNA targeting a target site for PRDM1 and comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 63 to 64 (k) a gRNA that targets a target site for MED12 and comprises a gRNA spacer sequence comprising a sequence as set forth in any one of SEQ ID NOs: 91 to 101 or a continuous portion thereof that is at least 14 nt; (l) a gRNA that targets a target site for CCNC and comprises a gRNA spacer sequence comprising a sequence as set forth in any one of SEQ ID NOs: 113 to 123 or a continuous portion thereof that is at least 14 nt; (m) a gRNA that targets a target site for FAS and comprises a gRNA spacer sequence comprising a sequence as set forth in any one of SEQ ID NOs: 212 to 217 and 296 to 299 or a continuous portion thereof that is at least 14 nt; (n) a gRNA that targets a target site for Fli1 and comprises a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 212 to 217 and 296 to 299 or a continuous portion thereof that is at least 14 nt. a gRNA comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 218 to 223 or a continuous portion thereof that is at least 14 nt;and (o) a gRNA that targets a target site for TGFBR2 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 303-305 and 309-311, or a contiguous portion thereof that is at least 14 nt;

[0057] In any of the embodiments herein, the gRNA is selected from the group consisting of: (a) a gRNA that targets a target site for CD5 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 35-37; (b) a gRNA that targets a target site for KDM1A and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 38-40; (c) a gRNA that targets a target site for CBLB and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 44-46; (d) a gRNA that targets a target site for DGKZ and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 47-49; (e) a gRNA that targets a target site for MYB and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 50-52; and (f) a gRNA that targets a target site for RASA2 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 51-52. (g) a gRNA targeting a target site for ELOB and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 56-58; (h) a gRNA targeting a target site for GATA3 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 59-61; (i) a gRNA targeting a target site for CISH and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 62-64; (j) a gRNA targeting a target site for PRDM1 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 65-67; ​​(k) a gRNA targeting a target site for MED12 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 91-101; (l) a gRNA targeting a target site for CCNC and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: (m) a gRNA targeting a target site for FAS and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 212-217 and 296-299;(n) a gRNA that targets a target site for Fli1 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOS: 218-223; and (o) a gRNA that targets a target site for TGFBR2 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOS: 303-305 and 309-311.

[0058] In any of the embodiments herein, the gRNA is selected from (a) a gRNA that targets a target site for CBLB and comprises a gRNA spacer sequence set forth in SEQ ID NO: 45; (b) a gRNA that targets a target site for MYB and comprises a gRNA spacer sequence set forth in SEQ ID NO: 52; (c) a gRNA that targets a target site for RASA2 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 53; (d) a gRNA that targets a target site for CISH and comprises a gRNA spacer sequence set forth in SEQ ID NO: 62; (e) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 67; and (f) a gRNA that targets a target site for MED12 and comprises a gRNA spacer sequence set forth in SEQ ID NO: 91.

[0059] In any of the embodiments herein, the gRNA comprises a spacer sequence that is 14 nt to 24 nt, or 16 nt to 22 nt in length, or 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0060] In any of the embodiments herein, the gRNA further comprises a scaffold sequence as shown in SEQ ID NO:69.

[0061] Also provided herein is a guide RNA (gRNA) that targets a target site for a gene selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the target site for the gene is in the gene or in a regulatory DNA element thereof. In some embodiments, the regulatory DNA element is an enhancer or promoter.

[0062] In any of the embodiments herein, the target site is within 1000 base pairs (bp) of the transcription start site of the gene. In any of the embodiments herein, the target site is within 500 base pairs (bp) of the transcription start site of the gene.

[0063] In any of the embodiments herein, the target site is selected from the group consisting of: (a) a target site for VAV1 having the sequence set forth in any one of SEQ ID NOs: 7-9, 156, and 170, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for IL2 having the sequence set forth in SEQ ID NO: 78, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for BATF having the sequence set forth in any one of SEQ ID NOs: 172-174, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for CD28 having the sequence set forth in any one of SEQ ID NOs: 144-146 and 189-191, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for EOMES having the sequence set forth in any one of SEQ ID NOS: 175 to 177, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for LAT having the sequence set forth in any one of SEQ ID NOS: 184 to 186, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LCP2 having the sequence set forth in any one of SEQ ID NOS: 150 to 152 and 187 to 188, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (i) a target site for IRF4 having the sequence set forth in any one of SEQ ID NOS: 175 to 177, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. The target site for TBX21 is selected from the group consisting of a sequence set forth in any one of NOS: 153 to 155, a continuous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0064] In any of the embodiments herein, the target site is selected from the group consisting of: (a) a target site for VAV1 having the sequence set forth in any one of SEQ ID NOs: 7-9, 156, and 170, or a complementary sequence of any of the foregoing; (b) a target site for IL2 having the sequence set forth in SEQ ID NO: 78, or a complementary sequence of any of the foregoing; (c) a target site for BATF having the sequence set forth in any one of SEQ ID NOs: 172-174, or a complementary sequence of any of the foregoing; (d) a target site for CD28 having the sequence set forth in any one of SEQ ID NOs: 144-146 and 189-191, or a complementary sequence of any of the foregoing; (e) a target site for EOMES having the sequence set forth in any one of SEQ ID NOs: 147-149, or a complementary sequence of any of the foregoing; (g) a target site for IRF4 having the sequence set forth in any one of SEQ ID NOS: 175 to 177, or a complementary sequence of any of the foregoing; (g) a target site for LAT having the sequence set forth in any one of SEQ ID NOS: 184 to 186, or a complementary sequence of any of the foregoing; (h) a target site for LCP2 having the sequence set forth in any one of SEQ ID NOS: 150 to 152 and 187 to 188, or a complementary sequence of any of the foregoing; and (i) a target site for TBX21 having the sequence set forth in any one of SEQ ID NOS: 153 to 155, or a complementary sequence of any of the foregoing.

[0065] In any of the embodiments herein, the target site is selected from: (a) a target site for IL-2 having the sequence set forth in SEQ ID NO:78, or a complementary sequence of any of the foregoing; (b) a target site for EOMES having the sequence set forth in SEQ ID NO:149, or a complementary sequence of any of the foregoing; (c) a target site for LCP2 having the sequence set forth in SEQ ID NO:151, or a complementary sequence of any of the foregoing; and (d) a target site for TBX21 having the sequence set forth in SEQ ID NO:155, or a complementary sequence of any of the foregoing.

[0066] In any of the embodiments herein, the gRNA is selected from the group consisting of: (a) a gRNA that targets a target site for VAV1 and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 41-43, 169, and 171, or a continuous portion thereof that is at least 14 nt; (b) a gRNA that targets a target site for IL2 and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 79, or a continuous portion thereof that is at least 14 nt; (c) a gRNA that targets a target site for BATF and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 178-180, or a continuous portion thereof that is at least 14 nt; (d) a gRNA that targets a target site for CD28 and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 157-159 and 197-199, or a continuous portion thereof that is at least 14 nt; and (e) a gRNA that targets a target site for EOMES and comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 157-159 and 197-199. (f) a gRNA targeting a target site for IRF4 and comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 181 to 183 or a continuous portion thereof which is at least 14 nt; (g) a gRNA targeting a target site for LAT and comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 192 to 194 or a continuous portion thereof which is at least 14 nt; (h) a gRNA targeting a target site for LCP2 and comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 163 to 165 and 195 to 196 or a continuous portion thereof which is at least 14 nt; and (i) a gRNA targeting a target site for TBX21 and comprising a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 164 to 166 and 168 to 169; The gRNA is selected from gRNAs comprising a gRNA spacer sequence containing the sequence shown in any one of NOs: 166 to 168 or a continuous portion thereof that is at least 14 nt.

[0067] In any of the embodiments herein, the gRNA is selected from the group consisting of: (a) a gRNA that targets a target site for VAV1 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 41-43, 169, and 171; (b) a gRNA that targets a target site for IL2 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 79; (c) a gRNA that targets a target site for BATF and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 178-180; (d) a gRNA that targets a target site for CD28 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 157-159 and 197-199; (e) a gRNA that targets a target site for EOMES and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 160-162; and (f) a gRNA that targets a target site for IRF4 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 161-162. (g) a gRNA that targets a target site for LAT and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 192 to 194; (h) a gRNA that targets a target site for LCP2 and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 163 to 165 and 195 to 196; and (i) a gRNA that targets a target site for TBX21 and comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 166 to 168.

[0068] In any of the embodiments herein, the gRNA is selected from (a) a gRNA that targets a target site for IL-2 and comprises the gRNA spacer sequence set forth in SEQ ID NO:79; (b) a gRNA that targets a target site for EOMES and comprises the gRNA spacer sequence set forth in SEQ ID NO:162; (c) a gRNA that targets a target site for LCP2 and comprises the gRNA spacer sequence set forth in SEQ ID NO:164; and (d) a gRNA that targets a target site for TBX21 and comprises the gRNA spacer sequence set forth in SEQ ID NO:168.

[0069] In any of the embodiments herein, the gRNA comprises a spacer sequence that is 14 nt to 24 nt, or 16 nt to 22 nt in length. In some embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0070] In any of the embodiments herein, the gRNA further comprises a scaffold sequence as shown in SEQ ID NO:69.

[0071] Also provided herein are gRNA combinations comprising two or more gRNAs each selected from gRNAs targeting target sites for genes selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2, or two or more gRNAs each selected from gRNAs targeting target sites for genes selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the two gRNAs comprise the spacer sequences set forth in SEQ ID NOs: 92 and 45; 92 and 62; 45 and 52; and 45 and 53; 92 and 304; 92, 304, or 62. In some embodiments, the two gRNAs comprise spacer sequences as shown in SEQ ID NOs: 79 and 164; SEQ ID NOs: 79 and 168; SEQ ID NOs: 79 and 162.

[0072] Also provided herein are Cas-guide RNA (gRNA) combinations comprising (a) a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) protein or variant thereof and (b) at least one gRNA described herein. In some embodiments, the at least one gRNA targets a target site for a gene selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

[0073] Also provided herein are Cas-guide RNA (gRNA) combinations comprising (a) a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) protein or variant thereof and (b) at least one gRNA, in some embodiments, the at least one gRNA targets a target site for a gene selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

[0074] In any of the embodiments herein, the Cas protein or variant thereof is an inactivated (dCas) protein. In some embodiments, the dCas protein lacks nuclease activity. In some embodiments, the dCas protein is a dCas9 protein. In some embodiments, the dCas protein is a dCas12 protein. In some embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, based on the numbering of positions in SEQ ID NO: 124. In some embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 125, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the dSaCas9 is set forth in SEQ ID NO: 125. In some embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, based on the numbering of positions in SEQ ID NO: 126. In some embodiments, the dSpCas9 comprises the sequence set forth in SEQ ID NO: 127, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the dSpCas9 is set forth in SEQ ID NO: 127.

[0075] Also provided herein are polynucleotides that encode the epigenetic modification DNA targeting systems described herein.

[0076] Also provided herein are polynucleotides encoding at least one DNA targeting module of the epigenetic modification DNA targeting system described herein.

[0077] Also provided herein are polynucleotides encoding the fusion proteins of the epigenetic modification DNA targeting systems described herein and at least one gRNA.

[0078] Also provided herein are polynucleotides encoding the gRNAs described herein. In some embodiments, the polynucleotides encode combinations of gRNAs described herein.

[0079] Also provided herein are polynucleotides encoding the Cas-gRNA combinations described herein.

[0080] Also provided herein are two or more polynucleotides that together encode an epigenetic modification DNA targeting system described herein, at least one DNA targeting module of an epigenetic modification DNA targeting system described herein, a fusion protein of an epigenetic modification DNA targeting system described herein and at least one gRNA, a gRNA combination described herein, and / or a Cas-gRNA combination described herein.

[0081] Also provided herein is a combination of polynucleotide and gRNA, comprising: a) a polynucleotide encoding at least one fusion protein of a DNA targeting module for suppressing the transcription of one or more genes of the epigenetic modification DNA targeting system described herein, and one or more gRNAs selected from the gRNAs described herein; or b) a polynucleotide encoding at least one fusion protein of a DNA targeting module for increasing the transcription of one or more genes of the epigenetic modification DNA targeting system described herein, and one or more gRNAs selected from the gRNAs described herein. In some embodiments, the polynucleotide encoding the fusion protein is mRNA.

[0082] Also provided herein are vectors comprising the polynucleotides described herein.

[0083] Also provided herein are vectors comprising two or more polynucleotides described herein.

[0084] Also provided herein are vectors comprising a combination of the polynucleotides and gRNAs described herein.

[0085] Also provided herein are vectors comprising a combination of the polynucleotides and gRNAs described herein.

[0086] In any embodiment herein, the vector is a viral vector.In some embodiments, the vector is an adeno-associated virus (AAV) vector.In some embodiments, the vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9.

[0087] In any of the embodiments herein, the vector is a non-viral vector. In some embodiments, the non-viral vector is selected from a lipid nanoparticle, a liposome, an exosome, or a cell-penetrating peptide. In some embodiments, the non-viral vector is a lipid nanoparticle.

[0088] In any of the embodiments herein, the vector exhibits immune cell tropism, and optionally, the vector exhibits T cell tropism.

[0089] Also provided herein are modified T cells comprising a DNA targeting system described herein, a combination of DNA targeting systems described herein, a gRNA described herein, a combination of gRNAs described herein, a CRISPR Cas-gRNA combination described herein, a polynucleotide described herein, two or more polynucleotides described herein, or a combination of a polynucleotide and a gRNA described herein.

[0090] Also provided herein are modified T cells comprising an epigenetic or phenotypic modification resulting from contact with a DNA targeting system described herein, a DNA targeting combination described herein, a gRNA described herein, a gRNA combination described herein, a CRISPR Cas-gRNA combination described herein, a polynucleotide described herein, two or more polynucleotides described herein, a polynucleotide and gRNA combination described herein, or a vector described herein.

[0091] In any of the embodiments herein, the modified T cells are derived from cells from a subject. In any of the embodiments herein, the modified T cells are derived from primary T cells. In any of the embodiments herein, the modified T cells are derived from T cell precursors, pluripotent stem cells, or induced pluripotent stem cells. In any of the embodiments herein, the modified T cells further comprise an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR).

[0092] Also provided herein are methods of silencing transcription of one or more genes in a T cell, the method comprising introducing into a T cell a DNA targeting system described herein, a combination of DNA targeting systems described herein, a gRNA described herein, a combination of gRNAs described herein, a Cas-gRNA combination described herein, a polynucleotide described herein, two or more polynucleotides described herein, a combination of a polynucleotide and a gRNA described herein, or a vector described herein. In some embodiments, silencing the transcription of the one or more genes promotes increased T cell effector function upon T cell stimulation compared to T cell effector function in the absence of T cell stimulation.

[0093] Also provided herein are methods of increasing transcription of one or more genes in a T cell, the method comprising introducing into a T cell a DNA targeting system described herein, a combination of DNA targeting systems described herein, a gRNA described herein, a combination of gRNAs described herein, a Cas-gRNA combination described herein, a polynucleotide described herein, two or more polynucleotides described herein, a combination of a polynucleotide and a gRNA described herein, or a vector described herein. In some embodiments, increasing transcription of the one or more genes promotes increased T cell effector function upon T cell stimulation compared to T cell effector function in the absence of T cell stimulation.

[0094] Also provided herein is a method of increasing T cell effector function, the method comprising introducing into a T cell a DNA targeting system described herein, a combination of DNA targeting systems described herein, a gRNA described herein, a combination described herein, a CRISPR Cas-gRNA combination described herein, a polynucleotide described herein, two or more polynucleotides described herein, a combination of a polynucleotide and a gRNA described herein, or a vector described herein.

[0095] In any of the embodiments herein, T cell effector function is increased compared to a T cell not transfected with a DNA targeting system described herein, a combination of DNA targeting systems described herein, a gRNA described herein, a combination of gRNAs described herein, a CRISPR Cas-gRNA combination described herein, a polynucleotide described herein, two or more polynucleotides described herein, a combination of a polynucleotide and a gRNA described herein, or a vector described herein.

[0096] In any of the embodiments herein, the T cells are T cells in a subject, and the method is carried out in vivo. In any of the embodiments herein, the T cells are T cells from a subject or are derived from cells from a subject, and the method is carried out ex vivo. In any of the embodiments herein, the T cells are primary T cells. In some embodiments herein, the T cells are derived from T cell precursors, pluripotent stem cells, or induced pluripotent stem cells.

[0097] In any of the embodiments herein, the introducing step is by transient delivery to the T cells. In some embodiments, transient delivery includes electroporation, transfection, or transduction.

[0098] In any of the embodiments herein, a DNA targeting system described in any one of claims 1-95, a combination of DNA targeting systems described herein, a gRNA described herein, a combination of gRNAs described herein, a CRISPR Cas-gRNA described herein, a polynucleotide described herein, two or more polynucleotides described herein, a combination of a polynucleotide and a gRNA described herein, or a vector described herein is transiently expressed and / or transiently present in a T cell.

[0099] In any of the aspects herein, the introducing step suppresses transcription of one or more genes in the T cell selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

[0100] In any of the embodiments herein, the introducing step increases transcription of one or more genes in the T cell selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

[0101] Also provided herein are modified T cells produced by the methods described herein.

[0102] Also provided herein is a method of treating a disease or condition in a subject, the method comprising administering to the subject an engineered T cell described herein.

[0103] Also provided herein are methods of increasing T cell persistence in a subject's T cells, comprising administering to the subject or its T cells a DNA targeting system described herein, a combination of DNA targeting systems described herein, a gRNA described herein, a combination of gRNAs described herein, a CRISPR Cas-gRNA combination described herein, a polynucleotide described herein, two or more polynucleotides described herein, a combination of polynucleotides and gRNAs described herein, or a vector described herein. In some embodiments, the T cells are from adoptive T cell therapy for treating a disease or condition in the subject. In some embodiments, the T cell therapy comprises T cells expressing a recombinant receptor specific for a target antigen. In some embodiments, the administration is performed before, concurrently with, or after administration of adoptive T cell therapy. In some embodiments, the administration is performed after administration of adoptive T cell therapy in the subject, at a time after the number or effector function of T cells from the adoptive T cell therapy has decreased or is suspected to have decreased.

[0104] Also provided herein are methods of treating a disease or condition in a subject, the method comprising administering to the subject a T cell therapy comprising cells expressing a recombinant receptor specific for a target antigen associated with the disease or condition, and a DNA targeting system described herein, a combination of DNA targeting systems described herein, a gRNA described herein, a combination of gRNAs described herein, a CRISPR Cas-gRNA combination described herein, a polynucleotide described herein, two or more polynucleotides described herein, a combination of a polynucleotide and gRNA described herein, or a vector described herein. In some embodiments, the recombinant receptor is an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR).

[0105] In any of the embodiments herein, the target antigen is a tumor antigen. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a blood cancer or a solid tumor. In any of the embodiments herein, the disease or condition is an autoimmune condition and / or an inflammatory condition.

[0106] In any of the aspects herein, the administering step results in transient delivery to the T cell of: a DNA targeting system, a combination of DNA targeting systems, a gRNA, a combination of gRNAs, a CRISPR Cas-gRNA combination, a polynucleotide, two or more polynucleotides, a combination of a polynucleotide and a gRNA, or a vector.

[0107] In any of the aspects herein, the administering step suppresses transcription of one or more genes in T cells selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

[0108] In any of the embodiments herein, the administering step suppresses transcription of one or more genes in T cells selected from the group consisting of CBLB, CISH, MED12, MYB, PRDM1, and RASA2.

[0109] In any of the aspects herein, the administering step increases transcription of one or more genes in T cells selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

[0110] In any of the embodiments herein, the administering step increases transcription of one or more genes in T cells selected from the group consisting of EOMES, IL-2, LCP2, and TBX21. [Brief explanation of the drawings]

[0111] [Figure 1] Figure 1A shows an exemplary workflow for transient CRISPR screening. Cells are transfected with a gRNA library. After enrichment of transfected cells, the cells are transiently transfected with an epi-editor, such as dCas fused to an effector domain (e.g., a transcriptional repressor, a transcriptional activator, etc.), for example, by electroporation. The cells are then screened for the desired phenotype. Figure 1B shows an exemplary plot showing IL-2 and IFNg expression in cells as assessed by flow cytometry. The boxes indicate the populations sorted according to phenotype. [Figure 2] 1 shows a plot of an exemplary CRISPRi screen for gRNAs and genes that regulate IL-2 expression, IFNg expression, and / or proliferation. Specifically, the plot represents gRNAs that affect IL-2 expression. Dots represent individual gRNAs. The gRNAs on the left target genes whose inhibition results in decreased IL-2 expression, while the gRNAs on the right target genes whose inhibition results in upregulation of IL-2 expression. Dots represent individual gRNAs. The X-axis represents the log2 fold change in gRNA abundance in IL-2+ sorted cells compared to unsorted cells. The Y-axis represents significance (-log10 adjusted p-value). [Figure 3]Figure 1 shows plots from an exemplary CRISPRi screen for gRNAs and genes that regulate IL-2 expression, IFNg expression, and / or proliferation. Specifically, the plots represent gRNAs that affect proliferation. Dots represent individual gRNAs. The gRNAs on the left target genes whose inhibition results in decreased proliferation, while the gRNAs on the right target genes whose inhibition results in increased proliferation. The x-axis represents the log2 fold change in gRNA abundance in unsorted cells 6 days after electroporation with a transiently expressed epieditor for targeted transcriptional repression compared to unsorted cells before electroporation. The y-axis represents significance (-log10 adjusted p-value). [Figure 4] The number of gRNA hits from the indicated conditions is represented on days 9, 12, or both 9 and 12 of a CRISPRi screen for gRNAs and genes that modulate T cell phenotype. [Figure 5] 1 shows plots from an exemplary CRISPRa screen for gRNAs and genes that regulate IL-2 expression, IFNg expression, and / or proliferation. Specifically, the plots represent gRNAs that affect IL-2 expression. Dots represent individual gRNAs. The gRNAs on the left target genes whose activation results in decreased IL-2 expression, while the gRNAs on the right target genes whose activation results in increased IL-2 expression. The X-axis represents the log2 fold change in gRNA abundance in IL-2+ sorted cells relative to unsorted cells. The Y-axis represents significance (-log10 adjusted p-value). [Figure 6A] Figure 1 shows the percent knockdown (%KD) of MED12 expression assessed by RT-qPCR after transient delivery of dSpCas9-KRAB and the indicated MED12-targeting or non-targeting gRNAs (NT1, NT2), or dSpCas9-KRAB alone. Results are shown for T cells from two different donors at 48 hours and 6 days post-transfection. [Figure 6B]MED12 expression was assessed by RT-qPCR in T cells (left) and CAR T cells (right) after transient delivery of dSpCas9-KRAB and the indicated MED12-targeting gRNAs. For the left panel, control conditions included cells delivered with dSpCas9-KRAB and non-targeting gRNAs (NT1, NT2) and cells delivered with dSpCas9-KRAB alone. For the right panel, control conditions included cells not expressing CAR (mock) and cells not delivered with the DNA targeting system. Results are shown 48 hours (left) and 72 hours and 7 days after transfection. Results are normalized to the dSpCas9-KRAB-only condition (left panel) or mock condition (right panel). [Figure 6C] Figure 1 shows the percent knockdown (%KD) of CCNC expression assessed by RT-qPCR after transient delivery of dSpCas9-KRAB with the indicated CCNC-targeting gRNA or non-targeting gRNA (NT1, NT2), or dSpCas9-KRAB alone. Results are shown for T cells from two different donors 48 hours and 6 days after transfection. [Figure 6D] Figure 1 shows the percent knockdown (%KD) of FAS expression assessed by intracellular cytokine staining (ICS) and flow cytometry after transient delivery of dSpCas9-KRAB and the indicated FAS-targeting gRNA, or dSpCas9-KRAB alone, or mock delivery. Results are shown for T cells from two different donors at 72 hours and 7 days after delivery. The top panel shows exemplary flow cytometry plots for assessing FAS expression in CD3+ T cells after delivery of dSpCas9-KRAB alone or dSpCas9-KRAB and the FAS-targeting gRNA. [Figure 7A]Figure 1 depicts flow cytometry plots to assess IL-2 expression in CD3+ Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 with an IL-2-targeting gRNA (IL-2_1, targeting SEQ ID NO:78). Results are shown for CAR T cells derived from two different donors. CAR T cells were stimulated with SKOV3 or 143B cells. Various control conditions omitted the CAR, gRNA, or stimulation, as indicated. [Figure 7B] Quantification of IL-2 expression (% IL-2+ cells assessed by flow cytometry) in Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 with an IL-2-targeting gRNA (IL-2_1, targeting SEQ ID NO:78) is shown. Control conditions omitted the CAR (mock transduction), or omitted the gRNA, or stimulation, as indicated. [Figure 7C] Figure 1 shows IL-2 expression in CAR T cells after delivery of a DNA targeting system for IL-2 activation. The left panel shows IL-2 expression in Her2 CAR T cells assessed by RT-qPCR 72 hours and 7 days after transient delivery of a DNA targeting system containing dSpCas9-2xVP64 and an IL-2-targeting gRNA (IL-2_1, targeting SEQ ID NO:78). Control conditions included cells not expressing a CAR (mock) or CAR T cells not delivered with a DNA targeting system (CAR only). Expression levels were normalized to mock control cells. The right panel shows IL-2 expression in control cells (CAR only) and CAR T cells delivered with a DNA targeting system for IL-2 activation after stimulation with Her2 antigen-expressing tumor cells, assessed by ICS and flow cytometry. [Figure 8A]Figures 8A-8C represent flow cytometry plots to assess IL-2 (Figure 8A), IFNg (Figure 8B), or TNFa (Figure 8C) expression in CD3+ Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 and VAV1-targeting gRNA (VAV1_5, targeting SEQ ID NO:170). Results are shown for CAR T cells derived from two different donors. CAR T cells were stimulated with SKOV3 or 143B cells. Various control conditions omitted the CAR, gRNA, or stimulation, as indicated. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 9A] Figure 1 depicts the percentage of multifunctional cells (IL-2+ / IFNg+ / TNFa+ cells) assessed by flow cytometry in Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 with IL-2-targeting gRNA (IL-2_1, targeting SEQ ID NO:78) or VAV1-targeting gRNA (VAV1_5, targeting SEQ ID NO:170). Control conditions omitted the CAR (mock transduction), or omitted the gRNA, or stimulation, as indicated. [Figure 9B] Figure 1 depicts the percentage of IL-2-expressing Her2 CAR T cells over a number of days after stimulation and transient delivery of dSpCas9-2xVP64 with IL-2-targeting gRNA (IL-2_1, targeting SEQ ID NO:78) or VAV1-targeting gRNA (VAV1_5, targeting SEQ ID NO:170). [Figure 9C] Figure 1 depicts flow cytometry plots to assess IL-2 expression in Her2 CAR T cells before and after stimulation and transient delivery of gRNA with effector systems. [Figure 10A]Figures 10A-10B present flow cytometry plots and mean fluorescence intensity (MFI) for assessing IL-2 and IFNg expression in CD4+ (Figure 10A) or CD8+ (Figure 10B) Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 with the indicated CBLB-targeting gRNA or a control non-targeting gRNA. As shown in the bottom panels, flow cytometry plots allow quantification of the percentage of cells with a particular phenotype (e.g., IL-2+), as well as quantification of the mean fluorescence intensity (MFI; corresponding to the average expression level). [Figure 10B] See legend to Figure 10A. [Figure 11A] Heatmaps depicting the relative levels of intracellular cytokine expression (assessed by ICS and flow cytometry) in Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 with the indicated gRNAs. Cytokine expression was quantified according to the % Her2 CAR T cells with the indicated phenotype (e.g., CD4+ / IL-2+, CD4+ / IFNg+ / TNFa+) assessed by flow cytometry, and the log2 fold change was calculated relative to the control condition using a non-targeting gRNA (non-targeting_sp_1). Darker shading indicates higher cytokine expression, as indicated in the legend. Each condition was assigned a cumulative score based on all cytokine expression measurements and ranked from low to high cytokine expression. [Figure 11B]Exemplary results from a sequential stimulation-killing assay are shown. Using an Incucyte automated tracking system, growth of antigen-expressing target cells expressing a fluorescent marker was quantified over time based on total fluorescence. Antigen-expressing target cells were cocultured with CAR T cells transiently delivered with a DNA targeting system for IL-2 activation, CAR T cells transiently delivered with a DNA targeting system for MED12 inhibition, CAR T cells without a DNA targeting system (CAR only), or cells not expressing a CAR (mock). Sequential stimulations (shown as Stim 1, Stim 2, and Stim 3) were performed by replating CAR T cells with fresh target cells at a 1:4 CAR T cell:target cell ratio. [Figure 11C] Quantification of fold expansion (left) and IL-2 secretion (right) by CAR T cells and control cells after the second stimulation from the experiment depicted in Figure 11B is shown. [Figure 12A] Flow cytometry plots of intracellular cytokine staining (ICS) for IL-2 and IFN-γ expression after transient transfection of T cells with control dSpCas9-2xVP64 effector alone (no targeting gRNA) or dSpCas9-2xVP64 individually combined with gRNAs targeting either VAV1 (VAV1_5) or IL-2 (IL-2_1). [Figure 12B] 10 depicts ICS flow cytometry plots for IL-2 and IFN-g after transient transfection of T cells with dSpCas9-2xVP64 effector and gRNAs targeting both VAV1 and IL-2. [Figure 12C]Shown are the percentages of IL-2+ cells (left panel) or IL-2+, IFNg+, and TNFalpha+ cells (right panel) after transient transfection of T cells from either of two donors with dSpCas9-2xVP64 effector alone (no targeting gRNA), dSpCas9-2xVP64 combined with VAV1 or IL-2, or dSpCas9-2xVP64 combined with gRNAs targeting both VAV1 and IL-2. [Figure 13A] Figures 13A-13B present heat maps showing the relative levels of intracellular cytokine expression, cytokine secretion, proliferation, and target cell killing activity in Her2 CAR T cells derived from a first donor (Figure 13A) and a second donor (Figure 13B) after stimulation and transient delivery of dSpCas9-2xVP64 or dSpCas9-KRAB with the indicated gRNAs. As described in Example 5, multiple stimulations were performed and cells electroporated with various DNA targeting systems were evaluated based on multiple readouts of T cell effector function after stimulation, including intracellular cytokine expression (by ICS), cytokine secretion, proliferation, and target cell killing. Readouts of T cell effector function were quantified after the first, second, and / or third stimulation (denoted as Stim 1, Stim 2, or Stim 3 in the figures). ICS was performed only after the first stimulation. Results are shown as Log2 fold change compared to control cells delivered with non-targeting gRNA (NT). As indicated in the legend, darker shading indicates increased measured T cell effector function. Each condition was assigned a cumulative score across all measurements and ranked from low to high T cell effector function. [Figure 13B] See legend to Figure 13A. [Figure 14]Heatmaps depicting the relative levels of intracellular cytokine expression in Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 with a combination of two gRNAs targeting the indicated genes are shown. The gRNAs targeting each gene are indicated next to the heatmap. When the targeting gene was the same, the same gRNA was delivered at twice the concentration. Cytokine expression was quantified according to the % Her2 CAR T cells with the indicated phenotype (e.g., CD8+ / IL-2+ / IFNg+ / TNFa+) as assessed by ICS and flow cytometry, and the log2 fold change was calculated relative to the control condition using a non-targeting gRNA (NT+NT). As indicated in the legend, darker shading indicates higher cytokine expression. Each condition was assigned a cumulative score based on all cytokine expression measurements and ranked from low to high cytokine expression. [Figure 15A] Figures 15A-15B present heat maps showing the relative levels of proliferation and cytokine expression in Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 or dSpCas9-KRAB with gRNAs targeting the indicated genes or their combinations. The gRNAs targeting each gene are listed in the accompanying tables. Cytokine expression was quantified according to the % Her2 CAR T cells with the indicated phenotype (e.g., CD4+ / IL-2+ / IFNg+ / TNFa+) as assessed by ICS and flow cytometry. Proliferation was quantified according to the fold change in Her2 CAR T cell numbers before and after stimulation, as described herein. For each cytokine or proliferation phenotype value, the log2 fold change was calculated relative to the control condition using a non-targeting gRNA (non-targeting_sp_1 for Figure 15A; dCas only for Figure 15B). As indicated in the legend, darker shading indicates increased proliferation and cytokine expression. Each condition was assigned a cumulative score for all cytokine and proliferation measurements and ranked from low to high cytokine expression and proliferation. [Figure 15B] See legend to Figure 15A. [Figure 16] Figure 1 depicts the fold expansion (i.e., proliferation) of Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 or dSpCas9-KRAB with the indicated gRNA. Results are shown for Her2 CAR T cells from two different donors after the first stimulation with antigen-expressing target cells. Negative control conditions included CAR T cells delivered with dCas-effector but no gRNA, CAR T cells delivered without the DNA targeting system (CAR only), and cells not expressing a CAR (mock). [Figure 17] Figure 1 depicts the levels of secreted cytokines IL-2 or IFNg in Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 or dSpCas9-KRAB with the indicated gRNA. Results are shown for Her2 CAR T cells from two different donors after the first stimulation with antigen-expressing target cells. Negative control conditions included CAR T cells delivered with dCas-effector but no gRNA, CAR T cells delivered without the DNA targeting system (CAR only), and cells not expressing a CAR (mock). [Figure 18] Figure 1 shows the killing activity based on the calculated killing index in Her2 CAR T cells after transient delivery of dSpCas9-2xVP64 or dSpCas9-KRAB with the indicated gRNAs after the third stimulation with antigen-expressing target cells. Negative control conditions included CAR T cells delivered with dCas-effector but no gRNA, CAR T cells delivered without the DNA targeting system (CAR only), and cells not expressing a CAR (mock). [Figure 19A]Figures 19A-19B present heat maps showing the relative levels of quantified T cell effector function in Her2 CAR T cells after stimulation and transient delivery of dSpCas9-2xVP64 or dSpCas9-KRAB with the indicated gRNA. Results are shown for cells derived from the first donor (Figure 19A) and the second donor (Figure 19B). T cell effector functions assessed included intracellular cytokine expression, secreted cytokine expression, proliferation, and killing, which were measured as described in Example 5. T cell effector function was quantified after the first, second, and / or third stimulation with antigen-expressing target cells, as indicated. For each quantified T cell effector function, results are presented as the log2 fold change calculated relative to the negative control condition (CAR only, i.e., CAR T cells without the DNA targeting system delivered). As indicated in the legend, darker shading indicates increased measured T cell effector function. Each condition was assigned a cumulative score across all measures and ranked from low to high T cell effector function. [Figure 19B] See legend to Figure 19A. [Figure 20A] Figures 20A-20C show results from CAR T cells electroporated with gRNAs targeting the indicated genes and mRNA encoding either dSpCas9-2xVP64 for activation or dSpCas9-KRAB for inhibition. For each result, experiments were performed using CAR T cells from two different donors, and results are plotted for each of the two donors (triangles). The mean values ​​are indicated by vertical lines. Figure 20A shows the CAR T expansion fold after electroporation of the DNA targeting system (preparation) and after the first, second, and third stimulations with Her2 antigen-expressing target cells. Figure 20B shows the CAR T cell target cell killing index after the first and second stimulations. Figure 20C shows CAR T cell cytokine production, assessed by ICS and flow cytometry, 9 days after electroporation of the DNA targeting system and after the first stimulation with Her2 antigen-expressing target cells. [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 21-1]Figures 21A-21C show results from stimulated CAR T cells delivered with a DNA targeting system to suppress TGF-beta receptor 2 (TGFBR2). The DNA targeting system contained the indicated TGFBR2-targeting gRNA and either dSpCas9-KRAB (SEQ ID NO:332) or DNMT3A / L-XTEN80-dSpCas9-KRAB (SEQ ID NO:337). Control cells included dSpCas9 alone, cells not expressing a CAR (mock), or CAR T cells not stimulated with antigen-expressing cells (CAR only). Figure 21A shows TGFBR2 expression 48 hours after electroporation with the DNA targeting system. Figure 21B shows secreted IFNg from CAR T cells 24 hours after the second stimulation with Her2 antigen-expressing cells in the presence of 10 ng / mL TGFb. Figure 21C shows the fold expansion of stimulated CAR T cells after a second stimulation with Her2 antigen-expressing cells in the presence of 10 ng / mL TGFb. Figures 21D-21G show results from stimulated CAR T cells delivered with a DNA targeting system to inhibit TGF-beta receptor 2 (TGFBR2). CAR T cells were electroporated for transient expression of the DNA targeting system composed of dSpCas9-KRAB-DNMT3A / L and the indicated gRNA targeting TGFBR2. Negative control cells were electroporated with dSpCas9-KRAB-DNMT3A / L and a non-targeting gRNA (non-targeting), or without the DNA targeting system (CAR only), or negative control cells did not express a CAR (mock). Figure 21D shows the % TGFBR2-negative cells at the indicated days after electroporation. Figure 21E shows the fold cell expansion of CAR T cells after stimulation with anti-CD3 / anti-CD28 coated wells and exposure to the indicated concentrations of TGF-beta. Expansion is normalized to the 0 ng / mL TGF-beta condition. Figure 21F shows the production of IFNg by CAR T cells in response to exposure to 10 ng / mL TGF-beta. The dotted horizontal line indicates the CAR-only control condition.Figure 21G shows the levels of secreted cytokines in stimulated CAR T cells after exposure to the indicated concentrations of TGF-beta. Secreted cytokine levels are normalized to the 0 ng / mL condition. [Figure 21-2] See description of Figure 21-1. [Figure 21-3] See description of Figure 21-1. [Figure 22A] Figures 22A-22D show the time course (Figure 22A) and results (Figures 22B-22D) from an experiment to evaluate in vivo CAR T cells delivered with a DNA targeting system for IL-2 activation or MED12 or CBLB inhibition. This in vivo experiment was performed by injecting CAR T cells into immunodeficient mice implanted with antigen-expressing tumor cells (NCI-H1975). Figure 22A shows the timing and details of tumor implantation, CAR T cell injection, and tracking. Figure 22B shows the results of the experiment, including animal survival (left panel), tumor growth (middle panel), and circulating CAR T cell levels (right panel), for CAR T cells delivered with a DNA targeting system for IL-2 activation (dSpCas9-2xVP64 and gRNA IL-2_1, targeting SEQ ID NO:78). Figure 22C shows experimental results for CAR T cells delivered with a DNA targeting system for MED12 inhibition (dSpCas9-KRAB and gRNA MED12_2, targeting SEQ ID NO:81), including animal survival (left panel), tumor growth (middle panel), and circulating CAR T cell levels (right panel). Figure 22D shows experimental results for CAR T cells delivered with a DNA targeting system for CBLB inhibition (dSpCas9-KRAB and gRNA CBLB_2, targeting SEQ ID NO:11), including tumor growth (left panel), and circulating CAR T cell levels (right panel). Control mice were injected with CAR T cells without the DNA targeting system (CAR only), T cells that do not express a CAR (mock T cells), or no T cells (tumor only). [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D] See legend to Figure 22A. [Figure 23] Figure 1 shows MED12 expression assessed by RT-qPCR at days 4 and 21 after electroporation with MED12-targeting gRNA (MED12_2, targeting SEQ ID NO:81) and mRNA encoding dSpCas9 (control; no transcriptional repression effector domain), dSpCas9-KRAB (SEQ ID NO:332), or DNMT3A / L-XTEN80-dSpCas9-KRAB (SEQ ID NO:337). Expression levels (shown as fold change in expression) are normalized to the expression level in T cells electroporated with the same fusion protein together with a non-targeting gRNA (dotted line of 1.0). [Figure 24-1]Figures 24A-24F show results from CAR T cells delivered with a DNA targeting system containing the indicated MED12-targeting gRNA and dSpCas9-KRAB (SEQ ID NO:332) or DNMT3A / L-XTEN80-dSpCas9-KRAB (SEQ ID NO:337). Control cells included cells delivered with DNMT3A / L-XTEN80-dSpCas9-KRAB without gRNA, cells delivered with DNMT3A / L-XTEN80-dSpCas9-KRAB and a non-targeting gRNA, and T cells not expressing a CAR (mock). Figure 24A shows MED12 expression assessed by qRT-PCR at 10 days after electroporation of the DNA targeting system. Figure 24B shows MED12 expression at 2, 7, 10, and 14 days after electroporation. Figure 24C represents CD25 expression assessed by flow cytometry on CAR T cells 3, 7, 10, and 14 days after electroporation. Figure 24D shows secreted IFN-γ expression and Figure 24E represents secreted IL-2 expression 24 hours after the second stimulation of CAR T cells with Her2-positive NCI-H1975 tumor cells. Figure 24F represents proliferation (fold expansion normalized to CAR-only control) after the second stimulation with antigen-expressing cells. [Figure 24-2] See description of Figure 24-1. [Figure 24-3] See description of Figure 24-1. [Figure 25] Figure 1 shows the time course of expression of an exemplary dSpCas9 protein after electroporation of mRNA encoding that protein. Results are shown for electroporation of dSpCas9 protein with a non-targeting gRNA or a gRNA targeting a gene in the cell, or no electroporation (control). Expression was assessed based on the associated GFP tag. Results show the percentage of GFP+ cells assessed by flow cytometry at the indicated time points. [Figure 26]Figures 26A-B show configurations of fusion proteins for targeted transcriptional repression (Figure 26A) and the results of experiments testing the ability of the fusion proteins to mediate sustained target gene repression (Figure 26B). Figure 26A shows four different configurations of dSpCas9 fusion proteins, from N- to C-terminus. These fusion proteins contain various domains selected from DNMT3A, DNMT3B, DNMT3L, and the KRAB or EZH2 domain (denoted as [KRAB]). Figure 26B shows MED12 expression at 4 and 21 days in T cells electroporated with a gRNA targeting MED12 (MED12_2, targeting SEQ ID NO:81) and mRNA encoding fusion proteins with each of the four different configurations and containing a repression domain (shown as [KRAB]) selected from the KRAB domain of KOX1 (KOX1(2-99)) (SEQ ID NO:355; the KRAB domain used in the dSpCas9 fusion proteins in the previous examples), the KRAB domain of KOX1 (KOX1(1-72)) (SEQ ID NO:356), the KRAB domain of ZIM3 (SEQ ID NO:357), the KRAB domain of ZNF324 (SEQ ID NO:358), and the EZH2 domain (SEQ ID NO:359). The mRNA encoding the fusion protein further contained an N-terminal FLAG epitope and a C-terminal P2A-mCherry domain to assess fusion protein expression. For each experimental condition, MED12 expression was normalized to the expression level in T cells electroporated with the same fusion protein together with a non-targeting gRNA. [Figure 27A] Figure 1 depicts expression of the indicated genes, assessed by RT-qPCR, in CAR T cells 72 hours after transient delivery of a dSpCas9 fusion protein for inhibition (e.g., dSpCas9-KRAB-DNMT3A / L) with gRNAs targeting the indicated genes and / or a control non-targeting gRNA ("NT guide"). [Figure 27B]IL-2 expression measured by ICS and flow cytometry was determined based on the percentage of viable T cells that were IL-2+ after each indicated stimulation and quantified as fold change relative to control cells delivered with a non-targeting gRNA. [Figure 27C] IL-2 expression before and after TGF-beta treatment and transient delivery of a DNA targeting system for multiplexed inhibition of MED12 and TGFBR2. [Figure 28A] Figures 28A-B depict tumor cell killing over time by Her2 CAR T cells delivering either mRNA encoding the dSpCas9-2xVP64 effector fusion protein, SpCas9 IL-2 targeting gRNA, and SpCas9 TBX21 targeting gRNA, or mRNA encoding the dSpCas9-KRAB-DNMT3A / L effector fusion protein, SpCas9 MED12 targeting gRNA, and SpCas9 CBLB targeting gRNA. [Figure 28B] See legend to Figure 28A. [Figure 29A] Figures 29A and 29B represent results from experiments in mouse tumor models for the in vivo evaluation of CAR T cells transiently delivered with single or multiple DNA targeting systems for IL-2, LCP2, EOMES, or TBX21 activation, where CAR T cells were administered to mice at low doses (Figure 29A) or high doses (Figure 29B). [Figure 29B] See legend to Figure 29A. [Figure 30A] Figures 30A and 30B represent results from experiments in mouse tumor models for the in vivo evaluation of CAR T cells transiently delivered with single or multiple DNA targeting systems for MED12, CBLB, and / or CISH suppression, where CAR T cells were administered to mice at low doses (Figure 30A) or high doses (Figure 30B). [Figure 30B] See legend to Figure 30A. DETAILED DESCRIPTION OF THE INVENTION

[0112] Detailed Description Provided herein is an epigenetic modification DNA targeting system, wherein the DNA targeting system comprises at least one DNA targeting module composed of a fusion protein comprising (a) a DNA binding domain capable of targeting to a target site in one or more genes or their regulatory DNA elements in T cells, and (b) at least one effector domain capable of regulating the transcription of the one or more genes. In some embodiments, the target site is in a gene or its regulatory region found herein to be a negative regulator of T cell function after transient transcriptional regulation of the gene, such as a target site in CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and / or RASA2. In some such embodiments, the at least one effector domain is a transcriptional repression domain, such as KRAB or DNMT3A / 3L, or a combination thereof. In some embodiments, the target site is in a gene or regulatory region thereof found herein to be a positive regulator of T cell function following transient transcriptional regulation of the gene, such as target sites in BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and / or VAV1. In some such embodiments, the at least one effector domain is a transcription activation domain, e.g., VP64. In some embodiments, the target site is within 1000 base pairs of the transcription start site (TSS) of any such gene, e.g., the target site can be within a regulatory region, such as a promoter or enhancer, of any such gene.

[0113] In some embodiments, the DNA targeting system is a synthetic transcription factor that can target and regulate, for example, decrease (or down-regulate) or increase (or up-regulate), gene transcription. In some embodiments, the DNA-binding domain of the DNA targeting system is a nuclease-inactive CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) protein (e.g., a dCas protein) or a variant thereof complexed with a guide RNA (gRNA). Also provided is a gRNA for targeting a target site in a gene or its regulatory DNA element in a T cell, where the gene is any gene provided herein whose transient epigenetic regulation of transcription has been found to promote T cell function. Also provided are CRISPR-Cas / gRNA combinations composed of a gRNA and a nuclease-inactivated Cas, such as dCas9. Also provided herein are polynucleotides encoding the DNA targeting system or a fusion protein of the DNA targeting system, as well as vectors and cells containing the same. Also provided herein are methods of using the epigenetic modification DNA targeting system to modulate T cell transcription or phenotype or function, and the resulting modified cells.

[0114] In some embodiments, DNA targeting system comprises at least one DNA targeting module, and each DNA targeting module of the system is a component of the DNA targeting system that can independently target one target site for the provided target gene.In some embodiments, each DNA targeting module comprises (a) a DNA binding domain that can be targeted to the target site for the provided target gene, and (b) an effector domain that can regulate (for example, repress or activate) the transcription of gene.

[0115] In some embodiments, the DNA targeting system comprises a single DNA targeting module for targeting the suppression of a single gene.In some embodiments, the gene is CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2 or RASA2.In some embodiments, the gene is CBLB, CISH, MED12, MYB, PRDM1 or RASA2.In some embodiments, the DNA targeting module comprises (a) a DNA binding domain that can be targeted to the target site of target gene or regulatory element, and (b) an effector domain that can reduce the transcription of gene.

[0116] In some embodiments, DNA targeting system comprises multiple DNA targeting modules, and each DNA targeting module is a module for targeting the suppression of different genes.In some embodiments, DNA targeting system is a multiple DNA targeting system, that is, it targets the target site for multiple genes.Therefore, the term DNA targeting system can include the multiple epigenetic modification DNA targeting system that comprises multiple DNA targeting modules.The multiple epigenetic modification DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 DNA targeting modules. In some embodiments, the multiple DNA targeting modules target multiple target sites for one or more genes, e.g., two, three, four or more genes, selected from CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

[0117] In some embodiments, the DNA targeting system comprises a single DNA targeting module for targeting the activation or increased expression of a single gene. In some embodiments, the gene is BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21 or VAV1. In some embodiments, the gene is EOMES, IL-2, LCP2 or TBX21. In some embodiments, the DNA targeting module comprises (a) a DNA binding domain that can be targeted to a target site of a target gene or regulatory element, and (b) an effector domain that can activate the transcription of the gene.

[0118] In some embodiments, the DNA targeting system comprises multiple DNA targeting modules, and each DNA targeting module is a module for targeting the activation of expression or increased transcription of different genes.In some embodiments, the DNA targeting system is a multiplex DNA targeting system, i.e., it targets the target sites of multiple genes.Therefore, the term DNA targeting system can include a multiplex epigenetic modification DNA targeting system that comprises multiple DNA targeting modules.The multiplex epigenetic modification DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 DNA targeting modules.In some embodiments, the multiple DNA targeting modules target multiple target sites for one or more genes, for example, two or three genes, selected from BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

[0119] In some embodiments, any two DNA targeting modules of the DNA targeting system comprise separate (i.e., non-overlapping) components. In some embodiments, different DNA targeting modules of the DNA targeting system comprise separate (i.e., non-overlapping) components. For example, the DNA targeting system may comprise a first DNA targeting module comprising a first fusion protein comprising a DNA binding domain (e.g., a ZFN or TALE-based DNA binding domain) that targets a first target site, and a second DNA targeting module comprising a second fusion protein comprising a second DNA binding domain (e.g., a ZFN or TALE-based DNA binding domain) that targets a second target site.

[0120] In some embodiments, any two DNA targeting modules of a DNA targeting system may contain shared (i.e., overlapping) components. In some embodiments, different DNA targeting modules of a DNA targeting system may contain shared (i.e., overlapping) components. For example, in one aspect, a DNA targeting system may include: (a) a fusion protein comprising a Cas protein and a transcription effector (e.g., repressor) domain; and (b) a first gRNA that forms a complex with the Cas protein and targets a first target site; and (a) a fusion protein of the first DNA targeting module; and (b) a second gRNA that forms a complex with the Cas protein and targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the same Cas protein to target the target sites of those two or more gRNAs. Conversely, different Cas protein variants (e.g., SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, making it possible to engineer a single DNA targeting system containing multiple non-overlapping CRISPR / Cas-based DNA targeting modules.

[0121] Provided embodiments relate to compositions and methods for promoting T cell function, e.g., one or more T cell effector functions, by epigenetically modifying target sites in one or more target genes. In some embodiments, these methods can be used in the context of T cell therapy, e.g., adoptive T cell therapy. In some embodiments, modulating the transcription of the one or more genes increases or improves one or more T cell phenotypes or functions. In some embodiments, a T cell effector function is increased, such as the ability to produce cytokines, e.g., IL-2 or IFN-gamma (IFNg), the ability of T cells to proliferate, the ability of T cells to kill target cells, or the ability of T cells to mount a sustained immune response. In certain embodiments, modulating the one or more genes improves T cell effector function after or upon T cell stimulation, including after successive stimulations that mimic the repeated antigen encounters that occur in vivo.

[0122] The administration of T cells targeting specific antigens, also known as adoptive cell therapy (ACT), is a promising approach for treating diseases such as cancer. However, current ACT treatments face challenges, including suboptimal T cell function, expansion, and persistence. Furthermore, the persistence and functionality of transferred T cells can vary significantly between different T cell subsets and between T cells from different patients. Recent clinical trials of ACT suggest that the ability to persist long-term in the circulation depends on the differentiation stage of T cells, including their ability to retain a network of transcription factors and metabolic regulators (Pilipow K. et al., Journal of Clinical Investigation Insight 2018;3(18):e122299). T cells transferred into patients are often terminally differentiated and therefore unable to persist long-term, ultimately limiting effective antitumor responses. For example, although the first CAR T-cell therapy was approved by the FDA as a cell and gene therapy in 2017, patients whose cancer recurs or who do not respond to treatment often suffer from a lack of persistence of CAR T cells (Mueller et al., Blood (2018)). Furthermore, durable benefits have yet to be observed for CAR T-cell therapy in solid tumors.

[0123] Strategies to mitigate these challenges and enhance the persistence, expansion, and antitumor activity of chimeric antigen receptor (CAR)-engineered T cells are being tested both preclinically and clinically. Strategies for optimizing ex vivo T cell culture conditions have been explored, including, for example, the addition of cytokines during production (Besser MJ, Cytotherapy 2009;11(2):206-17), cytokine and / or receptor expression by CAR T cells (Krenciute G., Cancer Immunol Res. 2017 07;5(7):571-581), the use of pharmacological inhibitors during expansion, such as AKT (Urak R. et. al., Journal of Immunotherapy Cancer 2017 Mar 21;5:26) or PI3K (Peterson CT et. al., Blood Advances 2018 Feb 13;2(3):210-223), immune depletion, and checkpoint blockade (Cherkassky L. et. al., Journal of clinical investigation 2016 Aug 1;126(8):3130-44). However, existing strategies have not been entirely satisfactory, and in some cases concerns regarding cytokine-induced toxicity or the emergence of lymphoproliferative disorders as a result of the above strategies have raised questions about alternative approaches.

[0124] Provided embodiments relate to identifying genomic locations that are epigenetically modified in T cells to influence or promote T cell effector function (including TCR- and / or CAR-induced or -dependent induced) upon T cell stimulation, as demonstrated, for example, by assessing cells that produce IL-2 and / or IFNg, have the capacity to proliferate, or have the capacity to kill target cells. In some embodiments, the stimulatory condition or agent comprises one or more agents, e.g., ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the agent activates or initiates the TCR / CD3 intracellular signaling cascade in the T cell. Such agents can include, for example, antibodies bound to a solid support such as beads, e.g., antibodies specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, and / or one or more cytokines. In some embodiments, the one or more agents are PMA and ionomycin. In some embodiments, the T cell stimulation is antigen-specific stimulation, and the cells are stimulated with an agent that provides an antigen or an epitope thereof specific to or recognized by an antigen receptor (e.g., CAR) expressed on the T cell. For example, the stimulating agent can include an antigen-expressing target cell. In certain embodiments, the phenotype is or includes the production or secretion of cytokines such as IL-2 or IFN-g in response to T cell stimulation. Cytokine production and / or secretion contribute to the immune response and are involved in various processes, including the induction of antiviral proteins and the induction of T cell proliferation. Cytokines are not preformed factors, but are rapidly produced and secreted in response to cell activation. Cytokine production or secretion can be measured, detected, and / or quantified by any suitable technique known in the art.

[0125] In certain embodiments, T cell function is the production of one or more cytokines. In particular embodiments, the production of one or more cytokines is measured, detected, and / or quantified by intracellular cytokine staining. Intracellular cytokine staining by flow cytometry (ICS) is a well-suited technique for examining cytokine production at the single-cell level. It detects cytokine production and accumulation within cells (e.g., in the endoplasmic reticulum) after cell stimulation, allowing for the identification of cell populations that are positive or negative for the production of a particular cytokine, or the separation of high- and low-producing cells, based on a threshold value. ICS can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or MHC multimers to assess cytokine production in specific subgroups of cells, making it a flexible and versatile method. Other single-cell techniques for measuring or detecting cytokine production include, but are not limited to, ELISPOT, limiting dilution, and T cell cloning.

[0126] Notably, the target genes and target sites therein of the present disclosure were identified by a screening method involving transient delivery. In this screening method, a DNA binding domain-effector fusion protein (also referred to as an "epieditor") was transiently delivered to T cells (i.e., delivered by a method that results in the transient expression and / or presence of the fusion protein in T cells), followed by primary or continuous stimulation of the T cells, and the effect on functional T cell cytokines was evaluated. It was found that transient delivery of an epigenetic modification DNA targeting system allowed the identification of genomic targets whose modulation substantially affects T cell function, without requiring the permanent presence of the epigenetic modification DNA targeting system and / or stable knockdown or knockout of the target gene. This approach is advantageous because it allows the identification of target genes and target sites that offer a better safety profile, since their modulation does not rely on the permanent integration of an editor, such as by lentiviral transduction. Furthermore, this transient screening strategy allows for the identification of target genes and target sites within them, with the persistence of the effect of the epigenetic modification DNA targeting system unmasked as a result of permanent integration into and expression from the genome, in contrast to other screening approaches that have utilized lentiviral delivery of DNA (Schmidt et al. 2022 Science, 375, DOI:10.1126 / science.abj4008; Freimer et al. 2022 Nature Genetics, 54:1133-1144).

[0127] The provided embodiments can be used to target genes that, when transcriptionally altered by epigenetic modification, can significantly enhance or promote T cell function, including effector activity required for T cell persistence and function. Such T cell profiles result in durable effector function, have a better fitness / growth advantage, and have the ability to produce growth-promoting cytokines (e.g., IL-2) and / or cytotoxic cytokines (e.g., IFNg) upon TCR or antigen stimulation. In particular, the provided embodiments provide epigenetic modification DNA targeting systems (i.e., "epi-editing systems") and methods that can provide long-lasting effector function with improved fitness. This approach offers a practical clinical solution for avoiding challenges associated with T cell persistence, suboptimal functionality, and / or exhaustion. Furthermore, cellular epigenetic modifications do not alter DNA at the sequence level, thereby avoiding safety concerns associated with gene editing approaches. Epigenetic control of the differentiation fate of T cells would be an advantageous approach to increase the percentage or number of T cells in a T cell population.

[0128] All publications mentioned in this application, including patent documents, scientific articles, and databases, are incorporated herein in their entirety for all purposes to the same extent as if each individual publication was individually incorporated herein by reference. To the extent that a definition set forth herein conflicts or is otherwise inconsistent with a definition set forth in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0129] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0130] I. DNA Targeting Systems In some embodiments, a DNA targeting system is provided that can specifically target a target site for at least one gene (i.e., a target gene) and regulate the transcription of the at least one gene. In some embodiments, the at least one gene is one or more genes in lymphoid cells, such as T cells. In some embodiments, the target site for a gene is a target site in the gene or in its regulatory DNA element. In some embodiments, the transcriptional modulation is a decrease in the transcription of each target gene. In some embodiments, the transcriptional modulation is an increase in the transcription of each target gene. In provided embodiments, for each targeted target gene, the DNA targeting system comprises a fusion protein comprising a DNA binding domain that binds to the target site for that gene and an effector domain that regulates the transcription of that gene. In some embodiments, the provided DNA targeting system can regulate, for example, suppress or increase, the transcription of the at least one gene in a cell. In some embodiments, transcriptional modulation of gene expression by the DNA targeting system provided herein can promote or improve the function of lymphoid cells. In certain embodiments, the provided DNA targeting systems promote T cell function, e.g., one or more T cell effector functions, by epigenetically modifying target sites in one or more target genes.

[0131] In some embodiments, the at least one effector domain is a transcriptional repression effector domain, e.g., any effector domain for transcriptional repression described in Section IE1, for repressing transcription of each of the at least one gene (e.g., inhibiting or reducing transcription of the gene compared to transcription of the gene in the absence of the DNA targeting system). In some embodiments, the effector domain is a transcriptional repression effector domain and the one or more genes are selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the effector domain is a transcriptional repression effector domain and the one or more genes are selected from the group consisting of CBLB, CISH, MED12, MYB, PRDM1, and RASA2.

[0132] In some embodiments, the effector domain directly or indirectly reduces transcription of a gene. In some embodiments, the effector domain induces, catalyzes, or causes transcriptional repression. In some embodiments, the effector domain induces transcriptional repression. In some aspects, the effector domain is selected from a KRAB domain, an ERF repression domain, an MXI1 domain, a SID4X domain, a MAD-SID domain, a DNMT family protein domain (e.g., DNMT3A or DNMT3B), a fusion of one or more DNMT family proteins or domains thereof (e.g., DNMT3A / L, which comprises a fusion of the DNMT3A domain and the DNMT3L domain), LSD1, EZH2, a partially or fully functional fragment or domain of any of the foregoing, or a combination of any of the foregoing. In some embodiments, the effector domain is KRAB. In some embodiments, the effector domain is DNMT3A / L.

[0133] In some embodiments, the at least one effector domain is a transcription activation effector domain, e.g., any effector domain for transcription activation described in Section IE2, for increasing transcription of each of the at least one gene (e.g., activating or increasing transcription of the gene compared to transcription of the gene in the absence of the DNA targeting system). In some embodiments, the effector domain is a transcription activation effector domain and the one or more genes are selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the effector domain is a transcription activation effector domain and the one or more genes are selected from the group consisting of EOMES, IL-2, LCP2, and TBX21.

[0134] In some embodiments, the effector domain directly or indirectly increases gene transcription. In some embodiments, the effector domain induces, catalyzes, or causes transcriptional activation. In some embodiments, the effector domain induces transcriptional activation. In some aspects, the effector domain comprises a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain (optionally the TET protein is TET1), a SunTag domain, or a domain, portion, mutant, or truncation of any of the foregoing. In some embodiments, the effector domain is VP64.

[0135] In some embodiments, the DNA targeting system comprises a fusion protein comprising (a) at least one DNA binding domain capable of being targeted to a target site and (b) at least one effector domain capable of regulating gene transcription. In some embodiments, the at least one effector domain is a transcriptional repression effector domain. In some embodiments, the at least one effector domain is a transcriptional activation effector domain. The fusion protein can be any suitable fusion protein, for example, as described in Section IF.

[0136] In some embodiments, the DNA binding domain comprises or is derived from a CRISPR-associated (Cas) protein, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, an I-SceI enzyme, or a mutant thereof. In some embodiments, the DNA binding domain comprises a catalytically inactive (e.g., nuclease-inactive or nuclease-inactivated) mutant of any of the above. In some embodiments, the DNA binding domain comprises an inactivated Cas9 (dCas9) protein or a mutant thereof that is catalytically inactivated so that it is inactive in terms of nuclease activity and cannot cleave DNA. The DNA binding domain can be any suitable DNA binding domain, for example, as described in sections IC and ID.

[0137] In some embodiments, the DNA-binding domain comprises or is derived from a Cas protein or a variant thereof, e.g., a nuclease-inactive Cas or dCas (e.g., dCas9), and the DNA targeting system comprises one or more guide RNAs (gRNAs), e.g., a combination of gRNAs (e.g., two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence capable of targeting and / or hybridizing to a target site. In some embodiments, the gRNA is capable of forming a complex with a Cas protein or a variant thereof. In some aspects, the gRNA targets or recruits the Cas protein or a variant thereof to the target site. The gRNA can be any suitable gRNA, e.g., as described in section IC2.

[0138] In some embodiments, the DNA targeting system is for repressing transcription of at least one gene, such as any of those described in Section IB2, and the fusion protein of the DNA targeting module is a dCas9-KRAB fusion protein. In some embodiments, the fusion protein is a dCas9-KRAB-DNMT3A / L fusion protein. In some embodiments, the fusion protein is any of those described herein, such as those described in Section IF.

[0139] In some embodiments, the DNA targeting system is for increasing transcription of at least one gene, e.g., any of those described in Section IB3, and the fusion protein of the DNA targeting module is a dCas9-VP64 fusion protein, e.g., a dCas9-2xVP64 fusion protein. In some embodiments, the fusion protein is any of those described herein, e.g., those described in Section IF.

[0140] Exemplary components and features of DNA targeting systems are listed in the following subsections.

[0141] A. DNA targeting modules and multiplexed DNA targeting systems In some embodiments, DNA targeting system comprises at least one DNA targeting module, and each DNA targeting module of the system is a component of the DNA targeting system that can independently target one target site for target gene.In some embodiments, each DNA targeting module comprises (a) a DNA binding domain that can be targeted to target site, and (b) an effector domain for regulating the transcription of gene.In some embodiments, the DNA targeting system comprises a single DNA targeting module for the targeted transcription regulation of a single gene.

[0142] In some embodiments, the DNA targeting module is a CRISPR / Cas-based DNA targeting module.In some embodiments, in the CRISPR / Cas-based DNA targeting module, the DNA binding domain of fusion protein is Cas protein or its variant (for example, dCas protein such as dCas9), and the DNA targeting module further comprises gRNA for targeting the DNA binding domain to target site.

[0143] In some embodiments, the DNA targeting module is a zinc finger protein (ZFP)-based DNA targeting module. In some embodiments, in a ZFP-based DNA targeting module, the DNA binding domain of the fusion protein is an engineered zinc finger protein (eZFP).

[0144] In some embodiments, the DNA targeting module is a transcription activator-like effector (TALE)-based DNA targeting module. In some embodiments, in a TALE-based DNA targeting module, the DNA binding domain of the fusion protein is an engineered TALE.

[0145] In some embodiments, the DNA targeting system comprises multiple DNA targeting modules, each targeting a different target site. In some embodiments, one or more target sites are target sites for different genes. In some embodiments, one or more target sites are target sites for the same gene. In some embodiments, the DNA targeting system is a multiplexed DNA targeting system, i.e., it targets target sites for multiple genes. Therefore, the term DNA targeting system can include a multiplexed epigenetic modification DNA targeting system comprising multiple DNA targeting modules. In some embodiments, the multiplexed epigenetic modification DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more DNA targeting modules. In some embodiments, the multiplexed epigenetic modification DNA targeting system comprises two DNA targeting modules. In some embodiments, the multiplexed epigenetic modification DNA targeting system comprises three DNA targeting modules.

[0146] In some embodiments, any two DNA targeting modules of a DNA targeting system can comprise separate (i.e., non-overlapping) components. For example, a DNA targeting system can comprise a first DNA targeting module comprising a first fusion protein with a DNA binding domain (e.g., a ZFN- or TALE-based DNA binding domain) that targets a first target site, and a second DNA targeting module comprising a second fusion protein with a second DNA binding domain (e.g., a ZFN- or TALE-based DNA binding domain) that targets a second target site.

[0147] In some embodiments, any two DNA targeting modules of a DNA targeting system can contain shared (i.e., overlapping) components. For example, a DNA targeting system can include: i) a first DNA targeting module comprising (a) a fusion protein comprising a Cas protein and an effector domain, and (b) a first gRNA that forms a complex with the Cas protein and targets a first target site; and ii) a second DNA targeting module comprising (a) a fusion protein of the first DNA targeting module, and (b) a second gRNA that forms a complex with the Cas protein and targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the Cas protein to target the target sites of those two or more gRNAs. Conversely, as described herein, different Cas protein variants (e.g., SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs. It is therefore possible to engineer a single DNA targeting system that contains multiple non-overlapping CRISPR / Cas-based DNA targeting modules.

[0148] In some aspects, the present disclosure provides an epigenetic modification DNA targeting system, comprising a plurality of DNA targeting modules for regulating the transcription of one or more genes.In some embodiments, the plurality of DNA targeting modules comprises a first DNA targeting module for regulating the transcription of a first gene among the one or more genes, and a second DNA targeting module for regulating the transcription of a second gene among the one or more genes.In some embodiments, each DNA targeting module comprises a fusion protein comprising (a) a DNA binding domain for targeting the target site of the target gene for the DNA targeting module, and (b) at least one effector domain.In some embodiments, each DNA targeting module comprises a transcription repression effector domain for repressing the transcription of the at least one gene.In some embodiments, each DNA targeting module comprises a transcription activation effector domain for increasing the transcription of the at least one gene.

[0149] B. Target Genes and Sites to Promote Lymphocyte (e.g., T Cell) Activation and Function In some aspects, provided herein are target sites in one or more target genes, where modulation of the target genes promotes T cell activation or function. In some embodiments, the target sites are targeted using any of the provided DNA targeting systems.

[0150] In some embodiments, the target site is among genes whose reduced gene expression promotes T cell activation or function, such as any one or more of the target genes described in Section IB2. In some embodiments, the target site is a target site in a gene selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the target site is a target site in the CBLB gene. In some embodiments, the target site is a target site in the CISH gene. In some embodiments, the target site is a target site in the MED12 gene. In some embodiments, the target site is a target site in the MYB gene. In some embodiments, the target site is a target site in the PRDM1 gene. In some embodiments, the target site is a target site in the RASA2 gene.

[0151] In some embodiments, the target site is among genes whose increased gene expression promotes T cell activation or function, such as any one or more of the target genes described in Section IB3. In some embodiments, the target site is a target site in a gene selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the target site is a target site in the EOMES gene. In some embodiments, the target site is a target site in the IL-2 gene. In some embodiments, the target site is a target site in the LCP2 gene. In some embodiments, the target site is a target site in the TBX21 gene.

[0152] In some embodiments, the target site is targeted by a DNA targeting system, for example, by a DNA targeting module of a DNA targeting system such as any of those described herein. In some embodiments, the target site is a target site for a gene (e.g., a target gene). In some embodiments, the target site for a gene is in the gene or in its regulatory DNA element. In some embodiments, the target site is a target site in a gene. In some aspects, the gene is a target gene. In some embodiments, the target gene is a gene in a cell. In some embodiments, the cell is an immune cell such as a T cell. In some embodiments, provided herein is a multiple epigenetic modification DNA targeting system that targets a combination of at least two target genes described herein or their regulatory DNA elements.

[0153] In some embodiments, the DNA targeting system targets or binds to a target site in a gene, such as any of those described herein. In some embodiments, the target site is located in a gene and / or in a regulatory DNA element of the gene. In some embodiments, the regulatory DNA element is a sequence to which a gene control protein can bind and affect the transcription of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of the gene. In some embodiments, the regulatory DNA element is a promoter or enhancer of the gene. In some embodiments, the target site is located within a promoter, enhancer, exon, intron, untranslated region (UTR), 5'UTR, or 3'UTR of the gene. In some embodiments, the regulatory DNA element is a promoter. In some embodiments, a promoter is a nucleotide sequence to which RNA polymerase binds and initiates transcription of the gene. In some embodiments, a promoter is a nucleotide sequence located within about 100 bp, about 500 bp, about 1000 bp, or more of the transcription start site of the gene. In some embodiments, the promoter is within 500 bp of the transcription start site of the gene. In some embodiments, the target site is located within a sequence of unknown or known function suspected to be capable of controlling expression of the gene.

[0154] 1. Lymphoid cells and their regulated effector functions In some embodiments, the provided DNA targeting systems and / or DNA targeting provide transcriptional regulation to suppress or increase expression of at least one target gene. In some embodiments, the target gene is a gene whose expression controls a cellular phenotype. In some embodiments, the target gene can regulate a phenotype in T cells. In some embodiments, regulated expression of the gene, e.g., increased or decreased transcription, regulates the phenotype. In some embodiments, regulated expression of the gene promotes increased T cell effector function upon T cell stimulation. In some embodiments, the increased T cell effector function is increased compared to T cells in which gene expression is not regulated by the provided DNA targeting system. Methods for regulating T cell function or other lymphoid cell function using the provided DNA targeting systems are further described below and in Section IV.

[0155] In some embodiments, gene is regulated by DNA targeting system, for example, by any DNA targeting system provided herein.In some embodiments, DNA targeting system is transiently delivered to cell.In some embodiments, delivery of DNA targeting system, for example, by transient delivery, promotes the increase of T cell effector function when T cell is stimulated.In some embodiments, T cell effector function is increased compared with the equivalent T cell that is not delivered by DNA targeting system.

[0156] In some aspects, transient delivery refers to any delivery method that causes the expression and / or existence of one or more components of DNA targeting system in cells for a limited duration.For example, the delivery of mRNA encoding a fusion protein of a DNA targeting system into cells (for example, by electroporation) can cause the transient expression of the fusion protein in the cell, for example, until the mRNA is degraded.In another example, the DNA targeting system can be expressed from one or more nucleic acids that encode the DNA targeting system, and in this case, the nucleic acid that encodes the DNA targeting system is not incorporated into the genome of the cell, and is eventually degraded and / or removed from the cell, so that the expression of the DNA targeting system does not persist.In another example, one or more components of the DNA targeting system, for example, fusion protein and optionally gRNA, can be synthesized in vitro and delivered to cells (for example, by electroporation) without the need for an expression vector, so that the DNA targeting system can exist transiently, for example, until the fusion protein and / or gRNA are degraded. In some aspects, transient delivery differs from non-transient delivery methods that result in stable expression, such as methods that involve integrating an expression vector for a DNA targeting system or components thereof into the genome of a cell.

[0157] In some embodiments, delivery of a DNA targeting system to a cell (e.g., a T cell), such as by transient delivery, promotes a phenotype in the cell (e.g., a T cell). In some embodiments, the phenotype is increased activation or function in the cell (e.g., a T cell). In some embodiments, delivery of a DNA targeting system to a cell (e.g., a T cell), such as by transient delivery, promotes increased activation or function in the cell (e.g., a T cell). In some embodiments, the phenotype is increased T cell effector function upon T cell stimulation. In some embodiments, T cell effector function is increased compared to T cells not delivered with the epigenetically modified DNA targeting system. In some embodiments, decreased expression (e.g., transcription) of the one or more target genes, such as a target gene described in Section IB2, leads to increased T cell effector function upon T cell stimulation. In some embodiments, increased expression (e.g., transcription) of the one or more target genes, such as a target gene described in Section IB3, leads to increased T cell effector function upon T cell stimulation. In some embodiments, the T cell effector function is characterized by an activity selected from the group consisting of IL-2 production, IFN-gamma production, TNF-alpha production, T cell proliferation, or a combination of any of the foregoing.

[0158] In some embodiments, the provided DNA targeting systems promote or increase improved T cell effector function, such as may occur after in vitro, ex vivo, or in vivo stimulation. In some embodiments, the T cell stimulation is polyclonal T cell stimulation. In some embodiments, the T cell stimulation is with anti-CD3 and anti-CD28 activating reagents. In some embodiments, the T cell stimulation is antigen-specific activity mediated or induced by specific binding of an antigen to an antigen receptor on the T cell surface. In some embodiments, the T cells express a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR) for the antigen, and the T cell stimulation is antigen-specific stimulation of the CAR or eTCR. In some embodiments, the T cell stimulation is with antigen-expressing target cells. In some embodiments, the T cell stimulation occurs when the T cells contact cells expressing the antigen. In some embodiments, the T cell stimulation is restimulation of the T cells after at least one prior T cell stimulation. In some embodiments, the T cells are stimulated, then the provided DNA targeting systems are transiently delivered, before the effector function or phenotype of the T cells is evaluated.

[0159] In certain embodiments, a cell composition containing T cells is stimulated with an anti-CD3 / anti-CD28 activating reagent for a period of time, and effector function is measured at one or more time points during or after incubation. In some embodiments, such an activating reagent comprises anti-CD3 / anti-CD28 coated on a support, such as a magnetic bead or other matrix. Exemplary activating reagents include Dynabeads™ or T cell TransAct™. In some embodiments, T cells are incubated with the activating reagent for 3 to 72 hours, e.g., 12 to 48 hours, e.g., 12, 18, 24, 36, or 48 hours, or any time in between. In some embodiments, cells can be directly assessed for effector function, e.g., cytokine production or proliferative capacity. In some embodiments, culture supernatants can be collected, and the amount of soluble factors, e.g., cytokines, detected. In some embodiments, T cells can be collected and re-exposed to the activating reagent to monitor cytolytic activity. In some embodiments, cells can be restimulated one or more times, eg, by serial stimulation, and serially assessed for effector function after each stimulation.

[0160] In certain embodiments, antigen-specific activity is measured by incubating a cell composition containing T cells expressing an antigen receptor, e.g., a CAR, with antigen-expressing cells for a period of time, and effector function is measured at one or more time points during or after incubation. In some embodiments, T cells are incubated with an antigen-specific agent, e.g., antigen-expressing cells, for 3 hours to 96 hours, e.g., 12 hours to 72 hours, e.g., 12 hours, 24 hours, 48 ​​hours, 72 hours, or any time in between. In some embodiments, cells can be directly assessed for effector function, e.g., cytokine production or proliferative capacity. In some embodiments, culture supernatant can be collected, and the amount of soluble factors, e.g., cytokines, is detected. In some embodiments, T cells can be collected and re-exposed to antigen-expressing target cells to monitor cell death (cytolytic activity) of the target cells. In some embodiments, cells can be restimulated one or more times, e.g., by serial stimulation, and effector function can be continuously assessed after each stimulation. In some embodiments, T cells bearing an engineered antigen receptor (e.g., a CAR) are incubated with a number of antigen-expressing cells at an effector to target (E:T) ratio of, for example, 1:4 to 4:1, e.g., 1:4, 1:3, 1:2, or 1:1.

[0161] In some embodiments, the cells (e.g., T cells) exhibit increased cytokine production. In some embodiments, increased cytokine production occurs upon T cell stimulation. In some embodiments, T cell effector function is characterized by cytokine production. In some embodiments, cytokine production is increased by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold or more compared to cells not delivered with the epigenetic modification DNA targeting system. In some embodiments, cytokine production is production of IL-2, IFN-gamma, TNF-alpha, or a combination thereof. In some embodiments, T cell effector function is characterized by IL-2 production. In some embodiments, the cells (e.g., T cells) exhibit increased IL-2 production. In some embodiments, T cell effector function is characterized by IFN-gamma production. In some embodiments, the cells (e.g., T cells) exhibit increased IFN-gamma production. In some embodiments, T cell effector function is characterized by IL-2 production and IFN-gamma production. In some embodiments, the cells (e.g., T cells) exhibit increased IL-2 production and increased IFN-gamma production. In some embodiments, T cell effector function is characterized by multi-functional production of IL-2, IFN-gamma, and TNF-alpha. In some embodiments, the cells (e.g., T cells) exhibit increased IL-2, IFN-gamma, and TNF-alpha production.

[0162] Suitable techniques for measuring the production or secretion of soluble factors, such as cytokines, are known in the art. The production and / or secretion of soluble factors can be measured by determining the concentration or amount of the extracellular amount of the factor or by determining the amount of transcriptional activity of the gene encoding the factor. Suitable techniques include, but are not limited to, immunoassays, aptamer-based assays, histological or cytological assays, mRNA expression level assays, enzyme-linked immunosorbent assays (ELISAs), immunoblotting, immunoprecipitation, radioimmunoassays (RIAs), immunostaining, flow cytometry assays, surface plasmon resonance (SPR), chemiluminescence assays, lateral flow immunoassays, inhibition or avidity assays, protein microarrays, high-performance liquid chromatography (HPLC), Meso Scale Discovery (MSD) electrochemiluminescence, and bead-based multiplex immunoassays (MIAs). In some embodiments, suitable techniques may use detectable binding reagents that specifically bind to soluble factors.

[0163] In some embodiments, cytokine production is measured as the percentage of cells positive for the cytokine (e.g., as measured by intracellular cytokine staining (ICS) and flow cytometry). Intracellular cytokine staining (ICS) by flow cytometry is a well-suited technique for examining cytokine production at the single-cell level. It detects cytokine production and accumulation in the endoplasmic reticulum after cell stimulation and allows for the identification of cell populations positive or negative for the production of a specific cytokine, or the separation of high- and low-producing cells, based on a threshold value. ICS can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or MHC multimers to access cytokine production in specific subgroups of cells, making it a highly flexible and versatile method. Other single-cell techniques for measuring or detecting cytokine production include, but are not limited to, ELISPOT, limiting dilution, and T cell cloning.

[0164] In some embodiments, cytokine production is measured as the amount of cytokine secreted from cells (e.g., as measured by ELISA (enzyme-linked immunosorbent assay)). ELISA is a plate-based assay technique designed to detect and quantify substances such as peptides, cytokines, antibodies, and hormones. In ELISA, a soluble factor such as a cytokine must be immobilized on a solid surface and then complexed with an antibody linked to an enzyme. Detection is achieved by assessing the activity of the conjugated enzyme through incubation with a substrate to generate a detectable signal.

[0165] In some embodiments, T cell effector function is characterized by an activity further comprising T cell proliferation. In some embodiments, cells (e.g., T cells) exhibit increased proliferation. In some embodiments, increased proliferation occurs upon T cell stimulation. In some embodiments, proliferation is increased by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold or more compared to cells to which the epigenetic modification DNA targeting system has not been delivered. In some embodiments, proliferation is measured as an increase in cell number before and after stimulation. In some embodiments, increased proliferation is measured as the number of cells after stimulation in a cell population to which the epigenetic modification DNA targeting system has been delivered compared to the number of cells after stimulation in a cell population to which the epigenetic modification DNA targeting system has not been delivered. In some embodiments, cells (e.g., T cells) do not exhibit increased proliferation.

[0166] In some embodiments, T cell effector function is characterized by an activity that further includes target cell killing. In some embodiments, the cells (e.g., T cells) exhibit increased target cell killing. In some embodiments, increased target cell killing occurs upon T cell stimulation. In some embodiments, stimulation is performed by contacting the cells (e.g., T cells) with target cells. In some embodiments, T cells are incubated with antigen-expressing target cells at a ratio of 4:1 to 1:4, inclusive, such as a ratio of 1:4, 1:3, 1:2, or 1:1. In some embodiments, target cell killing is increased by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more, compared to cells not delivered with the epigenetic modification DNA targeting system. In some embodiments, killing is measured as the ability of cells to kill target cells upon contact with the target cells. The death of target cells can be measured by any suitable assay, for example, by the assay described in the Examples herein. In some embodiments, the death is measured in an in vitro assay in which cells into which the epigenetic modified DNA targeting system has been delivered are co-cultured with target cells and the number of target cells is measured over time. In some embodiments, a reduction in the number and / or proliferation of target cells indicates target cell death. Cytolytic activity can be measured by directly or indirectly measuring the number of target cells over time. For example, target cells can be incubated with a detectable marker, such as a marker that becomes detectable when target cells are lysed or a detectable marker that is detectable in viable target cells, before incubation with antigen receptor (e.g., CAR)-expressing cells. These readouts directly or indirectly provide target cell number and / or target cell death and can be measured at different time points during the assay. A reduction in target cell number and / or an increase in target cell death indicates the cytolytic activity of the cells.Suitable methods for performing cytolytic assays are known in the art and include, but are not limited to, chromium-51 release assays, non-radioactive chromium assays, and flow cytometry assays using fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE), PKH-2, and PKH-26.

[0167] In some embodiments, T cell effector function is characterized by an activity that further includes T cell persistence. In some embodiments, the cells (e.g., T cells) exhibit increased persistence (e.g., T cell persistence). In some embodiments, persistence relates to the ability of the cells to remain present in the presence of target cells and / or maintain an immune response. In some embodiments, persistence can be measured in vitro or in vivo, e.g., after administration of the cells to a subject. Persistence can be measured by any suitable method, e.g., as described in Section IV.

[0168] In certain embodiments, the persistence of T cells can be measured as a pharmacokinetic profile of the cell composition after administration to a subject. In some embodiments, pharmacokinetic parameters can include exposure, number, concentration, persistence, and proliferation. Optionally, pharmacokinetics can be measured as a maximum (peak) plasma concentration (C) after administration. max ), peak time (i.e., maximum plasma concentration (C max ) appears;T max ), minimum plasma concentration (i.e., the minimum plasma concentration between administrations of a therapeutic agent, e.g., CAR+ T cells; C min ), elimination half-life (T 1 / 2The efficacy of the administered engineered T cells can be assessed by measuring parameters such as the CAR T cell concentration (AUC) and the area under the curve (i.e., the area under the curve generated by plotting the plasma concentration of the therapeutic agent versus time; AUC). Parameters of the administered engineered T cells can be measured in a blood sample from the subject. For example, nucleic acid-based methods such as quantitative PCR (qPCR) or flow cytometry-based methods, or other assays such as immunoassays, ELISA, or chromatography / mass spectrometry-based assays can be used.

[0169] In some aspects, a nucleic acid-based method such as quantitative PCR (qPCR) is used to assess the amount of cells expressing an antigen receptor (e.g., CAR-expressing cells administered for T cell-based therapy) in a subject's blood or serum sample or organ or tissue sample (e.g., a disease site, e.g., a tumor sample). In some aspects, persistence is quantified as the number of copies of the DNA or plasmid encoding the receptor, e.g., CAR, per microgram of DNA, or as the number of antigen receptor-expressing (e.g., CAR-expressing) cells per microliter of sample, such as blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of sample. In some embodiments, the primers or probes used for qPCR or other nucleic acid-based methods are specific for binding, recognizing, and / or amplifying the nucleic acid encoding the antigen receptor and / or other components or elements of the plasmid and / or vector (including regulatory elements, e.g., promoters, transcriptional and / or post-transcriptional regulatory or response elements, or markers, e.g., surrogate markers). In some embodiments, the primers can be specific for a regulatory element such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0170] In some embodiments, any of the phenotypes described herein, e.g., increased IL-2 production, increased IFN-gamma production, increased IL-2 and IFN-gamma production, increased IL-2, IFN-gamma and TNF-alpha production, increased or non-increased proliferation, increased target cell killing, and / or increased persistence, are observed following stimulation (e.g., T cell stimulation).

[0171] In some embodiments, any phenotype described herein, including, for example, an increase in T cell effector function, is observed 48 hours or later after transient delivery of an epigenetic modified DNA targeting system to T cells. In some embodiments, the phenotype, for example, an increase in T cell effector function, occurs within 6 days, 9 days, 12 days, 15 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 71 days, or later after transient delivery of an epigenetic modified DNA targeting system to T cells.

[0172] In some aspects, the phenotype is a phenotype characterized by the cell surface phenotype of the cells. In some embodiments, the phenotype comprises the expression of one or more cell surface markers selected from IL-2+, TNFα+, IFNg+, or any combination thereof. In some embodiments, the phenotype is a phenotype in T cells, such as CD3+ T cells, which may be CD4+ T cells or CD8+ T cells. Thus, in some embodiments, the phenotype comprises the expression of one or more cell surface markers selected from CD3+, CD4+, CD8+, IL-2+, TNFα+, IFNg+, or any combination thereof. In some aspects, the phenotype comprises the expression of IL-2+. In some embodiments, the phenotype comprises the expression of IL-2− and IFNg+.

[0173] It is understood that the embodiments of the provided epigenetic modification DNA targeting system are not limited to regulating target gene expression and promoting phenotype in T cells, but can also be used to regulate any one or more of the target genes described herein in any lymphoid cell. In addition to T cells, lymphoid cells can include NK cells, NKT cells, any cells differentiated from stem cells into such lymphoid cells and / or from precursor cells such as common lymphoid progenitors (CLPs). In some embodiments, lymphoid cells are differentiated from stem cells, such as hematopoietic stem cells, hematopoietic progenitor cells, or precursor cells. In some embodiments, lymphoid cells are transdifferentiated from non-pluripotent cells of non-hematopoietic lineages.

[0174] In some embodiments, the regulated lymphoid cells are an isolated or enriched population of lymphoid immune cells, such as an isolated or enriched population of T, NK, and / or NKT cells. In some embodiments, the regulated cells are isolated or enriched T cells. In some embodiments, the regulated cells are isolated or enriched NK cells. In some embodiments, the regulated cells are isolated or enriched NKT cells. In some embodiments, the regulated isolated or enriched population or subpopulation of immune cells, including T, NK, and / or NKT cells, can be obtained from a unit of blood using several techniques known to those skilled in the art, such as Ficoll™ separation. In one embodiment, T, NK, or NKT cells from an individual's circulating blood are obtained by apheresis and separated from other nucleated leukocytes, red blood cells, and platelets, such as by Ficoll™ separation or affinity-based selection. In some embodiments, the cells are primary cells. In some embodiments, the primary cells are isolated or enriched from a peripheral blood sample of a subject, such as a human subject.

[0175] In some embodiments, the lymphoid cells to be regulated are differentiated in vitro from stem cells or progenitor cells. In some embodiments, lymphoid cells, such as T, NK, or NKT cells, or their lineages, can be differentiated from stem cells, hematopoietic stem cells or hematopoietic progenitor cells (HSCs), or progenitor cells. The progenitor cells can be CD34+ hemogenic endothelial cells, multipotent progenitor cells, T cell precursors, NK cell precursors, or NKT cell precursors. In some embodiments, the progenitor cells are lymphoid progenitor cells, such as common lymphoid progenitor cells, early thymic progenitor cells, pre-T cell precursors, pre-NK precursors, T cell precursors, NK precursors, or NKT precursors. The stem cells can be pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). iPSCs are reprogrammed pluripotent cells that do not exist in nature. Once the cells of a subject have been reprogrammed to a pluripotent state, they can be reprogrammed or differentiated into a desired cell type or cell subtype, such as a T, NK, or NKT cell.

[0176] In some embodiments, iPSCs are differentiated into T, NK, or NKT cells by a multi-stage differentiation platform that can differentiate cells from various developmental stages to adopt hematopoietic phenotypes ranging from mesodermal stem cells to fully differentiated T, NK, or NKT cells (see, e.g., U.S. Pat. No. 10,626,372).

[0177] In some embodiments, a population or subpopulation of lymphoid cells is transdifferentiated in vitro from non-pluripotent cells of non-hematopoietic fate to hematopoietic lineage cells, or from non-pluripotent cells of a first hematopoietic cell type to a different hematopoietic cell type (which can be T, NK, or NKT progenitor cells or fully differentiated specific types of immune cells such as T, NK, or NKT cells). (See, e.g., U.S. Patent Application No. 9,376,664 and U.S. Patent Application No. 15 / 072,769, the disclosures of which are incorporated herein in their entireties.) In some embodiments, the non-pluripotent cells of non-hematopoietic fate are somatic cells such as skin fibroblasts, adipose tissue-derived cells, and human umbilical vein endothelial cells (HUVECs). Somatic cells useful for transdifferentiation can be immortalized somatic cells.

[0178] To induce pluripotency or increase developmental potential in cells (Takahashi, K., and Yamanaka, S., Cell 126, 663-676 (2006); Takahashi et al., Cell 131, 861-872 (2007); Yu et al., Science 318, 1917-1920 (2007); Zhou et al., Cell Stem Cell 4, 381-384 (2009); Kim et al., Cell Stem Cell 4, 472-476 (2009); Yamanaka et al., 2009; Saha, K., Jaenisch, R., Cell Stem Cell 5, 584-595 (2009)), and to improve the efficiency of reprogramming (Shi et al., Cell Stem Cell 2,525-528(2008a);Shi et al.,Cell Stem Cell 3,568-574(2008b);Huangfu et al.,Nat Biotechnol 26,795-797(2008a);Huangfu et al.,Nat Biotechnol 26,1269-1275(2008b);Silva et al. al.,Plos Bio 6,e253.Doi:10.1371 / journal.Pbio.0060253(2008);Lyssiotis et al.,PNAS 106,8912-8917(2009);Ichida et al.,Cell Stem Cell 5,491-503(2009);Maherali, N., Hochedlinger, K., Curr Biol 19, 1718-1723 (2009b); Esteban et al., Cell Stem Cell 6, 71-79 (2010); and Feng et al., Cell Stem Cell 4, 301-312 (2009)), the disclosures of which are incorporated herein by reference in their entireties.

[0179] Cells that are positive (+) for a particular cell surface marker are understood to be cells that express that marker on their surface at detectable levels. Similarly, cells that are negative (-) for a particular cell surface marker are understood to be cells that express that marker on their surface at undetectable levels. A given cell surface marker can be identified or detected by detecting the expression level of the marker protein using antibodies and other binding entities. Suitable antibodies include polyclonal, monoclonal, fragments (e.g., Fab fragments), single-chain antibodies, and other forms of specific binding molecules. Antibody reagents for the above-mentioned cell surface markers are readily apparent to those skilled in the art. Several well-known methods can be used to assess the expression level of surface markers or surface proteins, such as affinity-based methods, e.g., immunoaffinity-based methods, e.g., by flow cytometry in the context of surface markers. In some embodiments, the label is a fluorophore, and the method for detecting or identifying cell surface markers on cells (e.g., T cells) is by flow cytometry. In some embodiments, a different label is used for each different marker using multicolor flow cytometry. In some embodiments, surface expression can be determined by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting binding of the antibody to the marker.

[0180] In some embodiments, a cell (e.g., a T cell) is positive (pos or +) for a particular marker if there is detectable presence of that marker (which can be an intracellular marker or a surface marker) on or in the cell. In some embodiments, surface expression is positive if staining by flow cytometry is detectable at a level substantially greater than that detected using an isotype-matched control running the same procedure under otherwise identical conditions, and / or at a level substantially similar to, or in some cases, higher than, and / or higher than that of cells known to be negative for that marker.

[0181] In some embodiments, a cell (e.g., a T cell) is negative (neg or -) for a particular marker if there is no detectable presence of that marker (which can be an intracellular or surface marker) on or in the cell. In some embodiments, surface expression is negative if staining cannot be detected by flow cytometry at a level substantially greater than that detected using an isotype-matched control and performing the same procedure under otherwise identical conditions, and / or at a level substantially lower than that of cells known to be positive for that marker and / or at a level substantially similar to that of cells known to be negative for that marker.

[0182] In some aspects, the phenotype can be characterized by one or more functions of the cell. In some aspects, the phenotype is characterized by the polyfunctional activity of T cells, producing multiple T cell stimulatory cytokines as determined in a polyfunctional cytokine secretion assay after stimulation of the T cells with a stimulatory agent. In some embodiments, the T cells are polyfunctional in that they produce two or more cytokines. In some embodiments, the T cells are polyfunctional in that they produce two or more cytokines selected from interferon-gamma (IFN-gamma), interleukin 2 (IL-2), and TNF-alpha. In some embodiments, polyfunctional T cells produce IFN-gamma, IL-2, and TNF-alpha. In some embodiments, the stimulatory agent is a non-specific or non-antigen-dependent T cell stimulatory agent. In some embodiments, the non-specific or non-antigen-dependent T cell stimulatory agent is a polyclonal stimulatory agent. In some embodiments, the non-specific or non-antigen-dependent stimulatory agent comprises PMA / ionomycin, anti-CD3 / anti-CD28, phytohemagglutinin (PHA), or concanavalin A (ConA). In some embodiments, the non-specific or non-antigen-dependent T cell stimulatory agent contains PMA / ionomycin.

[0183] In certain embodiments, the production of one or more cytokines is measured, detected, and / or quantified by intracellular cytokine staining. Intracellular cytokine staining by flow cytometry (ICS) is a well-suited technique for examining cytokine production at the single-cell level. It detects cytokine production and accumulation in the endoplasmic reticulum after cell stimulation, allowing for the identification of cell populations that are positive or negative for the production of a particular cytokine, or the separation of high- and low-producing cells, based on a threshold value. In some embodiments, as described above, stimulation can be performed using a non-specific stimulus, e.g., the stimulus is not antigen-specific. For example, PMA / ionomycin can be used for non-specific cell stimulation. ICS can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or MHC multimers to access cytokine production in specific subgroups of cells, making it a highly flexible and versatile method. Other single-cell techniques for measuring or detecting cytokine production include, but are not limited to, ELISPOT, limiting dilution, and T cell cloning. In some embodiments, assays for assaying multifunctional cytokine secretion of multiple cytokines can include multiplex assays or other assays to assess multifunctionality (see, e.g., Xue et al., (2017) Journal for ImmunoTherapy of Cancer 5:85).

[0184] 2. Genes and target sites for reducing transcription In some embodiments, delivery of the DNA targeting system suppresses (e.g., reduces) transcription of one or more target genes selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. Also provided herein in some embodiments is a target site for one or more genes whose reduced transcription promotes a phenotype in a cell. Also provided herein in some embodiments is a target site for one or more genes whose reduced transcription promotes increased T cell effector function. In some embodiments, reduced transcription promotes increased T cell effector function upon T cell stimulation. In some embodiments, the one or more genes are selected from CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the one or more genes are selected from CBLB, CISH, MED12, MYB, PRDM1, and RASA2.

[0185] In some embodiments, the DNA targeting system comprises a plurality of DNA targeting modules. In some embodiments, each DNA targeting module targets a target site. In some embodiments, the plurality of DNA targeting modules targets at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the plurality of DNA targeting modules targets a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

[0186] In some embodiments, the plurality of DNA targeting modules targets at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the plurality of DNA targeting modules targets a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

[0187] In some embodiments, the DNA targeting system targets a combination of genes shown in Table 1. In some embodiments, said plurality of DNA targeting modules targets a combination of genes shown in Table 1. In some embodiments, transcription of each gene in said combination is repressed by the DNA targeting system.

[0188] Table 1. Combinations of genes targeted by the multiplex epigenetic modification DNA targeting system to reduce transcription of target genes. TIFF2025531268000002.tif182165TIFF2025531268000003.tif234165TIFF2025531268000004.tif234165 TIFF2025531268000005.tif234165TIFF2025531268000006.tif234165TIFF2025531268000007.tif122165

[0189] In some embodiments, the DNA targeting system targets target sites for CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and / or RASA2. In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOs: 1-6, 10-33, 80-90, 102-112, 200-211, 292-295, 300-302, and 306-308, or at least a 14 nucleotide contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 1-6, 10-33, 80-90, 102-112, 200-211, 292-295, 300-302, and 306-308 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of the target site sequences described herein above. In some embodiments, the target site is a sequence set forth in any one of SEQ ID NOs: 1-6, 10-33, 80-90, 102-112, 200-211, 292-295, 300-302, and 306-308. In some embodiments, the target site is a sequence shown in Table 5.

[0190] In some embodiments, the DNA targeting system targets target sites for CBLB, CISH, MED12, MYB, PRDM1, and / or RASA2.

[0191] In some embodiments, the DNA targeting system targets a target site for a CBLB. In some embodiments, the target site for modulating transcription of a CBLB is within coordinates chr3:105868857-105868876. In some embodiments, the target site is within coordinates chr3:105868757-105868976. In some embodiments, the target site is within coordinates chr3:105868807-105868926. In some embodiments, the target site is within coordinates chr3:105868837-105868896. In some embodiments, the target site is at or includes coordinates chr3:105868857-105868876. In some embodiments, the target site for the CBLB is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr3:105,655,461 (e.g., a target site at +500 of 105,655,461, or at -500 of 105,655,461, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr3:105,655,461. In some embodiments, the target site is located within approximately 80 bp of genomic coordinate chr3:105,655,461. In some embodiments, the target site is within the region between -40 and +40 of genomic coordinate chr3:105,655,461. In some embodiments, the target site is located within 20 bp of genomic coordinate chr3:105,655,461. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr3:105,655,461. In some embodiments, any of such target sites includes or spans genomic coordinate chr3:105,655,461, which is the CBLB transcription start site (TSS). In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:11, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO:11 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above. In some embodiments, the target site is the sequence shown in SEQ ID NO:11.

[0192] In some embodiments, the DNA targeting system targets a target site for CISH. In some embodiments, the target site for modulating transcription of CISH is within coordinates chr3:50,611,749-50,611,768. In some embodiments, the target site is within coordinates chr3:50,611,649-50611868. In some embodiments, the target site is within coordinates chr3:50,611,699-50611818. In some embodiments, the target site is within coordinates chr3:50,611,729-50611788. In some embodiments, the target site is at or includes coordinates chr1:50,611,749-50,611,768. In some embodiments, the target site for CISH is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr3:50,606,489 (e.g., a target site at +500 of 50,606,489, or -500 of 50,606,489, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr3:50,606,489. In some embodiments, the target site is located within approximately 80 bp of genomic coordinate chr3:50,606,489. In some embodiments, the target site is within the region between -40 and +40 of genomic coordinate chr3:50,606,489. In some embodiments, the target site is located within 20 bp of genomic coordinate chr3:50,606,489. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr3:50,606,489. In some embodiments, any of such target sites includes or spans genomic coordinate chr3:50,606,489, which is the CISH transcription start site (TSS). In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:28, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO:28 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described hereinabove, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described hereinabove. In some embodiments, the target site is the sequence shown in SEQ ID NO:28.

[0193] In some embodiments, the DNA targeting system targets a target site for MED12. In some embodiments, the target site for modulating transcription of MED12 is within coordinates chrX:71,118,489-71,118,508. In some embodiments, the target site is within coordinates chrX:71,118,389-71,118,608. In some embodiments, the target site is within coordinates chrX:71,118,439-71,118,558. In some embodiments, the target site is within coordinates chrX:71,118,469-71,118,528. In some embodiments, the target site is at or includes coordinates chrX:71,118,489-71,118,508. In some embodiments, the target site for MED12 is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chrX:71,118,596 (e.g., a target site at +500 of 71,118,596, or -500 of 71,118,596, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr3:50,606,489. In some embodiments, the target site is located within approximately 80 bp of genomic coordinate chrX:71,118,596. In some embodiments, the target site is within the region between -40 and +40 of genomic coordinate 71,118,596. In some embodiments, the target site is located within 20 bp of genomic coordinate chrX:71,118,596. In some embodiments, the gRNA targets a target site within the region -10 to +10 of genomic coordinate chrX:71,118,596. In some embodiments, any of such target sites includes or spans genomic coordinate chrX:71,118,596, which is the MED12 transcription start site (TSS). In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:81, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 81 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above. In some embodiments, the target site is the sequence shown in SEQ ID NO: 81.

[0194] In some embodiments, the DNA targeting system targets a target site for MYB. In some embodiments, the target site for modulating transcription of MYB is within coordinates chr6:135,181,383-135,181,402. In some embodiments, the target site is within coordinates chr6:135,181,283-135,181,502. In some embodiments, the target site is within coordinates chr6:135,181,333-135,181,452. In some embodiments, the target site is within coordinates chr6:135,181,363-135,181,422. In some embodiments, the target site is at or includes coordinates chr6:135,181,383-135,181,402. In some embodiments, the target site for MYB is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr6:135,181,308 (e.g., a target site that is +500 of 135,181,308, or -500 of 135,181,308, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr6:135,181,308. In some embodiments, the target site is located within about 80 bp of genomic coordinate chr6:135,181,308. In some embodiments, the target site is within the region from -40 to +40 of genomic coordinate 135,181,308. In some embodiments, the target site is located within 20 bp of genomic coordinate chr6:135,181,308. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr6:135,181,308. In some embodiments, any of such target sites includes or spans the MYB transcription start site (TSS) at genomic coordinate chr6:135,181,308. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:18, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 18 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 18.

[0195] In some embodiments, the DNA targeting system targets a target site for RASA2. In some embodiments, the target site for modulating transcription of RASA2 is within coordinates chr3:141,487,065-141,487,084. In some embodiments, the target site is within coordinates chr3:141,486,965-141,487,184. In some embodiments, the target site is within coordinates chr3:141,487,015-141,487,134. In some embodiments, the target site is within coordinates chr3:141,487,045-141,487,104. In some embodiments, the target site is at or includes coordinates chr3:141,487,065-141,487,084. In some embodiments, the target site for RASA2 is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr3:141,487,027 (e.g., a target site at +500 of 141,487,027, or -500 of 141,487,027, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr3:141,487,027. In some embodiments, the target site is located within approximately 80 bp of genomic coordinate chr3:141,487,027. In some embodiments, the target site is within the region between -40 and +40 of genomic coordinate 141,487,027. In some embodiments, the target site is located within 20 bp of genomic coordinate chr3:141,487,027. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr3:141,487,027. In some embodiments, any of such target sites includes or spans genomic coordinate chr3:141,487,027, which is the RASA2 transcription start site (TSS). In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:19, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 19 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 19.

[0196] In some embodiments, the DNA targeting system targets a target site for PRDM1. In some embodiments, the target site for modulating transcription of PRDM1 is within coordinates chr6:106,086,371-106,086,390. In some embodiments, the target site is within coordinates chr6:106,086,271-106,086,490. In some embodiments, the target site is within coordinates chr6:106,086,321-106,086,440. In some embodiments, the target site is within coordinates chr6:106,086,351-106,086,410. In some embodiments, the target site is at or includes coordinates chr6:106,086,371-106,086,390. In some embodiments, the target site for PRDM1 is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr6:106,086,336 (e.g., a target site that is +500 of 106,086,336, or -500 of 106,086,336, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr6:106,086,336. In some embodiments, the target site is located within approximately 80 bp of genomic coordinate chr6:106,086,336. In some embodiments, the target site is within the region from -40 to +40 of genomic coordinate 106,086,336. In some embodiments, the target site is located within 20 bp of genomic coordinate chr6:106,086,336. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr6:106,086,336. In some embodiments, any of such target sites includes or spans genomic coordinate chr6:106,086,336, which is the PRDM1 transcription start site (TSS). In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:33, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 33 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described hereinabove, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described hereinabove. In some embodiments, the target site is the sequence shown in SEQ ID NO: 33.

[0197] In some embodiments, the DNA targeting system targets a combination of genes, such as any combination shown in Table 1. In some embodiments, the DNA targeting system targets a combination of genes, such as CBLB and CCNC; CBLB and CD5; CBLB and CISH; CBLB and DGKZ; CBLB and ELOB; CBLB and FAS; CBLB and Fli1; CBLB and GATA3; CBLB and KDM1A; CBLB and MED12; CBLB and MYB; CBLB and PRDM1; CBLB and RASA2; CD5 and CISH; CD5 and MYB; CISH and DGKZ; CISH and MYB; CISH and RASA2; GATA3 and CD5; GATA3 and CISH; GATA3 and MYB; MED12 and CBLB; MED12 and CD5; MED12 and CISH; MED12 and DGKZ; MED12 and ELOB; MED12 and GATA3; MED12 and MYB; MED12 and PRDM1; MED12 and RASA2; MYB and RASA2; PRDM1 and CISH; PRDM1 and GATA3; PRDM1 and MYB; PRDM1 and RASA2; CD5, CISH, and MYB; GATA3, CBLB, and MYB; GATA3, CD5, and MYB; PRDM1, GATA3, and CISH; TGBR2 and MED12; and TGFBR2, MED12, and CISH.

[0198] In some embodiments, the DNA targeting system targets CBLB and MYB. In some embodiments, the target site for targeting CBLB can be any of the above, and the target site for targeting MYB can be any of the above. In some embodiments, the DNA targeting system targets a target site for CBLB that comprises the sequence shown in SEQ ID NO: 11 and a target site for MYB that comprises the sequence shown in SEQ ID NO: 18.

[0199] In some embodiments, the DNA targeting system targets CBLB and MED12. In some embodiments, the target site for targeting CBLB can be any of the above, and the target site for targeting MED12 can be any of the above. In some embodiments, the DNA targeting system targets a target site for CBLB that comprises the sequence shown in SEQ ID NO: 11 and a target site for MED12 that comprises the sequence shown in SEQ ID NO: 81.

[0200] In some embodiments, the DNA targeting system targets CBLB and CCNC. In some embodiments, the target site for targeting CBLB can be any of the above, and the target site for targeting CCNC can be any of the above. In some embodiments, the DNA targeting system targets the target site for CBLB, comprising the sequence shown in SEQ ID NO: 11, and the target site for CCNC, comprising the sequence shown in SEQ ID NO: 104.

[0201] In some embodiments, the DNA targeting system targets MED12 and CISH. In some embodiments, the target site for targeting MED12 can be any of the above, and the target site for targeting CISH can be any of the above. In some embodiments, the DNA targeting system targets a target site for MED12 that comprises the sequence shown in SEQ ID NO: 81 and a target site for CISH that comprises the sequence shown in SEQ ID NO: 28.

[0202] In some embodiments, the DNA targeting system targets MED12, CBLB, and CISH. In some embodiments, the target site for targeting MED12 can be any of the above, the target site for targeting CBLB can be any of the above, and the target site for targeting CISH can be any of the above. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence shown in SEQ ID NO: 81, a target site for CBLB comprising the sequence shown in SEQ ID NO: 11, and a target site for CISH comprising the sequence shown in SEQ ID NO: 28.

[0203] In some embodiments, the DNA targeting system targets MED12 and RASA2. In some embodiments, the target site for targeting MED12 can be any of those described above, and the target site for targeting RASA2 can be any of those described above. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence shown in SEQ ID NO:81 and a target site for RASA2 comprising the sequence shown in SEQ ID NO:19.

[0204] In some embodiments, the DNA targeting system targets TGFBR2 and MED12. In some embodiments, the DNA targeting system targets a target site for TGBR2 comprising the sequence set forth in SEQ ID NO:301 and a target site for MED12 comprising the sequence set forth in SEQ ID NO:82.

[0205] In some embodiments, the DNA targeting system targets TGFBR2, MED12, and CISH. In some embodiments, the DNA targeting system targets a target site for TGBR2 comprising the sequence set forth in SEQ ID NO:301, a target site for MED12 comprising the sequence set forth in SEQ ID NO:82, and a target site for CISH comprising the sequence set forth in SEQ ID NO:28.

[0206] In some embodiments, the DNA targeting system targets a combination of target sites for a combination of genes for transcriptional repression as shown in Table 2.

[0207] Table 2. Gene and target site combinations for transcriptional repression TIFF2025531268000008.tif231170

[0208] In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for CCNC comprising the sequence set forth in SEQ ID NO:104. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for CD5 comprising the sequence set forth in SEQ ID NO:3. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for CISH comprising the sequence set forth in SEQ ID NO:30. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for DGKZ comprising the sequence set forth in SEQ ID NO:13. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for ELOB comprising the sequence set forth in SEQ ID NO:24. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for FAS comprising the sequence set forth in SEQ ID NO:204. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for Fli1 comprising the sequence set forth in SEQ ID NO:208. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for GATA3 comprising the sequence set forth in SEQ ID NO:26.In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for KDM1A comprising the sequence set forth in SEQ ID NO:4. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for MED12 comprising the sequence set forth in SEQ ID NO:81. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for MYB comprising the sequence set forth in SEQ ID NO:18. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for PRDM1 comprising the sequence set forth in SEQ ID NO:32. In some embodiments, the DNA targeting system targets a target site for CBLB comprising the sequence set forth in SEQ ID NO:11 and a target site for RASA2 comprising the sequence set forth in SEQ ID NO:19. In some embodiments, the DNA targeting system targets a target site for CD5 comprising the sequence set forth in SEQ ID NO:3 and a target site for CISH comprising the sequence set forth in SEQ ID NO:30. In some embodiments, the DNA targeting system targets a target site for CD5 comprising the sequence set forth in SEQ ID NO:3 and a target site for MYB comprising the sequence set forth in SEQ ID NO:18. In some embodiments, the DNA targeting system targets a target site for CISH comprising the sequence set forth in SEQ ID NO:30 and a target site for DGKZ comprising the sequence set forth in SEQ ID NO:13.In some embodiments, the DNA targeting system targets a target site for CISH comprising the sequence set forth in SEQ ID NO:30 and a target site for MYB comprising the sequence set forth in SEQ ID NO:18. In some embodiments, the DNA targeting system targets a target site for CISH comprising the sequence set forth in SEQ ID NO:30 and a target site for RASA2 comprising the sequence set forth in SEQ ID NO:19. In some embodiments, the DNA targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO:26 and a target site for CD5 comprising the sequence set forth in SEQ ID NO:3. In some embodiments, the DNA targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO:26 and a target site for CISH comprising the sequence set forth in SEQ ID NO:30. In some embodiments, the DNA targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO:26 and a target site for MYB comprising the sequence set forth in SEQ ID NO:18. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for CBLB comprising the sequence set forth in SEQ ID NO:11. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for CD5 comprising the sequence set forth in SEQ ID NO:3. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for CISH comprising the sequence set forth in SEQ ID NO:30.In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for DGKZ comprising the sequence set forth in SEQ ID NO:13. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for ELOB comprising the sequence set forth in SEQ ID NO:24. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for GATA3 comprising the sequence set forth in SEQ ID NO:26. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for MYB comprising the sequence set forth in SEQ ID NO:18. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for PRDM1 comprising the sequence set forth in SEQ ID NO:32. In some embodiments, the DNA targeting system targets a target site for MED12 comprising the sequence set forth in SEQ ID NO:81 and a target site for RASA2 comprising the sequence set forth in SEQ ID NO:19. In some embodiments, the DNA targeting system targets a target site for MYB comprising the sequence set forth in SEQ ID NO:18 and a target site for RASA2 comprising the sequence set forth in SEQ ID NO:19. In some embodiments, the DNA targeting system targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO:32 and a target site for CISH comprising the sequence set forth in SEQ ID NO:30.In some embodiments, the DNA targeting system targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO:32 and a target site for GATA3 comprising the sequence set forth in SEQ ID NO:26. In some embodiments, the DNA targeting system targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO:32 and a target site for MYB comprising the sequence set forth in SEQ ID NO:18. In some embodiments, the DNA targeting system targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO:32 and a target site for RASA2 comprising the sequence set forth in SEQ ID NO:19. In some embodiments, the DNA targeting system targets a target site for CD5 comprising the sequence set forth in SEQ ID NO:3, a target site for CISH comprising the sequence set forth in SEQ ID NO:30, and a target site for MYB comprising the sequence set forth in SEQ ID NO:18. In some embodiments, the DNA targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26, a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, the DNA targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26, a target site for CD5 comprising the sequence set forth in SEQ ID NO: 3, and a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, the DNA targeting system targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO: 32, a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26, and a target site for CISH comprising the sequence set forth in SEQ ID NO: 30.

[0209] In some embodiments, delivery of the DNA targeting system reduces (e.g., reduces or suppresses) the transcription of one or more genes.In some embodiments, the reduction in gene expression in cells (e.g., T cells) is a log2 fold change of less than about -1.0.For example, the log2 fold change is less than or about -1.5, less than or about -2.0, less than or about -2.5, less than or about -3.0, less than or about -4.0, less than or about -5.0, less than or about -6.0, less than or about -7.0, less than or about -8.0, less than or about -9.0, less than or about -10.0, or any value between any of the above, compared to the level of the gene in control cells.

[0210] 3. Genes and target sites for increasing transcription In some embodiments, delivery of the DNA targeting system increases transcription of one or more genes selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. Also provided herein in some embodiments is a target site for one or more genes whose increased transcription promotes a phenotype in a cell. Also provided herein in some embodiments is a target site for one or more genes whose increased transcription promotes increased T cell effector function. In some embodiments, increased transcription promotes increased T cell effector function upon T cell stimulation. In some embodiments, the one or more genes are selected from BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the one or more genes are selected from EOMES, IL-2, LCP2, and TBX21.

[0211] In some embodiments, the DNA targeting system comprises a plurality of DNA targeting modules. In some embodiments, each DNA targeting module targets a target site. In some embodiments, the plurality of DNA targeting modules targets at least a first gene and a second gene, and the first and second genes are independently selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the plurality of DNA targeting modules targets a first gene and a second gene, and the first and second genes are independently selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the multiple DNA targeting modules target a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of EOMES, IL-2, LCP2, and TBX21.

[0212] In some embodiments, the plurality of DNA targeting modules targets at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the plurality of DNA targeting modules targets a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

[0213] In some embodiments, the DNA targeting system targets a combination of genes shown in Table 3. In some embodiments, a plurality of DNA targeting modules targets a combination of genes shown in Table 3. In some embodiments, transcription of each gene in the combination is increased by the DNA targeting system.

[0214] Table 3. Combinations of genes targeted by the multiplex epigenetic modification DNA targeting system to increase transcription of target genes. TIFF2025531268000009.tif182170TIFF2025531268000010.tif187170

[0215] In some embodiments, the DNA targeting system targets target sites for BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and / or VAV1. In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOs: 7-9, 78, 144-156, 170, 172-177, and 184-191, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 7-9, 78, 144-156, 170, 172-177, and 184-191 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a consecutive portion of a target site sequence described herein above. In some embodiments, the target site is a sequence set forth in any one of SEQ ID NOs: 7-9, 78, 144-156, 170, 172-177, and 184-191. In some embodiments, the target site is a sequence set forth in Table 6.

[0216] In some embodiments, the DNA targeting system targets target sites for IL-2, EOMES, LCP2, and / or TBX21.

[0217] In some embodiments, the DNA targeting system targets a target site for IL-2. In some embodiments, the target site for modulating transcription of IL-2 is within coordinates chr4:122,456,711-122,456,729. In some embodiments, the target site is within coordinates chr4:122,456,611-122,456,829. In some embodiments, the target site is within coordinates chr4:122,456,661-122,456,779. In some embodiments, the target site is within coordinates chr4:122,456,691-122,456,749. In some embodiments, the target site is at or includes coordinates chr4:122,456,711-122,456,729. In some embodiments, the target site for IL-2 is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr4:122,451,470 (e.g., a target site that is +500 of 122,451,470, or -500 of 122,451,470, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr4:122,451,470. In some embodiments, the target site is located within about 80 bp of genomic coordinate chr4:122,451,470. In some embodiments, the target site is within the region from -40 to +40 of genomic coordinate 122,451,470. In some embodiments, the target site is located within 20 bp of genomic coordinate chr4:122,451,470. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr4:122,451,470. In some embodiments, any of such target sites includes or spans genomic coordinate chr4:122,451,470, which is the IL-2 transcription start site (TSS). In some embodiments, the target site comprises a sequence selected from SEQ ID NO:78, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 78 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above. In some embodiments, the target site is the sequence shown in SEQ ID NO: 78.

[0218] In some embodiments, the DNA targeting system targets a target site for EOMES. In some embodiments, the target site for modulating EOMES is within coordinates chr3:27,722,421-27,722,440. In some embodiments, the target site is within coordinates chr3:27,722,321-27,722,540. In some embodiments, the target site is within coordinates chr3:27,722,371-27,722,490. In some embodiments, the target site is within coordinates chr3:27,722,401-27,722,460. In some embodiments, the target site is at or includes coordinates chr3:27,722,421-27,722,440. In some embodiments, the target site for EOMES is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr3:27,715,953 (e.g., a target site at +500 of 27,715,953, or -500 of 27,715,953, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr3:50,606,489. In some embodiments, the target site is located within approximately 80 bp of genomic coordinate chr3:27,715,953. In some embodiments, the target site is within the region between -40 and +40 of genomic coordinate 27,715,953. In some embodiments, the target site is located within 20 bp of genomic coordinate chr3:27,715,953. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr3:27,715,953. In some embodiments, any of such target sites includes or spans the EOMES transcription start site (TSS) at genomic coordinate chr3:27,715,953. In some embodiments, the target site comprises a sequence selected from SEQ ID NO: 149, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 149 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 149.

[0219] In some embodiments, the DNA targeting system targets a target site for LCP2. In some embodiments, the target site for modulating LCP2 is within coordinates chr5:170,298,278-170,298,297. In some embodiments, the target site is within coordinates chr5:170,298,178-170,298,397. In some embodiments, the target site is within coordinates chr5:170,298,228-170,298,347. In some embodiments, the target site is within coordinates chr5:170,298,258-170,298,317. In some embodiments, the target site is at or includes coordinates chr5:170,298,278-170,298,297. In some embodiments, the target site for LCP2 is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr5:170,246,233 (e.g., a target site at +500 of 170,246,233, or -500 of 170,246,233, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr5:170,246,233. In some embodiments, the target site is located within approximately 80 bp of genomic coordinate chr5:170,246,233. In some embodiments, the target site is within the region between -40 and +40 of genomic coordinate 170,246,233. In some embodiments, the target site is located within 20 bp of genomic coordinate chr5:170,246,233. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr5:170,246,233. In some embodiments, any of such target sites includes or spans the LCP2 transcription start site (TSS), genomic coordinate chr5:170,246,233. In some embodiments, the target site comprises a sequence selected from SEQ ID NO: 151, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 151 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 151.

[0220] In some embodiments, the DNA targeting system targets a target site for TBX21. In some embodiments, the target site for modulating TBX21 is within coordinates chr17:47,733,109-47,733,128. In some embodiments, the target site is within coordinates chr17:47,733,009-47,733,228. In some embodiments, the target site is within coordinates chr17:47,733,059-47,733,178. In some embodiments, the target site is within coordinates chr17:47,733,089-47,733,148. In some embodiments, the target site is at or includes coordinates chr17:47,733,109-47,733,128. In some embodiments, the target site for TBX21 is located within 500 bp of human genome assembly GRCh38 (hg38) genomic coordinate chr17:41,733,236 (e.g., a target site at +500 of 41,733,236, or -500 of 41,733,236, or a position between the foregoing). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of genomic coordinate chr17:41,733,236. In some embodiments, the target site is located within approximately 80 bp of genomic coordinate chr17:41,733,236. In some embodiments, the target site is within the region between -40 and +40 of genomic coordinate 41,733,236. In some embodiments, the target site is located within 20 bp of genomic coordinate chr17:41,733,236. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of genomic coordinate chr17:41,733,236. In some embodiments, any of such target sites includes or spans genomic coordinate chr17:41,733,236, which is the TBX21 transcription start site (TSS). In some embodiments, the target site comprises a sequence selected from SEQ ID NO: 155, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 155 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a continuous portion of the target site sequence described herein above. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 155.

[0221] In some embodiments, the DNA targeting system targets a combination of genes, such as any combination shown in Table 1. In some embodiments, the DNA targeting system targets a combination of genes selected from BATF and IL-2; BATF and VAV1; CD28 and BATF; CD28 and EOMES; CD28 and IL-2; CD28 and LCP2; CD28 and TBX21; CD28 and VAV1; EOMES and BATF; EOMES and LCP2; EOMES and TBX21; EOMES and VAV1; LCP2 and BATF; LCP2 and IL-2; LCP2 and TBX21; LCP2 and VAV1; TBX21 and BATF; TBX21 and IL-2; TBX21 and TBX21; TBX21 and VAV1; and VAV1 and IL-2.

[0222] In some embodiments, the DNA targeting system targets IL-2 and VAV1. In some embodiments, the target site for targeting IL-2 can be any of those described above, and the target site for targeting VAV1 can be any of those described above. In some embodiments, the DNA targeting system targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:78 and a target site for VAV1 comprising the sequence set forth in SEQ ID NO:170.

[0223] In some embodiments, the DNA targeting system targets IL-2 and LCP2. In some embodiments, the target site for targeting IL-2 can be any of those described above, and the target site for targeting LCP2 can be any of those described above. In some embodiments, the DNA targeting system targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:78 and a target site for LCP2 comprising the sequence set forth in SEQ ID NO:151.

[0224] In some embodiments, the DNA targeting system targets IL-2 and TBX21. In some embodiments, the target site for targeting IL-2 can be any of those described above, and the target site for targeting TBX21 can be any of those described above. In some embodiments, the DNA targeting system targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:78 and a target site for TBX21 comprising the sequence set forth in SEQ ID NO:155.

[0225] In some embodiments, the DNA targeting system targets IL-2 and EOMES. In some embodiments, the target site for targeting IL-2 can be any of those described above, and the target site for targeting EOMES can be any of those described above. In some embodiments, the DNA targeting system targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO: 78 and a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149.

[0226] In some embodiments, the DNA targeting system targets IL2RB and VAV1.

[0227] In some embodiments, the DNA targeting system targets a combination of target sites for a combination of genes for transcriptional activation shown in Table 4.

[0228] Table 4. Gene and target site combinations for transcriptional activation TIFF2025531268000011.tif122128

[0229] In some embodiments, the DNA targeting system targets a target site for BATF comprising the sequence set forth in SEQ ID NO: 172 and a target site for IL-2 comprising the sequence set forth in SEQ ID NO: 78. In some embodiments, the DNA targeting system targets a target site for BATF comprising the sequence set forth in SEQ ID NO: 172 and a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170. In some embodiments, the DNA targeting system targets a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144 and a target site for BATF comprising the sequence set forth in SEQ ID NO: 172. In some embodiments, the DNA targeting system targets a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144 and a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149. In some embodiments, the DNA targeting system targets a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144 and a target site for IL-2 comprising the sequence set forth in SEQ ID NO: 78. In some embodiments, the DNA targeting system targets a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144 and a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151. In some embodiments, the DNA targeting system targets a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144 and a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, the DNA targeting system targets a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144 and a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170.In some embodiments, the DNA targeting system targets a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149 and a target site for BATF comprising the sequence set forth in SEQ ID NO: 172. In some embodiments, the DNA targeting system targets a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149 and a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151. In some embodiments, the DNA targeting system targets a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149 and a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, the DNA targeting system targets a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149 and a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170. In some embodiments, the DNA targeting system targets a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151 and a target site for BATF comprising the sequence set forth in SEQ ID NO: 172. In some embodiments, the DNA targeting system targets a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151 and a target site for IL-2 comprising the sequence set forth in SEQ ID NO: 78. In some embodiments, the DNA targeting system targets a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151 and a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, the DNA targeting system targets a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151 and a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170.In some embodiments, the DNA targeting system targets a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155 and a target site for BATF comprising the sequence set forth in SEQ ID NO: 172. In some embodiments, the DNA targeting system targets a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155 and a target site for IL-2 comprising the sequence set forth in SEQ ID NO: 78. In some embodiments, the DNA targeting system targets a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155 and a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, the DNA targeting system targets a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155 and a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170. In some embodiments, the DNA targeting system targets a target site for VAV1 comprising the sequence set forth in SEQ ID NO:170 and a target site for IL-2 comprising the sequence set forth in SEQ ID NO:78.

[0230] In some embodiments, delivery of the DNA targeting system increases the expression (e.g., transcription) of one or more genes. In some embodiments, the increase in gene expression in cells (e.g., T cells) is a log2 fold change of greater than about 1.0. For example, the log2 fold change is greater than or about 1.5, greater than or about 2.0, greater than or about 2.5, greater than or about 3.0, greater than or about 4.0, greater than or about 5.0, greater than or about 6.0, greater than or about 7.0, greater than or about 8.0, greater than or about 9.0, greater than or about 10.0, or any value between any of the above, compared to the level of the gene in control cells.

[0231] C. CRISPR / Cas-based DNA targeting systems and DNA-binding domains Provided herein is a multiplex epigenetic targeting DNA targeting system based on the CRISPR / Cas system, i.e., a CRISPR / Cas-based DNA targeting system that can bind to target sites for target genes or a combination of target sites, for example, a combination of target genes. In some embodiments, the CRISPR / Cas DNA binding domain is nuclease-inactive, for example, includes dCas (e.g., dCas9), so that the system binds to target sites for target genes without mediating nucleic acid cleavage at the target sites. The CRISPR / Cas-based DNA targeting system can be used to regulate the expression of target genes in cells such as T cells. In some embodiments, the target genes can include any of those described herein, including those described above in Section IB. In some embodiments, the target sites for target genes can include any of those described herein, including those described above in Section IB. In some embodiments, the CRISPR / Cas-based DNA targeting system can include any known Cas enzyme and generally nuclease-inactive or dCas. In some embodiments, the CRISPR / Cas-based DNA targeting system comprises a fusion protein of a nuclease-inactive Cas protein or a mutant thereof with an effector domain, and at least one gRNA. In some embodiments, the effector domain reduces the transcription of one or more genes (e.g., the effector domain is a transcriptional repressor such as any of those described in Section IE1). In some embodiments, the effector domain increases the transcription of one or more genes (e.g., the effector domain is a transcriptional activator such as any of those described in Section IE2).

[0232] The CRISPR system (also known as the CRISPR / Cas system or CRISPR-Cas system) refers to a conserved microbial nuclease system found in bacterial and archaeal genomes that provides a form of acquired immunity against invading phages and plasmids. Clustered regularly interspaced short palindromic repeats (CRISPR) refers to a locus containing multiple repetitive DNA elements separated by non-repetitive DNA sequences called spacers. Spacers are short sequences of foreign DNA integrated into the genome between CRISPR repeats and serve as a "memory" of past exposure. Spacers encode the DNA-targeting portion of an RNA molecule that confers specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes that can act as RNA-guided nucleases to mediate cleavage, as well as non-protein-coding DNA elements that encode RNA molecules that can program the specificity of CRISPR-mediated nucleic acid cleavage.

[0233] In the type II CRISPR / Cas system, which contains the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) is complementary to the target nucleic acid sequence (target site) and contains a spacer sequence that encodes the sequence specificity of the complex. The trans-activating crRNA (tracrRNA) base-pairs with a portion of the crRNA, forming a structure that complexes with the Cas9 protein, forming the Cas / RNA RNP complex.

[0234] Naturally occurring CRISPR / Cas system, such as that with Cas9, has been engineered to enable efficient programming of Cas / RNA RNP to target desired sequence in cells of interest for both gene editing and regulating gene expression.TracrRNA and crRNA are engineered to form a single chimeric guide RNA molecule, generally referred to as guide RNA (gRNA), as described in, for example, International Publication No. 2013 / 176772, International Publication No. 2014 / 093661, International Publication No. 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21(2012) or Cong, L. et al. Science 339(6121):819-23(2013). The spacer sequence of the gRNA can be selected by the user so that the Cas / gRNA RNP complex targets a desired locus, e.g., a desired target site in a target gene.

[0235] Cas proteins have also been engineered to be catalytically inactivated or nuclease inactive to allow targeting of Cas / gRNA RNP without inducing cleavage at target sites. Mutations in Cas proteins can reduce or eliminate the nuclease activity of Cas proteins, making them catalytically inactive. Cas proteins with reduced or eliminated nuclease activity are referred to herein interchangeably as inactivated Cas (dCas) proteins or nuclease-inactive Cas (iCas) proteins. Exemplary inactivated Cas9 (dCas9) derived from S. pyogenes contains silencing mutations in the RuvC nuclease domain and the HNH nuclease domain (D10A and H840A), as described in, for example, International Publication No. 2013 / 176772, International Publication No. 2014 / 093661, Jinek, M. et al. Science 337(6096):816-21(2012), and Qi, L. et al. Cell 152(5):1173-83(2013). Exemplary dCas mutants derived from the Cas12 system (i.e., Cpf1) are described, for example, in International Publication No. 2017 / 189308 and Zetsche, B. et al. Cell 163(3):759-71(2015). Conserved domains that mediate nucleic acid cleavage, such as the RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologs and can be mutated to produce inactive mutants, for example, as described in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0236] dCas fusion proteins with transcriptional and / or epigenetic regulatory elements have been used as a versatile platform for ectopically modulating gene expression in target cells. These include fusion of Cas with effector domains, such as transcriptional activators or repressors. For example, fusing dCas9 with a transcriptional activator such as VP64 (a polypeptide composed of four tandem copies of VP16, a 16-amino acid transactivation domain of herpes simplex virus) can result in robust induction of gene expression. Alternatively, fusing dCas9 with a transcriptional repressor such as KRAB (Kruppel-associated box) can result in robust repression of gene expression. Various dCas fusion proteins having effector domains are described, for example, in WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2021 / 247570, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), Mali, P. et al. al. Nat. Biotechnol. 31, 833-838 (2013), Maeder, ML et al. Nat. Methods 10, 977-979 (2013), Gilbert, LA et al. Cell 154(2):442-451 (2013), and Nunez, JK et al. Cell 184(9):2503-2519 (2021).

[0237] In some aspects, a DNA targeting system is provided, comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or a mutant thereof and at least one effector domain (i.e., a transcriptional repressor) for reducing transcription or inducing transcriptional repression when targeted to a target gene in a cell (e.g., a T cell). In some embodiments, the dCas protein is any suitable dCas protein, such as any described in Section IC. In some embodiments, the dCas protein is a dCas9 protein, such as dSpCas9 or dSaCas9. In some embodiments, the at least one effector domain is any suitable transcriptional repressor effector domain, such as any described in Section IE1, e.g., KRAB and / or DNMT3A / L. In some embodiments, the at least one effector domain is KRAB. In some embodiments, the fusion protein is a dCas9-KRAB fusion protein or a dCas9-KRAB-DNMT3A / L fusion protein, e.g., as described in Section IF. In such embodiments, the DNA targeting system also includes one or more gRNAs (e.g., as described in Section IC2.a) provided in combination with or complexed with a dCas protein or variant thereof, so that the DNA targeting system targets the target site of the target gene. In some embodiments, the fusion protein is guided by the guide RNA to a specific target site sequence of the target gene, and the effector domain mediates a targeted epigenetic modification that reduces or suppresses transcription of the target gene. In some embodiments, a combination of gRNAs guides the fusion protein to a combination of target site sequences in a combination of genes, and the effector domain mediates a targeted epigenetic modification that reduces or suppresses transcription of the combination of target genes. As further described below, any of a variety of effector domains that reduce or suppress transcription can be used.

[0238] In some aspects, a DNA targeting system is provided, comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or a mutant thereof and an effector domain (i.e., a transcriptional activator) for increasing transcription or inducing transcriptional activation when targeting a target gene in a cell (e.g., a T cell). In some embodiments, the dCas protein is any suitable dCas protein, such as any described in Section IC. In some embodiments, the dCas protein is a dCas9 protein, such as dSpCas9 or dSaCas9. In some embodiments, at least one effector domain is any suitable transcriptional activator effector domain, such as any described in Section IE.2, e.g., VP64. In some embodiments, at least one effector domain is VP64. In some embodiments, the fusion protein is a dCas9-VP64 fusion protein, e.g., as described in Section IF. In such embodiments, the DNA targeting system also includes one or more gRNAs (e.g., as described in Section IC2.b) provided in combination with or complexed with a dCas protein or variant thereof, so that the DNA targeting system targets the target site of the target gene. In some embodiments, the fusion protein is guided by the guide RNA to a specific target site sequence of the target gene, and the effector domain mediates a targeted epigenetic modification that increases or activates transcription of the target gene. In some embodiments, a combination of gRNAs guides the fusion protein to a combination of target site sequences in a combination of genes, and the effector domain mediates a targeted epigenetic modification that increases or activates transcription of the combination of target genes. As further described below, any of a variety of effector domains that increase or activate transcription can be used.

[0239] 1. CRISPR / Cas-based DNA-binding domains In some aspects, the DNA binding domain comprises or is derived from a CRISPR-associated (Cas) protein or a mutant thereof. In certain embodiments herein, the Cas protein is nuclease-inactive (i.e., a dCas protein).

[0240] In some embodiments, the Cas protein is derived from a class 1 CRISPR system (i.e., a multiple Cas protein system), such as a type I, type III, or type IV CRISPR system. In some embodiments, the Cas protein is derived from a class 2 CRISPR system (i.e., a single Cas protein system), such as a type II, type V, or type VI CRISPR system. In some embodiments, the Cas protein is derived from a type V CRISPR system. In some embodiments, the Cas protein is derived from a Cas12 protein (i.e., Cpf1) or a variant thereof, as described, for example, in WO 2017 / 189308 and Zetsche, B. et al. Cell. 163(3):759-71 (2015). In some embodiments, the Cas protein is derived from a type II CRISPR system. In some embodiments, the Cas protein is a Cas protein described in, for example, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21(2012), Mali, P. et al. Science 339(6121):823-6(2013), Cong, L. et al. Science 339(6121):819-23(2013), Perez-Pinera, P. et al. Nat. Methods 10,973-976(2013), or Mali, P. et al. The CRISPR / Cas system is derived from the Cas9 protein or its variants, as described in [CRISPR / Cas system (CRISPR / Cas) (2013)], ...

[0241] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule or a variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or a variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. aureus. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes.

[0242] Non-limiting examples of Cas9 orthologs from other bacterial strains include, but are not limited to, Acaryochloris marina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifex degensi degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis strain Paraca; Arthrospira species PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum strain A3 Loch Maree;Clostridium botulinum strain Ba4 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii delbrueckii subsp. bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya species PCC 8106; Marinobacter species ELB17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc species PCC 7120;Oscillatoria species PCC 6506; Pelotomaculum thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas species JS666; Pseudoalteromonas haloplanktis TAC125; Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus Cas proteins identified in Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; ​​Synechococcus species PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013;10(5):726-737).

[0243] In some aspects, the Cas protein is a mutant lacking nuclease activity (i.e., a dCas protein). In some embodiments, the Cas protein is mutated to reduce or eliminate nuclease activity. Such Cas proteins are referred to interchangeably herein as inactivated or dead Cas (dCas) proteins, or nuclease-inactive Cas (iCas) proteins. In some embodiments, the mutant Cas protein is a mutant Cas9 protein that lacks nuclease activity or is an inactivated Cas9 (dCas9 or iCas9) protein.

[0244] In some embodiments, the Cas9 protein or variant thereof is derived from a Staphylococcus aureus Cas9 (SaCas9) protein or variant thereof. In some embodiments, the mutant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) comprising at least one amino acid mutation selected from D10A and N580A, based on the numbering of positions in SEQ ID NO: 124. In some embodiments, the mutant Cas9 protein comprises the sequence set forth in SEQ ID NO: 125, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0245] In some embodiments, the Cas9 protein or variant thereof is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein or variant thereof. In some embodiments, the mutant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein containing at least one amino acid mutation selected from D10A and H840A, based on the numbering of positions in SEQ ID NO: 126. In some embodiments, the mutant Cas9 protein comprises the sequence set forth in SEQ ID NO: 127, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0246] 2. Guide RNA (gRNA) In some embodiments, a Cas protein (e.g., dCas9) is provided in combination with or complexed with one or more guide RNAs (gRNAs). In some aspects, the gRNA is a nucleic acid that facilitates specific targeting or homing of the gRNA / Cas RNP complex to a target site in a target gene, such as any of those described above in Section IB. In some embodiments, the target site of the gRNA can be referred to as a protospacer.

[0247] Provided herein are gRNAs, such as gRNAs that target or bind to a target site for a gene, such as a target gene or its regulatory DNA element, for example, any of those described herein, for example, in Section IB. In some embodiments, the gRNA can form a complex with a Cas protein or a variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e., a spacer sequence or guide sequence) that can hybridize to the target site or is complementary to the target site, for example, any target site described herein. In some embodiments, the gRNA comprises a scaffold sequence that forms a complex with or binds to a Cas protein.

[0248] In some embodiments, the gRNA provided herein is a chimeric gRNA. Generally, gRNAs can be unimolecular (i.e., composed of a single RNA molecule) or modular (comprising multiple, typically two separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, with sequences from separate modular RNA molecules included in a single gRNA molecule, sometimes referred to as a chimeric gRNA, synthetic gRNA, or single gRNA. In some embodiments, the chimeric gRNA is a fusion of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence, as described, for example, in International Publication No. 2013 / 176772 or Jinek, M. et al. Science 337(6096):816-21(2012). In some embodiments, the chimeric gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the type II effector system, and the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein.

[0249] In some aspects, the spacer sequence of the gRNA is a polynucleotide sequence that contains at least a portion that is complementary to the target site in the target gene, sufficient to hybridize with the target site and direct sequence-specific binding of the Cas / gRNA complex to the sequence of the target site. Full complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization. In some embodiments, the gRNA contains a spacer sequence that is complementary to the target site, for example, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., fully complementary). The strand of the target nucleic acid that contains the target site sequence may be referred to as the "complementary strand" of the target nucleic acid.

[0250] In some aspects, gRNA targets the target site of double-stranded DNA.Therefore, in some aspects, the sequence of target site can be defined by the sequence that gRNA spacer hybridizes with or the sequence that is complementary to the sequence that gRNA spacer hybridizes with.In some aspects, the sequence of target site can be defined by the sequence that gRNA spacer replaces to hybridize with DNA.In some embodiments, the sequence of target site is the sequence that gRNA hybridizes with.

[0251] In some embodiments, the gRNA spacer sequence is about 14 nucleotides (nt) to about 26 nt, or 16 nt to 22 nt in length. In some embodiments, the gRNA spacer sequence is 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt, 23 nt, 24 nt, 25 nt, or 26 nt in length. In some embodiments, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some embodiments, the gRNA spacer sequence is 20 nt in length.

[0252] The target site of the gRNA may be referred to as a protospacer. In some aspects, the spacer is designed to target a protospacer with a specific protospacer adjacent motif (PAM), i.e., a sequence immediately adjacent to the protospacer that contributes to and / or is required for Cas binding specificity. Different CRISPR / Cas systems have different PAM requirements for targeting. For example, in some embodiments, S. pyogenes Cas9 uses the PAM 5'-NGG-3' (SEQ ID NO: 224), where N is any nucleotide. In some embodiments, S. aureus Cas9 uses the PAM 5'-NNGRRT-3' (SEQ ID NO: 225), where N is any nucleotide and R is G or A. In some embodiments, N. meningitidis Cas9 uses the PAM 5'-NNNNGATT-3' (SEQ ID NO: 226), where N is any nucleotide. In some embodiments, C. jejuni Cas9 uses a PAM 5'-NNNNRYAC-3' (SEQ ID NO:227), where N is any nucleotide, R is G or A, and Y is C or T. In some embodiments, S. thermophilus uses a PAM 5'-NNAGAAW-3' (SEQ ID NO:228), where N is any nucleotide, and W is A or T. In some embodiments, F. novicida Cas9 uses a PAM 5'-NGG-3' (SEQ ID NO:224), where N is any nucleotide. In some embodiments, T. denticola Cas9 uses a PAM 5'-NAAAAC-3' (SEQ ID NO:229), where N is any nucleotide. In some embodiments, Cas12a (also known as Cpf1) from various species uses the PAM 5'-TTTV-3' (SEQ ID NO:230). In some embodiments, the Cas protein may use, or be engineered to use, a different PAM than those listed above.For example, mutant SpCas9 proteins may use the PAM 5'-NGG-3' (SEQ ID NO:224), 5'-NGAN-3' (SEQ ID NO:231), 5'-NGNG-3' (SEQ ID NO:232), 5'-NGAG-3' (SEQ ID NO:233), or 5'-NGCG-3' (SEQ ID NO:234). In some embodiments, the protospacer adjacent motif (PAM) of a gRNA for complexing with S. pyogenes Cas9 or a mutant thereof is NGG as set forth in SEQ ID NO:224. In some embodiments, the PAM of a gRNA for complexing with S. aureus Cas9 or a mutant thereof is NNGRRT as set forth in SEQ ID NO:225.

[0253] The spacer sequence may be selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0254] In some embodiments, the gRNA (including the guide sequence) contains the base uracil (U), while the DNA encoding the gRNA molecule contains the base thymine (T). Without wishing to be bound by theory, it is believed that in some embodiments, the complementarity between the guide sequence and the target sequence contributes to the specificity of the interaction between the gRNA molecule / Cas molecule complex and the target nucleic acid. It is understood that in a pair of guide sequence and target sequence, the uracil base in the guide sequence pairs with the adenine base in the target sequence.

[0255] In some embodiments, one, several, or all of the nucleotides of the gRNA can have modifications, for example, to make the gRNA less susceptible to degradation and / or to improve biocompatibility. As a non-limiting example, the backbone of the gRNA can be modified with phosphorothioate or other modifications. In some cases, the nucleotides of the gRNA can include 2' modifications, such as 2-acetylation, 2' methylation, or other modifications.

[0256] Methods for designing gRNAs and exemplary targeting domains include those described, for example, in International PCT Publication Nos. WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, ...4 / 197748, WO 2014 / 197748, WO 2014 / 197748, WO 2014 / 197748, WO 2014 / 197748, WO 2014 / 197748, WO 2014 / 197748, WO 2014 / 197748, WO and WO 2017 / 093969.

[0257] a. gRNA for transcriptional repression In some embodiments, the gRNA provided herein targets the target site of a gene for transcriptional repression, such as any of the target sites or target genes described in section IB2. In some embodiments, the gRNA provided herein targets the target site of a gene, such as a gene in T cells, and the gene is selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the gRNA targets the gene for transcriptional repression.

[0258] In some embodiments, the gRNA is selected from the group consisting of SEQ ID NOs. The target site comprises a sequence selected from any one of NOs: 1-6, 10-33, 80-90, 102-112, 200-211, 292-295, 300-302, and 306-308, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 1-6, 10-33, 80-90, 102-112, and 200-211 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs: 1-6, 10-33, 80-90, 102-112, 200-211, 292-295, 300-302, and 306-308.

[0259] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOs: 35-40, 44-67, 91-101, 113-123, 212-223, 296-299, 303-305, and 309-311 shown in Table 5, or at least a 14 nt contiguous portion thereof, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 35-40, 44-67, 91-101, 113-123, 212-223, 296-299, 303-305, and 309-311 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOs: 35-40, 44-67, 91-101, 113-123, 212-223, 296-299, 303-305, and 309-311.

[0260] In some embodiments, the gRNA targets a target site of a CBLB. In some embodiments, the gRNA targets a target site comprising SEQ ID NO: 11, a continuous portion thereof of at least 14 nucleotides, the complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 11 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 11. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 45, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 45 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is shown in SEQ ID NO:45.

[0261] In some embodiments, the gRNA targets a target site in MYB. In some embodiments, the gRNA targets a target site comprising SEQ ID NO: 18, a continuous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 18 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 18. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 52, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 52 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is shown in SEQ ID NO:52.

[0262] In some embodiments, the gRNA targets a target site of RASA2. In some embodiments, the gRNA targets a target site comprising SEQ ID NO: 19, a continuous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 19 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 19. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 53, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 53 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is shown in SEQ ID NO:53.

[0263] In some embodiments, the gRNA targets a target site for CISH. In some embodiments, the gRNA targets a target site comprising SEQ ID NO:28, a continuous portion thereof of at least 14 nucleotides, the complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO:28 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 28. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 62, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 62 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is shown in SEQ ID NO:62.

[0264] In some embodiments, the gRNA targets the target site of PRDM1. In some embodiments, the gRNA targets the target site comprising SEQ ID NO: 33, a continuous portion thereof of at least 14 nucleotides, the complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 33 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 33. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 67, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 67 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is shown in SEQ ID NO:67.

[0265] In some embodiments, the gRNA targets a target site in MED12. In some embodiments, the gRNA targets a target site comprising SEQ ID NO:81, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:81 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 81. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 92, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 92 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is shown in SEQ ID NO:92.

[0266] In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence is SEQ ID NO:69 In some embodiments, the scaffold sequence is set forth in SEQ ID NO:69.

[0267] In some embodiments, any of the provided gRNA sequences is complexed with or provided in combination with a fusion protein comprising Cas9. In some embodiments, the Cas9 is dCas9. In some embodiments, the dCas9 is dSpCas9, such as the dSpCas9 set forth in SEQ ID NO: 127.

[0268] In some embodiments, the present invention provides a combination of gRNAs that respectively target the target site of a gene for transcriptional repression.In some embodiments, the present invention provides a multiple epigenetic modification DNA targeting system that includes a combination of gRNAs.

[0269] In some embodiments, the gRNA combination comprises at least two gRNAs that target at least two different genes for transcriptional repression. In some embodiments, the gRNAs target a combination of genes selected from the gene combinations listed in Table 1. In some embodiments, each gRNA of the gRNA combination is selected from any of the gRNAs described herein for targeted transcriptional repression.

[0270] In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first gene and a second gRNA targeting a second gene. In some embodiments, the first gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2, and the second gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2, and the first and second gRNAs target different genes.

[0271] In some embodiments, the first gRNA targets CBLB, and the second gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the first gRNA targets CCNC, and the second gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the first gRNA targets MED12 and the second gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. In some embodiments, the first gRNA targets MYB and the second gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

[0272] In some embodiments, the first gRNA targets a CBLB and the second gRNA targets a MYB. In some embodiments, the gRNA targeting a CBLB can be any of those described, and the gRNA targeting a MYB can be any of those described. In some embodiments, the first gRNA targets a target site for a CBLB having the sequence shown in SEQ ID NO: 11, and the second gRNA targets a target site for a MYB having the sequence shown in SEQ ID NO: 18.

[0273] In some embodiments, the first gRNA targets the CBLB, and the second gRNA targets the CCNC. In some embodiments, the gRNA targeting the CBLB can be any of those described, and the gRNA targeting the CCNC can be any of those described. In some embodiments, the first gRNA targets the target site for the CBLB, which has the sequence shown in SEQ ID NO: 11, and the second gRNA targets the target site for the CCNC, which has the sequence shown in SEQ ID NO: 104.

[0274] In some embodiments, the first gRNA targets the CBLB and the second gRNA targets MED12. In some embodiments, the gRNA targeting the CBLB can be any of those described, and the gRNA targeting MED12 can be any of those described. In some embodiments, the first gRNA targets a target site for the CBLB having the sequence set forth in SEQ ID NO: 11, and the second gRNA targets a target site for MED12 having the sequence set forth in SEQ ID NO: 81.

[0275] In some embodiments, the first gRNA targets the CBLB and the second gRNA targets RASA2. In some embodiments, the gRNA targeting the CBLB can be any of those described, and the gRNA targeting RASA2 can be any of those described. In some embodiments, the first gRNA targets a target site for the CBLB having the sequence shown in SEQ ID NO: 11, and the second gRNA targets a target site for RASA2 having the sequence shown in SEQ ID NO: 19.

[0276] In some embodiments, the first gRNA targets CISH and the second gRNA targets MED12. In some embodiments, the gRNA targeting CISH can be any of those described, and the gRNA targeting MED12 can be any of those described. In some embodiments, the first gRNA targets a target site for CISH having the sequence set forth in SEQ ID NO:28, and the second gRNA targets a target site for MED12 having the sequence set forth in SEQ ID NO:81.

[0277] In some embodiments, the gRNA combination comprises at least three gRNAs targeting at least three different genes. In some embodiments, the gRNA combination comprises a first gRNA targeting a first gene, a second gRNA targeting a second gene, and a third gRNA targeting a third gene. In some embodiments, the first gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2, and the second gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2. The first gRNA targets a gene selected from the list consisting of DM1, TGFBR2, and RASA2, and the third gRNA targets a gene selected from the list consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2, and the first, second, and third gRNAs each target a different gene.

[0278] In some embodiments, the gRNA combination targets a combination of target sites for a combination of genes for transcriptional repression as shown in Table 2 and described in Section IB2.

[0279] In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO:11, and a second gRNA targeting a target site for CCNC comprising the sequence set forth in SEQ ID NO:104. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO:11, and a second gRNA targeting a target site for CD5 comprising the sequence set forth in SEQ ID NO:3. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO:11, and a second gRNA targeting a target site for CISH comprising the sequence set forth in SEQ ID NO:30. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for DGKZ comprising the sequence set forth in SEQ ID NO: 13. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for ELOB comprising the sequence set forth in SEQ ID NO: 24. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for FAS comprising the sequence set forth in SEQ ID NO: 204. In some embodiments, the gRNA combination comprises a first gRNA that targets a target site for CBLB, comprising the sequence set forth in SEQ ID NO:11, and a second gRNA that targets a target site for Fli1, comprising the sequence set forth in SEQ ID NO:208.In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for KDM1A comprising the sequence set forth in SEQ ID NO: 4. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for PRDM1 comprising the sequence set forth in SEQ ID NO: 32. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a second gRNA targeting a target site for RASA2 comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the gRNA combination comprises a first gRNA that targets a target site for CD5, comprising the sequence set forth in SEQ ID NO:3, and a second gRNA that targets a target site for CISH, comprising the sequence set forth in SEQ ID NO:30.In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CD5 comprising the sequence set forth in SEQ ID NO: 3, and a second gRNA targeting a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CISH comprising the sequence set forth in SEQ ID NO: 30, and a second gRNA targeting a target site for DGKZ comprising the sequence set forth in SEQ ID NO: 13. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CISH comprising the sequence set forth in SEQ ID NO: 30, and a second gRNA targeting a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CISH comprising the sequence set forth in SEQ ID NO: 30, and a second gRNA targeting a target site for RASA2 comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26, and a second gRNA targeting a target site for CD5 comprising the sequence set forth in SEQ ID NO: 3. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26, and a second gRNA targeting a target site for CISH comprising the sequence set forth in SEQ ID NO: 30. In some embodiments, the gRNA combination comprises a first gRNA that targets a target site for GATA3, comprising the sequence set forth in SEQ ID NO:26, and a second gRNA that targets a target site for MYB, comprising the sequence set forth in SEQ ID NO:18.In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81, and a second gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81, and a second gRNA targeting a target site for CD5 comprising the sequence set forth in SEQ ID NO: 3. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81, and a second gRNA targeting a target site for CISH comprising the sequence set forth in SEQ ID NO: 30. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81, and a second gRNA targeting a target site for DGKZ comprising the sequence set forth in SEQ ID NO: 13. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81, and a second gRNA targeting a target site for ELOB comprising the sequence set forth in SEQ ID NO: 24. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81, and a second gRNA targeting a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26. In some embodiments, the gRNA combination comprises a first gRNA that targets a target site for MED12, comprising the sequence set forth in SEQ ID NO:81, and a second gRNA that targets a target site for MYB, comprising the sequence set forth in SEQ ID NO:18.In some embodiments, a gRNA combination comprises a first gRNA that targets a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81, and a second gRNA that targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO: 32. In some embodiments, a gRNA combination comprises a first gRNA that targets a target site for MED12 comprising the sequence set forth in SEQ ID NO: 81, and a second gRNA that targets a target site for RASA2 comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, a gRNA combination comprises a first gRNA that targets a target site for MYB comprising the sequence set forth in SEQ ID NO: 18, and a second gRNA that targets a target site for RASA2 comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, a gRNA combination comprises a first gRNA that targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO: 32, and a second gRNA that targets a target site for CISH comprising the sequence set forth in SEQ ID NO: 30. In some embodiments, a gRNA combination comprises a first gRNA that targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO: 32, and a second gRNA that targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26. In some embodiments, a gRNA combination comprises a first gRNA that targets a target site for PRDM1 comprising the sequence set forth in SEQ ID NO: 32, and a second gRNA that targets a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, the gRNA combination comprises a first gRNA that targets a target site for PRDM1, comprising the sequence set forth in SEQ ID NO:32, and a second gRNA that targets a target site for RASA2, comprising the sequence set forth in SEQ ID NO:19.In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CD5 comprising the sequence set forth in SEQ ID NO: 3, a second gRNA targeting a target site for CISH comprising the sequence set forth in SEQ ID NO: 30, and a third gRNA targeting a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26, a second gRNA targeting a target site for CBLB comprising the sequence set forth in SEQ ID NO: 11, and a third gRNA targeting a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26, a second gRNA targeting a target site for CD5 comprising the sequence set forth in SEQ ID NO: 3, and a third gRNA targeting a target site for MYB comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for PRDM1 comprising the sequence set forth in SEQ ID NO: 32, a second gRNA targeting a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 26, and a third gRNA targeting a target site for CISH comprising the sequence set forth in SEQ ID NO: 30.

[0280] Table 5. Genes, target sites, and gRNAs for transcriptional repression TIFF2025531268000013.tif208170TIFF2025531268000014.tif235170TIFF2025531268000015.tif235170TIFF2025531268000016.tif100170

[0281] b. gRNA for transcriptional activation In some embodiments, provided herein is a gRNA that targets the target site of a gene for transcriptional activation, such as any of the target sites or target genes described in section IB3.In some embodiments, provided herein is a gRNA that targets the target site of a gene for transcriptional activation.In some embodiments, provided herein is a gRNA that targets the target site of a gene, such as a gene in T cell, and the gene is selected from the list shown in Table 6, consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21 and VAV1.

[0282] In some embodiments, the gRNA targets a target site comprising a sequence selected from any one of SEQ ID NOs:7-9, 78, 144-156, 170, 172-177, and 184-191 shown in Table 6, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs:7-9, 78, 144-156, 170, 172-177, and 184-191 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs:7-9, 78, 144-156, 170, 172-177, and 184-191.

[0283] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOs: 41-43, 79, 157-169, 171, 178-183, and 192-199 shown in Table 6, or at least a 14 nt contiguous portion thereof, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 41-43, 79, 157-169, 171, 178-183, and 192-199 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOs: 41-43, 79, 157-169, 171, 178-183, and 192-199.

[0284] In some embodiments, the gRNA targets an IL-2 target site. In some embodiments, the gRNA targets a target site comprising SEQ ID NO: 78, a continuous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a continuous portion of any one of SEQ ID NO: 78 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NO: 78. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 79 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is shown in SEQ ID NO:79.

[0285] In some embodiments, the gRNA targets the target site of EOMES. In some embodiments, the gRNA targets the target site comprising SEQ ID NO: 149, a continuous portion thereof of at least 14 nucleotides, the complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NO: 149 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NO: 149. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 162, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 162 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 162.

[0286] In some embodiments, the gRNA targets a target site of LCP2. In some embodiments, the gRNA targets a target site comprising SEQ ID NO: 151, a continuous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NO: 151 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NO: 151. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 164, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 164 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 164.

[0287] In some embodiments, the gRNA targets a target site of TBX21. In some embodiments, the gRNA targets a target site comprising SEQ ID NO: 155, a continuous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NO: 155 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NO: 155. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 168, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 168 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 168.

[0288] In some embodiments, the present invention provides a combination of gRNAs that each target the target site of a gene for transcription activation.In some embodiments, the present invention provides a multiple epigenetic modification DNA targeting system that includes a combination of gRNAs.

[0289] In some embodiments, the gRNA combination comprises at least two gRNAs that target at least two different genes for transcriptional activation. In some embodiments, the gRNAs target a combination of genes selected from the gene combinations listed in Table 3. In some embodiments, each gRNA of the gRNA combination is selected from any of the gRNAs described herein for targeted transcriptional activation.

[0290] In some embodiments, the gRNA combination comprises a first gRNA targeting a first gene and a second gRNA targeting a second gene. In some embodiments, the first gRNA targets a gene selected from the list consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1, and the second gRNA targets a gene selected from the list consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1, and the first and second gRNAs target different genes. In some embodiments, the first gRNA targets IL-2 and the second gRNA targets a gene selected from the list consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1. In some embodiments, the first gRNA targets VAV1 and the second gRNA targets a gene selected from the list consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

[0291] In some embodiments, the first gRNA targets IL-2 and the second gRNA targets VAV1. In some embodiments, the gRNA targeting the IL-2 target site can be any described, and the gRNA targeting the VAV1 target site can be any described. In some embodiments, the first gRNA targets the target site for IL-2 having the sequence set forth in SEQ ID NO:78, and the second gRNA targets the target site for VAV1 having the sequence set forth in SEQ ID NO:170.

[0292] In some embodiments, the first gRNA targets IL-2 and the second gRNA targets LCP2. In some embodiments, the gRNA targeting the IL-2 target site can be any of those described, and the gRNA targeting the LCP2 target site can be any of those described. In some embodiments, the first gRNA targets the target site for IL-2 having the sequence set forth in SEQ ID NO:78, and the second gRNA targets the target site for VAV1 having the sequence set forth in SEQ ID NO:151.

[0293] In some embodiments, the first gRNA targets IL-2 and the second gRNA targets TBX21. In some embodiments, the gRNA targeting the IL-2 target site can be any of those described, and the gRNA targeting the TBX21 target site can be any of those described. In some embodiments, the first gRNA targets the target site for IL-2 having the sequence set forth in SEQ ID NO:78, and the second gRNA targets the target site for TBX21 having the sequence set forth in SEQ ID NO:155.

[0294] In some embodiments, the first gRNA targets IL-2 and the second gRNA targets EOMES. In some embodiments, the gRNA targeting the IL-2 target site can be any of those described, and the gRNA targeting the EOMES target site can be any of those described. In some embodiments, the first gRNA targets the IL-2 target site having the sequence set forth in SEQ ID NO: 78, and the second gRNA targets the EOMES target site having the sequence set forth in SEQ ID NO: 149.

[0295] In some embodiments, the gRNA combination comprises at least three gRNAs that target at least three different genes. In some embodiments, the gRNA combination comprises a first gRNA that targets a first gene, a second gRNA that targets a second gene, and a third gRNA that targets a third gene. In some embodiments, the first gRNA targets a gene selected from the list consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1; the second gRNA targets a gene selected from the list consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1; and the third gRNA targets a gene selected from the list consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1, and the first, second, and third gRNAs each target a different gene.

[0296] In some embodiments, the gRNA combination targets a combination of target sites for a combination of genes for transcriptional activation as shown in Table 4 and described in Section IB3.

[0297] In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for BATF comprising the sequence set forth in SEQ ID NO: 172, and a second gRNA targeting a target site for IL-2 comprising the sequence set forth in SEQ ID NO: 78. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for BATF comprising the sequence set forth in SEQ ID NO: 172, and a second gRNA targeting a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144, and a second gRNA targeting a target site for BATF comprising the sequence set forth in SEQ ID NO: 172. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144, and a second gRNA targeting a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144, and a second gRNA targeting a target site for IL-2 comprising the sequence set forth in SEQ ID NO: 78. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144, and a second gRNA targeting a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151. In some embodiments, the gRNA combination comprises a first gRNA that targets a target site for CD28, comprising the sequence set forth in SEQ ID NO:144, and a second gRNA that targets a target site for TBX21, comprising the sequence set forth in SEQ ID NO:155.In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for CD28 comprising the sequence set forth in SEQ ID NO: 144, and a second gRNA targeting a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149, and a second gRNA targeting a target site for BATF comprising the sequence set forth in SEQ ID NO: 172. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149, and a second gRNA targeting a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149, and a second gRNA targeting a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for EOMES comprising the sequence set forth in SEQ ID NO: 149, and a second gRNA targeting a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151, and a second gRNA targeting a target site for BATF comprising the sequence set forth in SEQ ID NO: 172. In some embodiments, the gRNA combination comprises a first gRNA that targets a target site for LCP2, comprising the sequence set forth in SEQ ID NO:151, and a second gRNA that targets a target site for IL-2, comprising the sequence set forth in SEQ ID NO:78.In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151, and a second gRNA targeting a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for LCP2 comprising the sequence set forth in SEQ ID NO: 151, and a second gRNA targeting a target site for VAV1 comprising the sequence set forth in SEQ ID NO: 170. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155, and a second gRNA targeting a target site for BATF comprising the sequence set forth in SEQ ID NO: 172. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155, and a second gRNA targeting a target site for IL-2 comprising the sequence set forth in SEQ ID NO: 78. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155, and a second gRNA targeting a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, a gRNA combination comprises a first gRNA targeting a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 155, and a second ...

Claims

1. 1. An epigenetic modification DNA targeting system for suppressing transcription of one or more genes in a T cell, comprising at least one DNA targeting module, each of said at least one DNA targeting module comprising: (a) a DNA binding domain capable of being targeted to a target site for one of the one or more genes selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2; (b) at least one transcriptional repression effector domain for repressing transcription of said one or more genes in a T cell; and Included are fusion proteins comprising Epigenetic modification DNA targeting system.

2. 1. An epigenetic modification DNA targeting system for increasing transcription of one or more genes in a T cell, comprising at least one DNA targeting module, each of said at least one DNA targeting module comprising: (a) a DNA binding domain capable of being targeted to a target site for one of the one or more genes selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1; (b) at least one transcriptional activation effector domain to increase transcription of said one or more genes in T cells; and Included are fusion proteins comprising Epigenetic modification DNA targeting system.

3. The epigenetic modified DNA targeting system of claim 1 or claim 2, wherein transient delivery of the epigenetic modified DNA targeting system to a T cell promotes, optionally, an increase in T cell effector function upon T cell stimulation, compared to a T cell to which the epigenetic modified DNA targeting system has not been delivered.

4. The epigenetic modification DNA targeting system of any one of claims 1 to 3, wherein the T cell effector function is characterized by an activity selected from the group consisting of IL-2 production, IFN-gamma production, TNF-alpha production, T cell proliferation, or any combination thereof.

5. The epigenetic modification DNA targeting system of any one of claims 1 to 4, wherein the T cell effector function is characterized by IL-2 production.

6. The epigenetic modification DNA targeting system of any one of claims 1 to 4, wherein the T cell effector function is characterized by IFN-gamma production.

7. The epigenetic modification DNA targeting system of any one of claims 1 to 4, wherein the T cell effector function is characterized by IL-2 production and IFN-gamma production.

8. The epigenetic modification DNA targeting system of any one of claims 1 to 4, wherein the T cell effector function is characterized by multifunctional production of IL-2, IFN-gamma and TNF-alpha.

9. The epigenetic modification DNA targeting system of any one of claims 4 to 8, wherein the T cell effector function is characterized by an activity that further includes T cell proliferation.

10. The epigenetic modification DNA targeting system of any one of claims 4 to 9, wherein the T cell effector function is characterized by an activity that further includes killing of target cells.

11. The epigenetic modification DNA targeting system of any one of claims 4 to 10, wherein the T cell effector function is characterized by an activity that further includes T cell persistence.

12. The epigenetic modified DNA targeting system of any one of claims 3 to 11, wherein the increase in T cell effector function occurs 48 hours or more after transient delivery of the epigenetic modified DNA targeting system to the T cell.

13. The epigenetic modified DNA targeting system of any one of claims 3 to 12, wherein the increase in T cell effector function occurs by 6 days, by 9 days, by 12 days, by 15 days, by 21 days, by 28 days, by 35 days, by 42 days, by 49 days, by 56 days, by 63 days, by 71 days, or more after transient delivery of the epigenetic modified DNA targeting system to the T cell.

14. The epigenetic modified DNA targeting system of any one of claims 3 to 13, wherein T cell stimulation is with anti-CD3 and anti-CD28 activating reagents.

15. The epigenetic modification DNA targeting system of any one of claims 3 to 14, wherein the T cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor or T cell receptor (eTCR).

16. The epigenetically modified DNA targeting system of claim 15, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR) against an antigen, and the T cell stimulation is antigen-specific stimulation of the CAR or eTCR, and optionally, the T cell stimulation is by an antigen-expressing target cell.

17. The epigenetic modified DNA targeting system of any one of claims 3 to 16, wherein the T cells express a chimeric antigen receptor (CAR) against an antigen, and the T cell stimulation is antigen-specific stimulation of the CAR, and optionally, the T cell stimulation is by an antigen-expressing target cell.

18. The epigenetic modification DNA targeting system of any one of claims 3 to 17, wherein the T cell stimulation is restimulation of the T cells after at least one prior T cell stimulation.

19. The epigenetic modification DNA targeting system of any one of claims 1 to 18, which does not introduce gene disruption or DNA breaks.

20. The epigenetic modification DNA targeting system of any one of claims 1 to 19, wherein the fusion protein of each DNA targeting module comprises a DNA binding domain selected from a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated (Cas) protein or a mutant thereof, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or an I-SceI enzyme or a mutant thereof, and optionally, the DNA binding domain comprises a catalytically inactive mutant of any of the foregoing.

21. The epigenetic modification DNA targeting system of any one of claims 1 to 20, wherein the at least one DNA targeting module is a single DNA targeting module that targets a target site for one of the one or more genes.

22. the at least one DNA targeting module is a plurality of DNA targeting modules for inhibiting transcription of one or more genes in a T cell, each DNA targeting module targeting a target site for one of the one or more genes; or the at least one DNA targeting module is a plurality of DNA targeting modules for increasing transcription of one or more genes in a T cell, each DNA targeting module targeting a target site for one of the one or more genes; An epigenetic modification DNA targeting system according to any one of claims 1 to 20.

23. The epigenetic modification DNA targeting system of claim 22, wherein the plurality of DNA targeting modules is 2, 3, 4, 5 or 6 DNA targeting modules.

24. The epigenetic modification DNA targeting system of claim 22 or 23, wherein the multiple DNA targeting modules are two DNA targeting modules.

25. The epigenetic modification DNA targeting system of any one of claims 22 to 24, wherein the multiple DNA targeting modules for suppressing transcription of one or more genes in a T cell target at least a first gene and a second gene, and the first and second genes are independently selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

26. The epigenetic modification DNA targeting system of any one of claims 22 to 25, wherein the multiple DNA targeting modules for suppressing transcription of one or more genes in a T cell target at least a first gene and a second gene, and the first and second genes are independently selected from the group consisting of CBLB, CISH, MED12, MYB, PRDM1, and RASA2.

27. The epigenetic modification DNA targeting system of claim 22 or 23, wherein the multiple DNA targeting modules for suppressing transcription of one or more genes in T cells target at least a first gene, a second gene, and a third gene, and the first, second, and third genes are independently selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

28. 28. The epigenetic modification DNA targeting system of any one of claims 22 to 23 or 27, wherein the multiple DNA targeting modules for suppressing transcription of one or more genes in a T cell target at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of CBLB, CISH, MED12, MYB, PRDM1, and RASA2.

29. The epigenetic modification DNA targeting system of any one of claims 22 to 24, wherein the multiple DNA targeting modules for increasing transcription of one or more genes in T cells target at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

30. 30. The epigenetic modification DNA targeting system of any one of claims 22 to 24 or 29, wherein the multiple DNA targeting modules for increasing transcription of one or more genes in T cells target at least a first gene and a second gene, wherein the first and second genes are independently selected from the group consisting of EOMES, IL-2, LCP2, and TBX21.

31. 30. The epigenetic modification DNA targeting system of any one of claims 22, 23, and 29, wherein the multiple DNA targeting modules for increasing transcription of one or more genes in T cells target at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

32. The epigenetic modification DNA targeting system of any one of claims 22, 23, 29, or 31, wherein the multiple DNA targeting modules for increasing transcription of one or more genes in T cells target at least a first gene, a second gene, and a third gene, wherein the first, second, and third genes are independently selected from the group consisting of EOMES, IL-2, LCP2, and TBX21.

33. the plurality of DNA targeting modules are selected from the group consisting of CBLB and CCNC; CBLB and CD5; CBLB and CISH; CBLB and DGKZ; CBLB and ELOB; CBLB and FAS; CBLB and Fli1; CBLB and GATA3; CBLB and KDM1A; CBLB and MED12; CBLB and MYB; CBLB and PRDM1; CBLB and RASA2; CD5 and CISH; CD5 and MYB; CISH and DGKZ; CISH and MYB; CISH and RASA2; GATA3 and CD5; GATA3 and CISH; GATA3 and MYB; MED12 and CBLB; MED12 and CD5; MED12 and CISH; MED The epigenetic modification DNA targeting system of claim 25 or 27 targets a combination of genes selected from: 12 and DGKZ; MED12 and ELOB; MED12 and GATA3; MED12 and MYB; MED12 and PRDM1; MED12 and RASA2; MYB and RASA2; PRDM1 and CISH; PRDM1 and GATA3; PRDM1 and MYB; PRDM1 and RASA2; CD5, CISH, and MYB; GATA3, CBLB, and MYB; GATA3, CD5, and MYB; PRDM1, GATA3, and CISH, TGFBR2 and MED12; and TGFBR2, MED12, and CISH.

34. The epigenetic modification DNA targeting system of any one of claims 25 to 27, or 33, wherein the multiple DNA targeting modules target combinations of genes selected from MED12 and CBLB; MED12 and CISH; CBLB and MYB; and CBLB and RASA2.

35. 28. The epigenetic modification DNA targeting system of claim 25 or 27, wherein the first and second genes are CBLB and MYB.

36. 28. The epigenetic modification DNA targeting system of claim 25 or 27, wherein the first and second genes are CBLB and MED12.

37. The epigenetic modification DNA targeting system of claim 25 or 27, wherein the first and second genes are CBLB and CCNC.

38. The epigenetic modification DNA targeting system of claim 29 or 31, wherein the multiple DNA targeting modules target combinations of genes selected from BATF and IL-2; BATF and VAV1; CD28 and BATF; CD28 and EOMES; CD28 and IL-2; CD28 and LCP2; CD28 and TBX21; CD28 and VAV1; EOMES and BATF; EOMES and LCP2; EOMES and TBX21; EOMES and VAV1; EOMES and IL-2; LCP2 and BATF; LCP2 and IL-2; LCP2 and TBX21; LCP2 and VAV1; TBX21 and BATF; TBX21 and IL-2; TBX21 and TBX21; TBX21 and VAV1; and VAV1 and IL-2.

39. The epigenetic modification DNA targeting system of claim 29 or 31, wherein the first and second genes are IL2RB and VAV1.

40. 29 or 31 epigenetic modification DNA targeting systems, with the first and second genes being IL2 and VAV1.

41. 29 or 31 epigenetic modification DNA targeting systems, with the first and second genes being IL2 and LCP2.

42. 29 or 31 epigenetic modification DNA targeting systems, with the first and second genes being IL2 and TBX21.

43. 29 or 31 epigenetic modification DNA targeting systems, with the first and second genes being IL2 and EOMES.

44. The epigenetic modification DNA targeting system of any one of claims 1 to 43, wherein the target site for the gene or each of the one or more genes is located in the gene and / or in its regulatory DNA element.

45. The epigenetic modification DNA targeting system of claim 44, wherein the regulatory DNA element is an enhancer or promoter of the gene.

46. The epigenetic modification DNA targeting system of any one of claims 1 to 45, wherein the target site is within 1000 base pairs (bp) of the transcription start site of the gene.

47. The epigenetic modification DNA targeting system of any one of claims 1 to 46, wherein the target site is within 500 base pairs (bp) of the transcription start site of the gene.

48. The target site is (a) a target site for CD5 having a sequence set forth in any one of SEQ ID NOs: 1-3, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for KDM1A having a sequence set forth in any one of SEQ ID NOs: 4-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for a CBLB having a sequence set forth in any one of SEQ ID NOs: 10-12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for DGKZ having a sequence set forth in any one of SEQ ID NOs: 13-15, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for MYB having a sequence set forth in any one of SEQ ID NOs: 16-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for RASA2 having a sequence set forth in any one of SEQ ID NOs: 19-21, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for ELOB having a sequence set forth in any one of SEQ ID NOs: 22-24, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for GATA3 having a sequence set forth in any one of SEQ ID NOs: 25-27, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for CISH having a sequence set forth in any one of SEQ ID NOs:28-30, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (j) a target site for PRDM1 having a sequence set forth in any one of SEQ ID NOs: 31-33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (k) a target site for MED12 having a sequence set forth in any one of SEQ ID NOs: 80-90, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (l) a target site for CCNC having a sequence set forth in any one of SEQ ID NOs: 102-112, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (m) a target site for FAS having a sequence set forth in any one of SEQ ID NOs: 200-205 and 292-295, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (n) a target site for Fli1 having a sequence set forth in any one of SEQ ID NOs: 206-211, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (o) a target site for TGFBR2 having a sequence set forth in any one of SEQ ID NOs: 300-302 and 306-308, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, and 44-47, selected from:

49. The target site is (a) a target site for CD5 having the sequence shown in any one of SEQ ID NOs: 1-3, or its complementary sequence; (b) a target site for KDM1A having the sequence set forth in any one of SEQ ID NOs: 4-6, or its complementary sequence; (c) a target site for a CBLB having a sequence set forth in any one of SEQ ID NOs: 10-12 or its complementary sequence; (d) a target site for DGKZ having the sequence set forth in any one of SEQ ID NOs: 13-15 or the complementary sequence of any of the foregoing; (e) a target site for MYB1 having the sequence set forth in any one of SEQ ID NOs: 16-18, or the complementary sequence of any of the foregoing; (f) a target site for RASA2 having the sequence set forth in any one of SEQ ID NOs: 19-21 or the complementary sequence of any of the foregoing; (g) a target site for ELOB having the sequence set forth in any one of SEQ ID NOs: 22-24 or the complementary sequence of any of the foregoing; (h) a target site for GATA3 having the sequence set forth in any one of SEQ ID NOs:25-27, or the complementary sequence of any of the foregoing; (i) a target site for CISH having a sequence set forth in any one of SEQ ID NOs:28-30, or a complementary sequence of any of the foregoing; (j) a target site for PRDM1 having a sequence set forth in any one of SEQ ID NOs: 31-33, or a complementary sequence of any of the foregoing; (k) a target site for MED12 having a sequence set forth in any one of SEQ ID NOs: 80-90 or a complementary sequence of any of the foregoing; (l) a target site for CCNC having a sequence set forth in any one of SEQ ID NOs: 102-112 or a complementary sequence of any of the foregoing; (m) a target site for FAS having a sequence set forth in any one of SEQ ID NOs: 200-205 and 292-295, or a complementary sequence of any of the foregoing; (n) a target site for Fli1 having a sequence set forth in any one of SEQ ID NOs: 206-211 or a complementary sequence thereof; and (o) a target site for TGFBR2 having a sequence set forth in any one of SEQ ID NOs: 300-302 and 306-308, or a complementary sequence of any of the foregoing; The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, and 44-47, selected from:

50. (a) the target site is selected from target sites for CBLB having the sequence shown in SEQ ID NO:11 or its complementary sequence; (b) the target site is selected from target sites for MYB having the sequence set forth in SEQ ID NO:18 or its complementary sequence; (c) the target site is selected from target sites for RASA2 having the sequence set forth in SEQ ID NO: 19 or its complementary sequence; (d) the target site is selected from target sites for CISH having the sequence set forth in SEQ ID NO:28 or its complementary sequence; (e) the target site is selected from target sites for PRDM1 having the sequence set forth in SEQ ID NO:33 or its complementary sequence; and (f) the target site is selected from the target site for MED12 having the sequence set forth in SEQ ID NO: 81 or its complementary sequence; An epigenetic modification DNA targeting system according to any one of claims 1, 3-27, 33-37, and 44-47.

51. The target site is (a) a target site for VAV1 having a sequence set forth in any one of SEQ ID NOs: 7-9, 156, and 170, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for IL2 having the sequence set forth in SEQ ID NO:78, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for BATF having a sequence set forth in any one of SEQ ID NOs: 172-174, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for CD28 having a sequence set forth in any one of SEQ ID NOs: 144-146 and 189-191, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for EOMES having a sequence set forth in any one of SEQ ID NOs: 147-149, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for IRF4 having a sequence set forth in any one of SEQ ID NOs: 175-177, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for LAT having a sequence set forth in any one of SEQ ID NOs: 184-186, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LCP2 having a sequence set forth in any one of SEQ ID NOs: 150-152 and 187-188, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (i) a target site for TBX21 having a sequence set forth in any one of SEQ ID NOs: 153-155, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; The epigenetic modification DNA targeting system of any one of claims 2 to 24, 29, 31, and 38 to 47, selected from:

52. The target site is (a) a target site for VAV1 having a sequence set forth in any one of SEQ ID NOs: 7-9, 156, and 170, or a complementary sequence of any of the foregoing; (b) a target site for IL2 having the sequence shown in SEQ ID NO:78, or a complementary sequence of any of the foregoing; (c) a target site for BATF having a sequence set forth in any one of SEQ ID NOs: 172-174, or a complementary sequence of any of the foregoing; (d) a target site for CD28 having a sequence set forth in any one of SEQ ID NOs: 144-146 and 189-191, or a complementary sequence of any of the foregoing; (e) a target site for EOMES having a sequence set forth in any one of SEQ ID NOs: 147-149, or a complementary sequence of any of the foregoing; (f) a target site for IRF4 having a sequence set forth in any one of SEQ ID NOs: 175-177, or a complementary sequence of any of the foregoing; (g) a target site for LAT having a sequence set forth in any one of SEQ ID NOs: 184-186, or a complementary sequence of any of the foregoing; (h) a target site for LCP2 having a sequence set forth in any one of SEQ ID NOs: 150-152 and 187-188, or a complementary sequence of any of the foregoing; and (i) a target site for TBX21 having the sequence set forth in any one of SEQ ID NOs: 153-155, or a complementary sequence of any of the foregoing; The epigenetic modification DNA targeting system of any one of claims 2 to 24, 29, 31, 38 to 47, and 51, selected from:

53. The target site is (a) a target site for IL-2 having the sequence shown in SEQ ID NO:78, or a complementary sequence of any of the foregoing; (b) a target site for EOMES having the sequence set forth in SEQ ID NO:149, or a complementary sequence of any of the foregoing; (c) a target site for LCP2 having the sequence set forth in SEQ ID NO: 151, or a complementary sequence of any of the foregoing; and (d) a target site for TBX21 having the sequence set forth in SEQ ID NO:155, or a complementary sequence of any of the foregoing. The epigenetic modification DNA targeting system of any one of claims 2 to 24, 29, 31, 38 to 47, and 52, selected from the group consisting of:

54. The epigenetic modification DNA targeting system of any one of claims 1 to 53, wherein the DNA binding domain of each of the at least one DNA targeting module is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated (Cas) protein or a variant thereof, and each of the at least one DNA targeting module further comprises at least one gRNA for targeting the DNA binding domain to a target site in the one or more genes.

55. The epigenetic modification DNA targeting system of any one of claims 20 to 54, wherein the Cas protein or a mutant thereof is an inactivated (dCas) protein.

56. 56. The epigenetic modification DNA targeting system of claim 55, wherein the dCas protein lacks nuclease activity.

57. 57. The epigenetic modification DNA targeting system of claim 55 or 56, wherein the dCas protein is a dCas9 protein.

58. The epigenetic modification DNA targeting system of claim 55 or 56, wherein the dCas protein is a dCas12 protein.

59. 58. The epigenetic modification DNA targeting system according to 57, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

60. The epigenetic modification DNA targeting system of claim 59, wherein dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, according to the numbering of positions in SEQ ID NO:

124.

61. The epigenetic modification DNA targeting system of claim 59 or 60, wherein the dSaCas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

62. The epigenetic modification DNA targeting system of any one of claims 59 to 61, wherein dSaCas9 is set forth in SEQ ID NO:

125.

63. 58. The epigenetic modification DNA targeting system of claim 57, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

64. 64. The epigenetic modification DNA targeting system of claim 63, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, according to the numbering of positions in SEQ ID NO:

126.

65. The epigenetic modification DNA targeting system of claim 63 or 64, wherein the dSpCas9 comprises an amino acid sequence set forth in SEQ ID NO: 127 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

66. The epigenetic modification DNA targeting system of any one of claims 63 to 65, wherein dSpCas9 is set forth in SEQ ID NO:

127.

67. The epigenetic modification DNA targeting system of any one of claims 54 to 66, wherein the gRNA comprises a gRNA spacer complementary to a target site of the gene.

68. the DNA targeting module is for repressing transcription of the one or more genes, and the gRNA is (a) a gRNA that targets a target site for CD5 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 35-37, or a contiguous portion thereof that is at least 14 nt; (b) a gRNA that targets a target site for KDM1A and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 38-40, or a contiguous portion thereof that is at least 14 nt; (c) a gRNA that targets a target site for a CBLB and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 44 to 46 or a contiguous portion thereof that is at least 14 nt; (d) a gRNA that targets a target site for DGKZ and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 47-49 or a contiguous portion thereof that is at least 14 nt; (e) a gRNA that targets a target site for MYB and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 50-52, or a contiguous portion thereof that is at least 14 nt; (f) a gRNA that targets a target site for RASA2 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 53 to 55, or a contiguous portion thereof that is at least 14 nt; (g) a gRNA that targets a target site for ELOB and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 56-58 or a contiguous portion thereof that is at least 14 nt; (h) a gRNA that targets a target site for GATA3 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 59-61 or a contiguous portion thereof that is at least 14 nt; (i) a gRNA that targets a target site for CISH and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 62-64 or a contiguous portion thereof that is at least 14 nt; (j) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 65 to 67, or a contiguous portion thereof that is at least 14 nt; (k) a gRNA that targets a target site for MED12 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 91-101 or a contiguous portion thereof that is at least 14 nt; (l) a gRNA that targets a target site for CCNC and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 113 to 123 or a contiguous portion thereof that is at least 14 nt; (m) a gRNA that targets a target site for FAS and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 212-217 and 296-299, or a contiguous portion thereof that is at least 14 nt; (n) a gRNA that targets a target site for Fli1 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 218-223 or a contiguous portion thereof that is at least 14 nt; and (o) a gRNA that targets a target site for TGFBR2 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 303-305 and 309-311, or a contiguous portion thereof that is at least 14 nt; The epigenetic modification DNA targeting system of any one of claims 54 to 67, selected from:

69. the DNA targeting module is for repressing transcription of the one or more genes, and the gRNA is (a) a gRNA targeting a target site for CD5 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 35-37; (b) a gRNA targeting a target site for KDM1A and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 38-40; (c) a gRNA targeting a target site for a CBLB and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 44 to 46; (d) a gRNA targeting a target site for DGKZ and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 47-49; (e) a gRNA targeting a target site for MYB and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 50-52; (f) a gRNA targeting a target site for RASA2 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 53-55; (g) a gRNA targeting a target site for ELOB and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 56-58; (h) a gRNA targeting a target site for GATA3 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 59-61; (i) a gRNA targeting a target site for CISH and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 62-64; (j) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 65 to 67; (k) a gRNA targeting a target site for MED12 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 91-101; (l) a gRNA targeting a target site for CCNC and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 113 to 123; (m) a gRNA that targets a target site for FAS and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 212-217 and 296-299; (n) a gRNA that targets a target site for Fli1 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 218-223; and (o) a gRNA targeting a target site for TGFBR2 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 303-305 and 309-311; The epigenetic modification DNA targeting system of any one of claims 54 to 68, selected from:

70. the DNA targeting module is for repressing transcription of the one or more genes, and the gRNA is (a) a gRNA targeting a target site for a CBLB and comprising a gRNA spacer sequence as shown in SEQ ID NO:45; (b) a gRNA targeting a target site for MYB and comprising the gRNA spacer sequence shown in SEQ ID NO:52; (c) a gRNA targeting a target site for RASA2 and comprising the gRNA spacer sequence shown in SEQ ID NO:53; (d) a gRNA targeting a target site for CISH and comprising the gRNA spacer sequence shown in SEQ ID NO:62; (e) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence shown in SEQ ID NO:67; and (f) a gRNA targeting the target site for MED12 and comprising the gRNA spacer sequence shown in SEQ ID NO:92; The epigenetic modification DNA targeting system of any one of claims 54 to 68, selected from:

71. the DNA targeting module is for increasing transcription of the one or more genes, and the gRNA (a) a gRNA that targets a target site for VAV1 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 41-43, 169, and 171, or a contiguous portion thereof that is at least 14 nt; (b) a gRNA that targets a target site for IL2 and comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:79 or a contiguous portion thereof that is at least 14 nt; (c) a gRNA that targets a target site for BATF and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 178-180 or a contiguous portion thereof that is at least 14 nt; (d) a gRNA that targets a target site for CD28 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 157-159 and 197-199, or a contiguous portion thereof that is at least 14 nt; (e) a gRNA that targets a target site for EOMES and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 160-162 or a contiguous portion thereof that is at least 14 nt; (f) a gRNA that targets a target site for IRF4 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 181-183, or a contiguous portion thereof that is at least 14 nt; (g) a gRNA that targets a target site for LAT and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 192 to 194, or a contiguous portion thereof that is at least 14 nt; (h) a gRNA that targets a target site for LCP2 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 163-165 and 195-196, or a contiguous portion thereof that is at least 14 nt; and (i) a gRNA that targets a target site for TBX21 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 166 to 168, or a contiguous portion thereof that is at least 14 nt; The epigenetic modification DNA targeting system of any one of claims 54 to 67, selected from:

72. the DNA targeting module is for increasing transcription of the one or more genes, and the gRNA (a) a gRNA targeting a target site for VAV1 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 41-43, 169, and 171; (b) a gRNA targeting a target site for IL2 and comprising the gRNA spacer sequence shown in SEQ ID NO:79; (c) a gRNA targeting a target site for BATF and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 178-180; (d) a gRNA that targets a target site for CD28 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 157-159 and 197-199; (e) a gRNA targeting a target site for EOMES and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 160-162; (f) a gRNA targeting a target site for IRF4 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 181-183; (g) a gRNA targeting a target site for LAT and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 192-194; (h) a gRNA that targets a target site for LCP2 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 163-165 and 195-196; and (i) a gRNA targeting a target site for TBX21 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 166 to 168; The epigenetic modification DNA targeting system of any one of claims 54 to 67 and 71, selected from:

73. the DNA targeting module is for increasing transcription of the one or more genes, and the gRNA (a) a gRNA targeting a target site for IL-2 and comprising the gRNA spacer sequence shown in SEQ ID NO:79; (a) a gRNA targeting a target site for EOMES and comprising a gRNA spacer sequence as shown in SEQ ID NO: 162; (a) a gRNA targeting a target site for LCP2 and comprising the gRNA spacer sequence shown in SEQ ID NO:164; and (a) A gRNA targeting the target site for TBX21 and comprising the gRNA spacer sequence shown in SEQ ID NO:

168. The epigenetic modification DNA targeting system of any one of claims 54 to 67 and 71, selected from:

74. The epigenetic modification DNA targeting system of any one of claims 54 to 72, wherein the gRNA comprises a spacer sequence that is 14 nt to 24 nt, or 16 nt to 22 nt in length.

75. The epigenetic modification DNA targeting system of any one of claims 54 to 74, wherein the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

76. The epigenetic modification DNA targeting system of any one of claims 54 to 75, wherein the gRNA further comprises a scaffold sequence as set forth in SEQ ID NO:

69.

77. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 36-41, 54-69, and 74-76, wherein the at least one transcriptional repression effector domain is capable of reducing transcription of the one or more genes.

78. The epigenetic modification DNA targeting system of any one of claims 1, 3 to 27, 33 to 37, 44 to 49, 54 to 69, and 74 to 77, wherein the transcriptional repression effector domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3A-DNMT3L fusion protein domain, an ERF repression domain, an Mxi1 repression domain, a SID4X repression domain, a Mad-SID repression domain, an LSD1 repression domain, an EZH2 repression domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing.

79. The epigenetic modification DNA targeting system of any one of claims 1, 3 to 27, 33 to 37, 44 to 49, 54 to 69, and 74 to 78, wherein the transcriptional repression effector domain is a KRAB domain, a DNMT3A domain, or a DNMT3L domain, or any combination thereof.

80. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 44-49, 54-69, and 74-79, wherein the at least one transcriptional repression effector domain comprises a KRAB domain, or a variant or portion thereof that exhibits transcriptional repressor activity.

81. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 44-49, 54-69, and 74-80, wherein the at least one transcriptional repression effector domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences set forth in any one of SEQ ID NOs: 70, 235, and 355-358, a portion thereof, or any of the foregoing.

82. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 44-49, 54-69, and 74-79, wherein the at least one transcriptional repression effector domain comprises a DNMT3A domain or a variant or portion thereof that exhibits transcriptional repressor activity.

83. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 44-49, 54-69, 74-79 and 82, wherein the at least one transcriptional repression domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 131 or 238, a portion thereof, or any of the foregoing.

84. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 44-49, 54-69, and 74-79, wherein the at least one transcriptional repression domain comprises a DNMT3L domain or a variant or portion thereof that exhibits transcriptional repressor activity.

85. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 44-49, 54-69, and 74-79 and 84, wherein the at least one transcriptional repression domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences set forth in any one of SEQ ID NOs: 133 and 240-242, a portion thereof, or any of the foregoing.

86. The epigenetic modification DNA targeting system of any one of claims 1, 3 to 27, 33 to 37, 44 to 49, 54 to 69, and 74 to 79, wherein the at least one transcriptional repression domain is a DNMT3A-DNMT3L fusion protein domain, a DNMT3B-DNMT3L fusion protein domain, or a variant thereof that exhibits transcriptional repressor activity.

87. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 44-49, 54-69, 74-79 and 86, wherein the at least one transcriptional repression domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences set forth in SEQ ID NOs: 135, 137, or 363, a portion thereof, or any of the foregoing.

88. The epigenetic modification DNA targeting system of any one of claims 1, 3-27, 33-37, 44-49, 54-69, and 74-87, wherein the fusion protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, of any one of SEQ ID NOs: 138-141, 332-351, and 365-384.

89. The DNA targeting system of any one of claims 2-24, 29, 31, 38-47, 51-67, and 71-76, wherein said at least one transcriptional activation effector domain is capable of increasing transcription of said one or more genes.

90. 90. The DNA targeting system of any one of claims 2-24, 29, 31, 38-47, 51-67, 71-76, and 89, wherein the at least one transcription activation effector domain is selected from the group consisting of a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain (optionally the TET protein is TET1), a SunTag domain, or a domain, portion, mutant, or truncation of any of the foregoing.

91. The DNA targeting system of any one of claims 2 to 24, 29, 31, 38 to 47, 51 to 67, 71 to 76, 89 and 90, wherein the at least one transcriptional activation effector domain comprises at least one VP16 domain and / or VP16 tetramer ("VP64"), or a mutant thereof.

92. The DNA targeting system of any one of claims 2 to 24, 29, 31, 38 to 47, 51 to 67, 71 to 76, and 89 to 91, wherein the at least one transcriptional activation effector domain comprises a VP64 domain, or a variant or portion thereof that exhibits transcriptional activation activity.

93. The DNA targeting system of any one of claims 2 to 24, 29, 31, 38 to 47, 51 to 67, 71 to 76, and 89 to 92, wherein the at least one transcription activation effector domain is VP64.

94. 94. The DNA targeting system of any one of claims 2-24, 29, 31, 38-47, 51-67, 71-76, and 89-93, wherein the at least one transcription activation effector domain comprises the sequence set forth in SEQ ID NO: 142, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

95. The DNA targeting system of any one of claims 2-24, 29, 31, 38-47, 51-67, 71-76, and 89-94, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

96. A combination of epigenetic modification DNA targeting systems comprising at least two of the DNA targeting systems of any of claims 1, 3-27, 33-37, 44-49, 54-69, and 74-88, wherein each DNA targeting system suppresses transcription of a different gene among the one or more genes.

97. A combination of epigenetic modification DNA targeting systems comprising at least two of the DNA targeting systems of any of claims 2 to 24, 29, 31, 38 to 47, 51 to 67, 71 to 76, and 89 to 95, wherein each DNA targeting system increases transcription of a different gene among the one or more genes.

98. A guide RNA (gRNA) targeting a target site for a gene selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

99. 99. The gRNA of Claim 98, wherein the target site for said gene is within said gene or in a regulatory DNA element thereof.

100. 100. The gRNA of claim 99, wherein the regulatory DNA element is an enhancer or a promoter.

101. The gRNA of any one of claims 98-100, wherein the target site is within 1000 base pairs (bp) of the transcription start site of the gene.

102. The gRNA of any one of claims 98-101, wherein the target site is within 500 base pairs (bp) of the transcription start site of said gene.

103. The target site is (a) a target site for CD5 having a sequence set forth in any one of SEQ ID NOs: 1-3, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for KDM1A having a sequence set forth in any one of SEQ ID NOs: 4-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for a CBLB having a sequence set forth in any one of SEQ ID NOs: 10-12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for DGKZ having a sequence set forth in any one of SEQ ID NOs: 13-15, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for MYB having a sequence set forth in any one of SEQ ID NOs: 16-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for RASA2 having a sequence set forth in any one of SEQ ID NOs: 19-21, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for ELOB having a sequence set forth in any one of SEQ ID NOs: 22-24, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for GATA3 having a sequence set forth in any one of SEQ ID NOs: 25-27, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for CISH having a sequence set forth in any one of SEQ ID NOs:28-30, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (j) a target site for PRDM1 having a sequence set forth in any one of SEQ ID NOs: 31-33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (k) a target site for MED12 having a sequence set forth in any one of SEQ ID NOs: 80-90, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (l) a target site for CCNC having a sequence set forth in any one of SEQ ID NOs: 102-112, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (m) a target site for FAS having a sequence set forth in any one of SEQ ID NOs: 200-205 and 292-295, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (n) a target site for Fli1 having a sequence set forth in any one of SEQ ID NOs: 206-211, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (o) a target site for TGFBR2 having a sequence set forth in any one of SEQ ID NOs: 300-302 and 306-308, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; The gRNA of any one of claims 98 to 102, selected from:

104. The target site is (a) a target site for CD5 having the sequence shown in any one of SEQ ID NOs: 1-3, or its complementary sequence; (b) a target site for KDM1A having the sequence set forth in any one of SEQ ID NOs: 4-6, or its complementary sequence; (c) a target site for a CBLB having a sequence set forth in any one of SEQ ID NOs: 10-12 or its complementary sequence; (d) a target site for DGKZ having the sequence set forth in any one of SEQ ID NOs: 13-15 or the complementary sequence of any of the foregoing; (e) a target site for MYB having the sequence set forth in any one of SEQ ID NOs: 16-18, or the complementary sequence of any of the foregoing; (f) a target site for RASA2 having the sequence set forth in any one of SEQ ID NOs: 19-21 or the complementary sequence of any of the foregoing; (g) a target site for ELOB having the sequence set forth in any one of SEQ ID NOs: 22-24 or the complementary sequence of any of the foregoing; (h) a target site for GATA3 having the sequence set forth in any one of SEQ ID NOs:25-27, or the complementary sequence of any of the foregoing; (i) a target site for CISH having a sequence set forth in any one of SEQ ID NOs:28-30, or a complementary sequence of any of the foregoing; (j) a target site for PRDM1 having a sequence set forth in any one of SEQ ID NOs: 31-33, or a complementary sequence of any of the foregoing; (k) a target site for MED12 having a sequence set forth in any one of SEQ ID NOs: 80-90 or a complementary sequence of any of the foregoing; (l) a target site for CCNC having a sequence set forth in any one of SEQ ID NOs: 102-112 or a complementary sequence of any of the foregoing; (m) a target site for FAS having a sequence set forth in any one of SEQ ID NOs: 200-205 and 292-295, or a complementary sequence of any of the foregoing; (n) a target site for Fli1 having a sequence set forth in any one of SEQ ID NOs: 206-211 or a complementary sequence thereof; and (o) a target site for TGFBR2 having a sequence set forth in any one of SEQ ID NOs: 300-302 and 306-308, or a complementary sequence of any of the foregoing; The gRNA of any one of claims 98 to 103, selected from:

105. The target site is (a) a target site for a CBLB having the sequence shown in SEQ ID NO:11 or a complementary sequence of any of the foregoing; (b) a target site for MYB having the sequence shown in SEQ ID NO:18 or the complementary sequence of any of the foregoing. (c) a target site for RASA2 having the sequence shown in SEQ ID NO:19 or a complementary sequence of any of the foregoing; (d) a target site for CISH having the sequence set forth in SEQ ID NO:28 or the complementary sequence of any of the foregoing; (e) a target site for PRDM1 having the sequence set forth in SEQ ID NO:33 or the complementary sequence of any of the foregoing; and (f) a target site for MED12 having the sequence set forth in SEQ ID NO:81 or a complementary sequence of any of the foregoing; The gRNA of any one of claims 98 to 104, selected from:

106. (a) a gRNA that targets a target site for CD5 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 35-37, or a contiguous portion thereof that is at least 14 nt; (b) a gRNA that targets a target site for KDM1A and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 38-40, or a contiguous portion thereof that is at least 14 nt; (c) a gRNA that targets a target site for a CBLB and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 44 to 46 or a contiguous portion thereof that is at least 14 nt; (d) a gRNA that targets a target site for DGKZ and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 47-49 or a contiguous portion thereof that is at least 14 nt; (e) a gRNA that targets a target site for MYB and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 50-52, or a contiguous portion thereof that is at least 14 nt; (f) a gRNA that targets a target site for RASA2 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 53 to 55, or a contiguous portion thereof that is at least 14 nt; (g) a gRNA that targets a target site for ELOB and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 56-58 or a contiguous portion thereof that is at least 14 nt; (h) a gRNA that targets a target site for GATA3 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 59-61 or a contiguous portion thereof that is at least 14 nt; (i) a gRNA that targets a target site for CISH and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 62-64 or a contiguous portion thereof that is at least 14 nt; (j) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 65 to 67, or a contiguous portion thereof that is at least 14 nt; (k) a gRNA that targets a target site for MED12 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 91-101 or a contiguous portion thereof that is at least 14 nt; (l) a gRNA that targets a target site for CCNC and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 113 to 123 or a contiguous portion thereof that is at least 14 nt; (m) a gRNA that targets a target site for FAS and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 212-217 and 296-299, or a contiguous portion thereof that is at least 14 nt; (n) a gRNA that targets a target site for Fli1 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 218-223 or a contiguous portion thereof that is at least 14 nt; and (o) a gRNA that targets a target site for TGFBR2 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 303-305 and 309-311, or a contiguous portion thereof that is at least 14 nt; The gRNA of any one of claims 98 to 105, selected from:

107. (a) a gRNA targeting a target site for CD5 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 35-37; (b) a gRNA targeting a target site for KDM1A and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 38-40; (c) a gRNA targeting a target site for a CBLB and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 44 to 46; (d) a gRNA targeting a target site for DGKZ and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 47-49; (e) a gRNA targeting a target site for MYB and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 50-52; (f) a gRNA targeting a target site for RASA2 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 53-55; (g) a gRNA targeting a target site for ELOB and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 56-58; (h) a gRNA targeting a target site for GATA3 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 59-61; (i) a gRNA targeting a target site for CISH and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 62-64; (j) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 65 to 67; (k) a gRNA targeting a target site for MED12 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 91-101; (l) a gRNA targeting a target site for CCNC and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 113 to 123; (m) a gRNA that targets a target site for FAS and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 212-217 and 296-299; (n) a gRNA that targets a target site for Fli1 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 218-223; and (o) a gRNA targeting a target site for TGFBR2 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 303-305 and 309-311; The gRNA of any one of claims 98 to 106, selected from:

108. (a) a gRNA targeting a target site for a CBLB and comprising a gRNA spacer sequence as shown in SEQ ID NO:45; (b) a gRNA targeting a target site for MYB and comprising the gRNA spacer sequence shown in SEQ ID NO:52; (c) a gRNA targeting a target site for RASA2 and comprising the gRNA spacer sequence shown in SEQ ID NO:53; (d) a gRNA targeting a target site for CISH and comprising the gRNA spacer sequence shown in SEQ ID NO:62; (e) a gRNA that targets a target site for PRDM1 and comprises a gRNA spacer sequence shown in SEQ ID NO:67; and (f) a gRNA targeting a target site for MED12 and comprising the gRNA spacer sequence shown in SEQ ID NO:91; The gRNA of any one of claims 98 to 107, selected from:

109. The gRNA of any one of claims 98-108, comprising a spacer sequence that is 14 nt to 24 nt, or 16 nt to 22 nt in length.

110. 110. The gRNA of any one of claims 98-109, comprising a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

111. The gRNA of any one of claims 98-110, further comprising a scaffold sequence as set forth in SEQ ID NO:

69.

112. A guide RNA (gRNA) that targets a target site for a gene selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

113. 113. The gRNA of claim 112, wherein the target site for said gene is within said gene or in a regulatory DNA element thereof.

114. 114. The gRNA of claim 113, wherein the regulatory DNA element is an enhancer or a promoter.

115. 115. The gRNA of any one of claims 112-114, wherein the target site is within 1000 base pairs (bp) of the transcription start site of said gene.

116. 116. The gRNA of any one of claims 112-115, wherein the target site is within 500 base pairs (bp) of the transcription start site of said gene.

117. The target site is (a) a target site for VAV1 having a sequence set forth in any one of SEQ ID NOs: 7-9, 156, and 170, a contiguous portion thereof that is at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for IL2 having the sequence set forth in SEQ ID NO:78, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for BATF having a sequence set forth in any one of SEQ ID NOs: 172-174, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for CD28 having a sequence set forth in any one of SEQ ID NOs: 144-146 and 189-191, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for EOMES having a sequence set forth in any one of SEQ ID NOs: 147-149, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for IRF4 having a sequence set forth in any one of SEQ ID NOs: 175-177, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for LAT having a sequence set forth in any one of SEQ ID NOs: 184-186, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LCP2 having a sequence set forth in any one of SEQ ID NOs: 150-152 and 187-188, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (i) a target site for TBX21 having a sequence set forth in any one of SEQ ID NOs: 153-155, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; 117. The gRNA of any one of claims 112 to 116, selected from:

118. The target site is (a) a target site for VAV1 having a sequence set forth in any one of SEQ ID NOs: 7-9, 156, and 170, or a complementary sequence of any of the foregoing; (b) a target site for IL2 having the sequence shown in SEQ ID NO:78, or a complementary sequence of any of the foregoing; (c) a target site for BATF having a sequence set forth in any one of SEQ ID NOs: 172-174, or a complementary sequence of any of the foregoing; (d) a target site for CD28 having a sequence set forth in any one of SEQ ID NOs: 144-146 and 189-191, or a complementary sequence of any of the foregoing; (e) a target site for EOMES having a sequence set forth in any one of SEQ ID NOs: 147-149, or a complementary sequence of any of the foregoing; (f) a target site for IRF4 having a sequence set forth in any one of SEQ ID NOs: 175-177, or a complementary sequence of any of the foregoing; (g) a target site for LAT having a sequence set forth in any one of SEQ ID NOs: 184-186, or a complementary sequence of any of the foregoing; (h) a target site for LCP2 having a sequence set forth in any one of SEQ ID NOs: 150-152 and 187-188, or a complementary sequence of any of the foregoing; and (i) a target site for TBX21 having the sequence set forth in any one of SEQ ID NOs: 153-155, or a complementary sequence of any of the foregoing; 118. The gRNA of any one of claims 112-117, selected from:

119. The target site is (a) a target site for IL-2 having the sequence shown in SEQ ID NO:78, or a complementary sequence of any of the foregoing; (b) a target site for EOMES having the sequence set forth in SEQ ID NO:149, or a complementary sequence of any of the foregoing; (c) a target site for LCP2 having the sequence set forth in SEQ ID NO: 151, or a complementary sequence of any of the foregoing; and (d) a target site for TBX21 having the sequence set forth in SEQ ID NO:155, or a complementary sequence of any of the foregoing. The gRNA of any one of claims 112-118, selected from:

120. (a) a gRNA that targets a target site for VAV1 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 41-43, 169, and 171, or a contiguous portion thereof that is at least 14 nt; (b) a gRNA that targets a target site for IL2 and comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:79 or a contiguous portion thereof that is at least 14 nt; (c) a gRNA that targets a target site for BATF and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 178-180 or a contiguous portion thereof that is at least 14 nt; (d) a gRNA that targets a target site for CD28 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 157-159 and 197-199, or a contiguous portion thereof that is at least 14 nt; (e) a gRNA that targets a target site for EOMES and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 160-162 or a contiguous portion thereof that is at least 14 nt; (f) a gRNA that targets a target site for IRF4 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 181-183, or a contiguous portion thereof that is at least 14 nt; (g) a gRNA that targets a target site for LAT and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 192 to 194, or a contiguous portion thereof that is at least 14 nt; (h) a gRNA that targets a target site for LCP2 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 163-165 and 195-196, or a contiguous portion thereof that is at least 14 nt; and (i) a gRNA that targets a target site for TBX21 and comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 166 to 168, or a contiguous portion thereof that is at least 14 nt; 120. The gRNA of any one of claims 112-119, selected from:

121. (a) a gRNA targeting a target site for VAV1 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 41-43, 169, and 171; (b) a gRNA targeting a target site for IL2 and comprising the gRNA spacer sequence shown in SEQ ID NO:79; (c) a gRNA targeting a target site for BATF and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 178-180; (d) a gRNA that targets a target site for CD28 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 157-159 and 197-199; (e) a gRNA targeting a target site for EOMES and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 160-162; (f) a gRNA targeting a target site for IRF4 and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 181-183; (g) a gRNA targeting a target site for LAT and comprising a gRNA spacer sequence set forth in any one of SEQ ID NOs: 192-194; (h) a gRNA that targets a target site for LCP2 and comprises a gRNA spacer sequence set forth in any one of SEQ ID NOs: 163-165 and 195-196; and (i) a gRNA targeting a target site for TBX21 and comprising a gRNA spacer sequence as set forth in any one of SEQ ID NOs: 166 to 168; The gRNA of any one of claims 112 to 120, selected from:

122. (a) a gRNA targeting a target site for IL-2 and comprising the gRNA spacer sequence shown in SEQ ID NO:79; (b) a gRNA targeting a target site for EOMES and comprising the gRNA spacer sequence shown in SEQ ID NO: 162; (c) a gRNA targeting a target site for LCP2 and comprising the gRNA spacer sequence shown in SEQ ID NO: 164; and (d) a gRNA targeting the target site for TBX21 and comprising the gRNA spacer sequence shown in SEQ ID NO: 168; 122. The gRNA of any one of claims 112-121, selected from:

123. 123. The gRNA of any one of claims 112-122, comprising a spacer sequence that is 14 nt to 24 nt, or 16 nt to 22 nt in length.

124. 124. The gRNA of any one of claims 112-123, comprising a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

125. The gRNA of any one of claims 112-124, further comprising a scaffold sequence as set forth in SEQ ID NO:

69.

126. two or more gRNAs each selected from the gRNAs of any one of claims 98 to 111; or Two or more gRNAs each selected from the gRNAs of any one of claims 112 to 125. A gRNA combination comprising:

127. the two gRNAs comprise the spacer sequences set forth in SEQ ID NOs: 92 and 45; SEQ ID NOs: 92 and 62; SEQ ID NOs: 45 and 52; and SEQ ID NOs: 45 and 53; SEQ ID NOs: 92 and 304; SEQ ID NOs: 92, 304, or 62; or The two gRNAs comprise the spacer sequences shown in SEQ ID NOs: 79 and 164; SEQ ID NOs: 79 and 168; SEQ ID NOs: 79 and 162; The gRNA combination of claim 126.

128. (a) CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) proteins or their mutants (b) at least one gRNA according to any one of claims 98 to 111; A Cas-guide RNA (gRNA) combination comprising:

129. (a) CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) proteins or their mutants (b) at least one gRNA according to any one of claims 112 to 125; A Cas-guide RNA (gRNA) combination comprising:

130. 130. The Cas-guide RNA (gRNA) combination of claim 128 or 129, wherein the Cas protein or a variant thereof is an inactivated (dCas) protein.

131. 131. The Cas-guide RNA (gRNA) combination of claim 130, wherein said dCas protein lacks nuclease activity.

132. 132. The Cas-guide RNA (gRNA) combination of claim 130 or 131, wherein the dCas protein is a dCas9 protein.

133. 132. The Cas-guide RNA (gRNA) combination of claim 130 or 131, wherein the dCas protein is a dCas12 protein.

134. 133. The Cas-guide RNA (gRNA) combination of claim 132, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

135. 135. The Cas-guide RNA (gRNA) combination of claim 134, wherein said dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, according to the numbering of positions in SEQ ID NO:

124.

136. 136. The Cas-guide RNA (gRNA) combination of Claim 134 or 135, wherein the dSaCas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

137. The Cas-guide RNA (gRNA) combination of any one of claims 134-136, wherein dSaCas9 is set forth in SEQ ID NO:

125.

138. 133. The Cas-guide RNA (gRNA) combination of claim 132, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

139. 139. The Cas-guide RNA (gRNA) combination of claim 138, wherein said dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, according to the numbering of the positions in SEQ ID NO:

126.

140. 123. The Cas-guide RNA (gRNA) combination of Claim 121 or 122, wherein the dSpCas9 comprises an amino acid sequence set forth in SEQ ID NO: 127, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

141. 141. The Cas-guide RNA (gRNA) combination of any one of claims 138-140, wherein the dSpCas9 is set forth in SEQ ID NO:

127.

142. A polynucleotide encoding the epigenetic modification DNA targeting system of any one of claims 1 to 95.

143. A polynucleotide encoding at least one DNA targeting module of the epigenetic modification DNA targeting system of any one of claims 1 to 95.

144. A polynucleotide encoding a fusion protein of the epigenetic modification DNA targeting system of any one of claims 1 to 95 and at least one gRNA.

145. A polynucleotide encoding the gRNA of any one of claims 98 to 125.

146. A polynucleotide encoding the gRNA combination of claim 126.

147. 142. A polynucleotide encoding the Cas-gRNA combination of any one of claims 128 to 141.

148. The epigenetic modification DNA targeting system of any one of claims 1 to 95. At least one DNA targeting module of the epigenetic modification DNA targeting system according to any one of claims 1 to 95; The epigenetic modification DNA targeting system according to any one of claims 1 to 95, comprising a fusion protein and said at least one gRNA; a gRNA combination according to claim 126, and / or The Cas-gRNA combination of any one of claims 128 to 141. Two or more polynucleotides that together encode a

149. a) a polynucleotide encoding a fusion protein of at least one DNA targeting module for suppressing transcription of the one or more genes of the epigenetic modification DNA targeting system according to any one of claims 1, 3 to 26, 33 to 37, 44 to 49, 54 to 69, 74 to 88, and 96, and one or more gRNAs selected from the gRNAs according to any one of claims 98 to 111; or b） A polynucleotide encoding a fusion protein of at least one DNA targeting module for increasing the transcription of the one or more genes of the epigenetic modification DNA targeting system according to any one of claims 2 to 24, 29, 31, 38 to 47, 51 to 67, 71 to 76, 89 to 95, and 97, and one or more gRNAs selected from the gRNAs according to any one of claims 112 to 125. A combination of a polynucleotide and a gRNA comprising:

150. The combination of polynucleotide and gRNA of claim 149, wherein the polynucleotide encoding the fusion protein is mRNA.

151. 148. A vector comprising the polynucleotide of any one of claims 142 to 147.

152. 149. A vector comprising two or more polynucleotides of claim 148.

153. A vector comprising a combination of the polynucleotide of claim 149 and a gRNA.

154. A vector comprising a combination of the polynucleotide of claim 150 and a gRNA.

155. 155. The vector of any one of claims 151 to 154, which is a viral vector.

156. 156. The vector of claim 155, which is an adeno-associated virus (AAV) vector.

157. The vector of claim 156, wherein the vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

158. 155. The vector of any one of claims 151 to 154, which is a non-viral vector.

159. The vector of claim 158, wherein the non-viral vector is selected from a lipid nanoparticle, a liposome, an exosome, or a cell-penetrating peptide.

160. The vector of claim 158 or 159, wherein the non-viral vector is a lipid nanoparticle.

161. 161. The vector of any one of claims 151-160, which exhibits immune cell tropism, and optionally, which exhibits T cell tropism.

162. 150. A modified T cell comprising a DNA targeting system of any one of claims 1-95, a combination of DNA targeting systems of claims 96 or 97, a gRNA of any one of claims 98-125, a combination of gRNAs of claims 126 or 127, a CRISPR Cas-gRNA combination of any one of claims 128-141, a polynucleotide of any one of claims 142-147, two or more polynucleotides of claim 148, or a combination of a polynucleotide and gRNA of claim 149 or 150.

163. 162. An engineered T cell comprising an epigenetic or phenotypic modification resulting from contact with a DNA targeting system of any one of claims 1-95, a combination of DNA targeting systems of claims 96 or 97, a gRNA of any one of claims 98-125, a combination of gRNAs of claims 126 or 127, a CRISPR Cas-gRNA combination of any one of claims 128-141, a polynucleotide of any one of claims 142-147, two or more polynucleotides of claim 148, a combination of a polynucleotide and gRNA of claim 149 or 150, or a vector of any one of claims 151-161.

164. The modified T cell of claim 162 or 163, which is derived from a cell from a subject.

165. The modified T cell of any one of claims 162-164, wherein the modified T cell is derived from a primary T cell.

166. 166. The modified T cell of any one of claims 162-165, wherein the T cell is derived from a T cell precursor, a pluripotent stem cell, or an induced pluripotent stem cell.

167. The modified T cell of any one of claims 162-166, further comprising an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR).

168. 161. A method of suppressing transcription of one or more genes in a T cell, the method comprising introducing into a T cell a DNA targeting system of any one of claims 1, 3-26, 33-37, 44-49, 54-69, and 74-88, a combination of DNA targeting systems of claim 96, a gRNA of any one of claims 98-99, a combination of gRNA of claim 126 or claim 127, a Cas-gRNA combination of any one of claims 128 and 130-145, a polynucleotide of any one of claims 142-147, two or more polynucleotides of claim 148, a combination of polynucleotide and gRNA of claim 149 or 150, or a vector of any one of claims 151-161.

169. The method of claim 168, wherein inhibiting transcription of said one or more genes promotes increased T cell effector function upon T cell stimulation compared to T cell effector function in the absence of T cell stimulation.

170. 161. A method of increasing transcription of one or more genes in a T cell, the method comprising introducing into a T cell a DNA targeting system of any one of claims 2-24, 29, 31, 38-47, 51-67, 71-76, and 89-95, a combination of DNA targeting systems of claim 97, a gRNA of any one of claims 112-125, a combination of gRNA of claim 126 or claim 127, a Cas-gRNA combination of any one of claims 129 and 130-141, a polynucleotide of any one of claims 142-147, two or more polynucleotides of claim 148, a combination of polynucleotide and gRNA of claim 149 or 150, or a vector of any one of claims 151-161.

171. 171. The method of claim 170, wherein increasing transcription of said one or more genes promotes increased T cell effector function upon T cell stimulation compared to T cell effector function in the absence of T cell stimulation.

172. 161. A method of increasing T cell effector function, comprising introducing into a T cell a DNA targeting system of any one of claims 1 to 95, a combination of DNA targeting systems of claims 96 or 97, a gRNA of any one of claims 98 to 125, a combination of gRNA of claims 126 or 127, a CRISPR Cas-gRNA combination of any one of claims 128 to 141, a polynucleotide of any one of claims 142 to 147, two or more polynucleotides of claim 148, a combination of polynucleotide and gRNA of claim 149 or 150, or a vector of any one of claims 151 to 161.

173. 169, 171, or 172, wherein the method of claim 169, 171, or 172 increases T cell effector function compared to a T cell not introduced with the DNA targeting system of any one of claims 1 to 95, the combination of DNA targeting systems of claims 96 or 97, the gRNA of any one of claims 98 to 125, the combination of gRNA of claim 126 or claim 127, the CRISPR Cas-gRNA combination of any one of claims 128 to 141, the polynucleotide of any one of claims 142 to 147, two or more polynucleotides of claim 148, the combination of the polynucleotide and gRNA of claim 149 or 150, or the vector of any one of claims 151 to 161.

174. The method of any one of claims 168-173, wherein the T cell is a T cell in a subject, and wherein the method is carried out in vivo.

175. The method of any one of claims 168-173, wherein the T cell is a T cell from the subject or is derived from a cell from the subject, and wherein the method is performed ex vivo.

176. The method of claim 175, wherein the T cells are primary T cells.

177. The method of claim 176, wherein the T cell is derived from a T cell precursor, a pluripotent stem cell, or an induced pluripotent stem cell.

178. The method of any one of claims 168-177, wherein the introducing step is by transient delivery to T cells.

179. The method of claim 178, wherein the transient delivery comprises electroporation, transfection or transduction.

180. 179. The method of any one of claims 168-179, wherein the DNA targeting system of any one of claims 1-95, the combination of DNA targeting systems of claims 96 or 97, the gRNA of any one of claims 98-125, the combination of gRNA of claim 126 or claim 127, the CRISPR Cas-gRNA combination of any one of claims 128-141, the polynucleotide of any one of claims 142-147, two or more polynucleotides of claim 148, the combination of polynucleotide and gRNA of claim 149 or 150, or the vector of any one of claims 151-161 is transiently expressed and / or transiently present in the T cell.

181. The method of any one of claims 168, 169, and 272-180, wherein the introducing step suppresses transcription of one or more genes in the T cell selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

182. The method of any one of claims 170-180, wherein the introducing step increases transcription of one or more genes in the T cell selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

183. 183. A modified T cell produced by the method of any one of claims 168-182.

184. 184. A method of treating a disease or condition in a subject, said method comprising administering to the subject the modified T cell of any of claims 162-167 and 183.

185. 161. A method of increasing T cell persistence in T cells of a subject, the method comprising administering to a subject or T cells thereof a DNA targeting system of any one of claims 1-95, a combination of DNA targeting systems of claims 96 or 97, a gRNA of any one of claims 98-125, a combination of gRNA of claim 126 or claim 127, a CRISPR Cas-gRNA combination of any one of claims 128-141, a polynucleotide of any one of claims 142-147, two or more polynucleotides of claim 148, a combination of polynucleotide and gRNA of claim 149 or 150, or a vector of any one of claims 151-161.

186. The method of claim 185, wherein the T cells are from adoptive T cell therapy for treating a disease or condition in a subject.

187. The method of claim 186, wherein the T cell therapy comprises T cells expressing a recombinant receptor specific for the target antigen.

188. The method of claim 186 or 187, wherein said administering is carried out prior to administration of adoptive T cell therapy, concurrently with administration of adoptive T cell therapy, or after administration of adoptive T cell therapy.

189. The method of any one of claims 186-188, wherein administering is carried out in the subject following administration of adoptive T cell therapy in the subject, at a time after the number or effector function of T cells in the adoptive T cell therapy has decreased, or is suspected to have decreased.

190. 1. A method of treating a disease or condition in a subject, comprising: a T cell therapy comprising cells expressing a recombinant receptor specific for a target antigen associated with the disease or condition; A DNA targeting system according to any one of claims 1 to 95, a combination of DNA targeting systems according to claims 96 or 97, a gRNA according to any one of claims 98 to 125, a combination of gRNAs according to claims 126 or 127, a CRISPR Cas-gRNA combination according to any one of claims 128 to 141, a polynucleotide according to any one of claims 142 to 147, two or more polynucleotides according to claim 148, a combination of a polynucleotide and gRNA according to claim 149 or 150, or a vector according to any one of claims 151 to 161. to a subject.

191. The method of any one of claims 187-190, wherein the recombinant receptor is an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR).

192. 192. The method of any one of claims 187 to 191, wherein the target antigen is a tumor antigen.

193. The method of any one of claims 186-192, wherein the disease or condition is cancer.

194. 194. The method of claim 193, wherein the cancer is a blood cancer or a solid tumor cancer.

195. The method of any one of claims 186-192, wherein the disease or condition is an autoimmune and / or inflammatory condition.

196. The method of any one of claims 185-195, wherein the administering results in transient delivery to a T cell of: the DNA targeting system, combination of DNA targeting systems, gRNA, combination of gRNAs, CRISPR Cas-gRNA combination, polynucleotide, two or more polynucleotides, combination of polynucleotides and gRNA, or vector.

197. The method of any one of claims 185-196, wherein the administering step suppresses transcription of one or more genes in T cells selected from the group consisting of CBLB, CCNC, CD5, CISH, DGKZ, ELOB, FAS, Fli1, GATA3, KDM1A, MED12, MYB, PRDM1, TGFBR2, and RASA2.

198. The method of any one of claims 185-197, wherein the administering step suppresses transcription of one or more genes in T cells selected from the group consisting of CBLB, CISH, MED12, MYB, PRDM1, and RASA2.

199. The method of any one of claims 185-196, wherein the administering increases transcription of one or more genes in the T cell selected from the group consisting of BATF, CD28, EOMES, IL-2, IL2RB, IRF4, LAT, LCP2, TBX21, and VAV1.

200. The method of any one of claims 185-196 or 199, wherein the administering increases transcription of one or more genes in the T cell selected from the group consisting of EOMES, IL-2, LCP2, and TBX21.

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