Compositions and methods for modulating il-2 gene expression

Abstract

Provided are epigenetic-modifying DNA-targeting systems, such as CRISPR-Cas / guide RNA (gRNA) systems, that bind to or target one or multiple target sites in an IL-2 gene or regulatory element thereof in a cell, such as a lymphoid cell (e.g., T cell). In some aspects, the provided epigenetic modifying DNA-targeting systems modulate a lymphoid cell function, such as a lymphoid cell phenotype or activity, for example a T cell phenotype or activity. In some aspects, also provided herein are methods and uses related to the provided compositions, for example in modulating lymphoid cells, such as T cells, including in connection with methods of adoptive T cell therapy.

Description

22474-20029.40 COMPOSITIONS AND METHODS FOR MODULATING IL-2 GENE EXPRESSION Cross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No.63 / 530,054 filed July 31, 2023, U.S. provisional application No.63 / 570,730 filed March 27, 2024, and U.S. provisional application No.63 / 662,406 filed June 20, 2024, the contents of which are incorporated by reference in their entireties. Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 22474_2002940_SeqList.xml, created July 16, 2024, which is 548,241 bytes in size. The information in the electronic format of the Sequence Listing is herein incorporated by reference in its entirety. Field

[0003] The present disclosure relates in some aspects to epigenetic-modifying DNA- targeting systems, such as CRISPR-Cas / guide RNA (gRNA) systems, that bind to or target the interleukin-2 (IL-2) gene, or a regulatory element thereof in a lymphoid cell (e.g., T cell). In some aspects, the provided epigenetic modifying DNA-targeting systems of the present disclosure modulate a lymphoid cell function, such as a lymphoid cell phenotype or activity, for example T cell phenotype or activity. In some aspects, the provided epigenetic modifying- targeting systems of the present disclosure enables transcription control or modulation of interleukin (IL-2) expression. In some aspects, the present disclosure is directed to methods and uses related to the provided compositions, for example in modulating lymphoid cells, such as T cells, including in connection with methods of lymphoid cell therapies, e.g., adoptive T cell therapy. Background

[0004] The administration of lymphoid cells (e.g., T cells) targeting a specific antigen, 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, 6037971-sf22474-20029.40 expansion, and persistence. Therefore, there is a need for new and improved methods to overcome these challenges. The present disclosure addresses these and other needs. Summary

[0005] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising a plurality of DNA-targeting modules for increasing transcription of the interleukin (IL-2) gene, wherein each of the DNA- targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain for targeting to a target site of the IL-2 gene; and (b) at least one transcriptional activator effector domain. In some of any of the provided embodiments, the DNA-binding domain of each fusion protein comprises: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I- SceI enzyme or a variant thereof, optionally wherein the DNA- binding domain comprises a catalytically inactive variant of any of the foregoing, wherein when the DNA-binding domain of each fusion protein comprises a Cas protein, the DNA-targeting system further comprises at least two gRNAs, each capable of targeting the Cas protein to a target site.

[0006] In some of any of the provided embodiments, the plurality of DNA-targeting modules is 2-6 DNA targeting modules. In some of any of the provided embodiments, the plurality of DNA-targeting modules is 2 DNA-targeting modules. In some of any of the provided embodiments, the plurality of DNA-targeting modules is 3 DNA-targeting modules. In some of any of the provided embodiments, the plurality of DNA-targeting modules is 4 DNA- targeting modules or 5 DNA-targeting modules.

[0007] In some of any of the provided embodiments, each target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4:122,451,261-122,593,946. In some of any of the provided embodiments, each target site is in a putative regulatory region of the IL-2 gene, wherein the putative regulatory region is characterized by having one or more of an epigenetic mark, a regulatory feature or a transcription factor motif.

[0008] In some of any of the provided embodiments, the epigenetic mark includes Histone H3K27 acetylation. In some of any of the provided embodiments, the at least one transcriptional activator effector domain catalyzes acetylation of histone H3 lysine 27 at the target site or is able to recruit an enzyme that catalyzes acetylation of histone H3 lysine 27 at the target site. In some of any of the provided embodiments, the enzyme that catalyzes the acetylation is an 6037971-sf22474-20029.40 acetyltransferase. In some of any of the provided embodiments, the enzyme that catalyzes the acetylation is a histone acetyltransferase.

[0009] In some of any of the provided embodiments, the putative regulatory region is a promoter or an enhancer. In some of any of the provided embodiments, each target site is in a promoter or an enhancer.

[0010] In some of any of the provided embodiments, each target site is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840- 122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some of any of the provided embodiments, each target site is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000- 122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0011] In some of any of the provided embodiments, at least two of the plurality of DNA- targeting modules target a different target site. In some of any of the provided embodiments, the at least two different target sites are in two different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410- 122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some of any of the provided embodiments, the at least two different targets site are in two different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0012] In some of any of the provided embodiments, at least three of the plurality of DNA- targeting modules target a different target site. In some of any of the provided embodiments, the at least three different target sites are in three different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410- 122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 6037971-sf22474-20029.40 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some of any of the provided embodiments, the at least three different targets site are in three different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0013] In some of any of the provided embodiments, at least four of the plurality of DNA- targeting modules target a different target site or at least five of the plurality of DNA-targeting modules target a different target site. In some of any of the provided embodiments, the at least four different target sites or the at least five different target sites are in four different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840- 122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some of any of the provided embodiments, the at least four different targets site or the at least five different target sites are in four different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0014] In some of any of the provided embodiments, each of the plurality of DNA-targeting modules target a different target site. In some of any of the provided embodiments, each target site is in a different target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000- 122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some of any of the provided embodiments, each target site is in a different target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300- 122,544,050. In some of any of the provided embodiments, at least two of the DNA-targeting modules of the plurality of DNA-targeting modules target the same target region.

[0015] In some of any of the provided embodiments, the DNA-binding domain is a zinc finger protein. In some of any of the provided embodiments, each fusion protein of the plurality 6037971-sf22474-20029.40 of DNA-targeting modules is different. In some of any of the provided embodiments, each of the DNA-targeting modules share the same fusion protein and each comprise a different guide nucleic acid that is complementary to a different target site. In some of any of the provided embodiments, the guide nucleic acid is a guide RNA (gRNA).

[0016] In some of any of the provided embodiments, the DNA-binding domain of the fusion protein is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof. In some of any of the provided embodiments, at least one DNA-targeting module targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,451,000-122,460,000. In some of any of the provided embodiments, at least one DNA-targeting module targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,488,840-122,491,890. In some of any of the provided embodiments, at least one DNA-targeting module targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,507,000-122,508,985. In some of any of the provided embodiments, at least one DNA- targeting module targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,539,300-122,544,050.

[0017] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional activator effector domain; and (b) a plurality of guide RNAs (gRNAs) comprising at least two gRNAs that each target a target site of the interleukin-2 (IL-2) gene. In some of any of the provided embodiments, the DNA-targeting system increases transcription of the interleukin (IL-2) gene.

[0018] In some of any of the provided embodiments, the plurality of gRNAs is 2-6 gRNAs. In some of any of the provided embodiments, the plurality of gRNAs is 2 gRNAs. In some of any of the provided embodiments, the plurality of gRNAs is 3 gRNAs. In some of any of the provided embodiments, the plurality of gRNAs is 4 gRNAs or is 5 gRNAs.

[0019] In some of any of the provided embodiments, each target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4:122,451,261-122,593,946. In some of any of the provided embodiments, each target site is in a putative regulatory region of the IL-2 gene, wherein the putative regulatory region is characterized by having one or more of an epigenetic mark, a regulatory feature or a transcription factor motif. In some of any of the 6037971-sf22474-20029.40 provided embodiments, the epigenetic mark includes Histone H3K27 acetylation. In some of any of the provided embodiments, the at least one transcriptional activator effector domain catalyzes acetylation of histone H3 lysine 27 at the target site or is able to recruit an enzyme that catalyzes acetylation of histone H3 lysine 27 at the target site. In some of any of the provided embodiments, the enzyme that catalyzes the acetylation is an acetyltransferase. In some of any of the provided embodiments, the enzyme that catalyzes the acetylation is a histone acetyltransferase.

[0020] In some of any of the provided embodiments, the putative regulatory region is a promoter or an enhancer. In some of any of the provided embodiments, each target site is in a promoter or an enhancer.

[0021] In some of any of the provided embodiments, each target site is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840- 122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, and (7) chr4: 122,576,890-122,579,315. In some of any of the provided embodiments, each target site is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000- 122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0022] In some of any of the provided embodiments, the DNA-targeting system targets at least two different target sites, optionally, 2, 3, 4 or 5 different target sites, wherein each different target site is located within a different target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000- 122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0023] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional activator effector domain; and (b) at least one guide RNA (gRNA) that targets a target site of the interleukin-2 (IL-2) gene located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group 6037971-sf22474-20029.40 consisting of: (1) chr4: 122,465,000-122,472,000, (2) chr4: 122,479,410-122,482,750, (3) chr4: 122,488,840-122,491,890, (4) 122,507,000-122,508,985, (4) chr4: 122,539,300-122,544,050, and (6) chr4: 122,576,890-122,579,315. In some of any of the provided embodiments, the at least one gRNA targets a target site of the interleukin-2 (IL-2) gene located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,488,840-122,491,890, (2) 122,507,000-122,508,985, and (3) chr4: 122,539,300-122,544,050.

[0024] In some of any of the provided embodiments, the at least one gRNA is 1-6 gRNAs.

[0025] In some of any of the provided embodiments, the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any of the provided embodiments, the dCas protein is a dCas12 protein.

[0026] In some of any of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO:62. In some of any of the provided embodiments, the dSpCas9 comprises the sequence set forth in SEQ ID NO:63, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 is set forth in SEQ ID NO:63.

[0027] In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:64. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 is set forth in SEQ ID NO:65.

[0028] In some of any of the provided embodiments, each gRNA comprises a gRNA spacer sequence that is complementary to the target site of the respective gene. In some of any of the provided embodiments, each gRNA targets a target site in IL-2 comprising the sequence set 6037971-sf22474-20029.40 forth in any one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, a portion of any of the foregoing comprising at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, each gRNA targets a target site in IL-2 set forth in any one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or a complementary sequence of any of the foregoing.

[0029] In some of any of the provided embodiments, each gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt.

[0030] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a zinc finger protein (ZFP) or variant thereof, and at least one transcriptional activator effector domain; wherein the ZFP targets a target site of the interleukin-2 (IL-2) gene located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,465,000-122,472,000, (2) chr4: 122,479,410-122,482,750, (3) chr4: 122,488,840-122,491,890, (4) 122,507,000-122,508,985, (4) chr4: 122,539,300- 122,544,050, and (6) chr4: 122,576,890-122,579,315. In some of any of the provided embodiments, the ZFP targets a target site of the interleukin-2 (IL-2) gene located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,488,840-122,491,890, (2) 122,507,000- 122,508,985, and (3) chr4: 122,539,300-122,544,050. In some of any of the provided embodiments, the ZFP targets a target site in IL-2 comprising the sequence set forth in any one of SEQ ID NOs: 186-188.

[0031] In some of any of the provided embodiments, the ZFP targets a target site set forth in SEQ ID NO: 186. In some of any of the provided embodiments, the ZFP comprises a zinc finger recognition region comprising six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, selected from F1-F6 as follows: F1: QNAHRKT (SEQ ID NO: 195), F2: 6037971-sf22474-20029.40 RKYYLAK (SEQ ID NO: 196), F3: RSAHLSR (SEQ ID NO: 197), F4: QSGDLTR (SEQ ID NO: 198), F5: RSDHLTQ (SEQ ID NO: 199), and F6: DSANLSR (SEQ ID NO: 200). In some of any of the provided embodiments, the ZFP comprises the sequence set forth in SEQ ID NO: 189, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the ZFP comprises the sequence set forth in SEQ ID NO: 189. In some of any of the provided embodiments, the ZFP is encoded by the sequence set forth in SEQ ID NO: 192 or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the ZFP is encoded by the sequence set forth in SEQ ID NO: 192.

[0032] In some of any of the provided embodiments, the ZFP targets a target site set forth in SEQ ID NO: 187. In some of any of the provided embodiments, the ZFP comprises a zinc finger recognition region comprising six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, selected from F1-F6 as follows: F1: DSSHLEL (SEQ ID NO: 201), F2: DRSNLTR (SEQ ID NO: 202), F3: RSDNLSE (SEQ ID NO: 203), F4: VRRALSS (SEQ ID NO: 204), F5: QSGALAR (SEQ ID NO: 205), and F6: RLDWLPM (SEQ ID NO: 206). In some of any of the provided embodiments, the ZFP comprises the sequence set forth in SEQ ID NO: 190, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the ZFP comprises the sequence set forth in SEQ ID NO: 190. In some of any of the provided embodiments, the ZFP is encoded by the sequence set forth in SEQ ID NO: 193 or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the ZFP is encoded by the sequence set forth in SEQ ID NO: 193.

[0033] In some of any of the provided embodiments, the ZFP targets a target site set forth in SEQ ID NO: 188. In some of any of the provided embodiments, the ZFP comprises a zinc finger recognition region comprising six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, selected from F1-F6 as follows: F1: RSDNLSV (SEQ ID NO: 207), F2: RSAHLSR (SEQ ID NO: 208), F3: QNAHRKT (SEQ ID NO: 209), F4: LRHHLTR (SEQ ID NO: 210), F5: TSSNRKT (SEQ ID NO: 211), and F6: TSSNLSR (SEQ ID NO: 212). In some of any of the provided embodiments, the ZFP comprises the sequence set forth in SEQ ID NO: 191, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 6037971-sf22474-20029.40 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the ZFP comprises the sequence set forth in SEQ ID NO: 191. In some of any of the provided embodiments, the ZFP is encoded by the sequence set forth in SEQ ID NO: 194 or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the ZFP is encoded by the sequence set forth in SEQ ID NO: 194.

[0034] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a deactivated Cas9 from Streptococcus pyogenes (dSpCas9) and at least one transcriptional activator effector domain; and (b) at least one guide RNA (gRNA) that targets a target site of the interleukin-2 (IL-2) gene, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt.

[0035] In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO:62. In some of any of the provided embodiments, the dSpCas9 comprises the sequence set forth in SEQ ID NO:63, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 is set forth in SEQ ID NO:63.

[0036] In some of any of the provided embodiments, each gRNA targets a target site in IL-2 comprising the sequence set forth in any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59, a portion of any of the foregoing comprising at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, each gRNA targets a target site in IL-2 set forth in any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59, or a complementary sequence of any of the foregoing.

[0037] In some of any of the provided embodiments, each gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, 6037971-sf22474-20029.40 SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt.

[0038] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a deactivated Cas9 from Staphylococcus aureus (dSaCas9) and at least one transcriptional activator effector domain; and (b) at least one guide RNA (gRNA) that targets a target site of the interleukin-2 (IL-2) gene, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt.

[0039] In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:64. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 is set forth in SEQ ID NO:65.

[0040] In some of any of the provided embodiments, each gRNA independently comprises a spacer sequence between 14 nt and 24 nt. In some of any of the provided embodiments, each gRNA independent comprises a spacer sequence between 16 nt and 22 nt in length. In some of any of the provided embodiments, each gRNA independently comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0041] In some of any of the provided embodiments, each gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt of any of the foregoing.

[0042] In some of any of the provided embodiments, each gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt of any of the foregoing.

[0043] In some of any of the provided embodiments, the DNA-targeting system comprises at least two gRNAs that target the same target site. In some of any of the provided embodiments, 6037971-sf22474-20029.40 the DNA-targeting system comprises at least two copies of the same gRNA. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO: 23.

[0044] In some of any of the provided embodiments, the DNA-targeting system comprises at least two gRNAs that target different target sites. In some of any of the provided embodiments, each gRNA of the DNA-targeting system targets a different target site.

[0045] In some of any of the provided embodiments, at least one gRNA targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,451,000-122,460,000. In some of any of the provided embodiments, the at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:11, or a contiguous portion thereof of at least 14nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:11.

[0046] In some of any of the provided embodiments, the least one gRNA comprises a gRNA spacer comprising the sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42, or a contiguous portion thereof of at least 14nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:42.

[0047] In some of any of the provided embodiments, at least one gRNA targets a target site 50 to 150 kilobases (kb) upstream of the IL-2 transcriptional start site (TSS). In some of any of the provided embodiments, at least one gRNA targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,488,840-122,491,890. In some of any of the provided embodiments, the at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:23, or a contiguous portion thereof of at least 14nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:23. In some of any of the provided embodiments, at least 6037971-sf22474-20029.40 one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:25 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:25.

[0048] In some of any of the provided embodiments, the least one gRNA comprises a gRNA spacer comprising the sequence set forth in SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:50, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:50.

[0049] In some of any of the provided embodiments, at least one gRNA targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,507,000-122,508,985. In some of any of the provided embodiments, the at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:27, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:27.

[0050] In some of any of the provided embodiments, the least one gRNA comprises a gRNA spacer comprising the sequence set forth in SEQ ID NO:56, SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:56, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:56. In some of any of the provided embodiments, at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:58.

[0051] In some of any of the provided embodiments, at least one gRNA targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,539,300-122,544,050. In some of any of the provided embodiments, the at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, at least one gRNA comprises a gRNA spacer 6037971-sf22474-20029.40 sequence comprising the sequence set forth in SEQ ID NO:37, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:37.

[0052] In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:23. In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:25. In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:27. In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:37. In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:23 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:37. In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:23 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:25. In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:23 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:27. In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA, a second gRNA, and a third gRNA set forth by three gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence set forth in SEQ ID NO:11, a gRNA comprising the spacer sequence set forth in SEQ ID NO:23, a gRNA comprising the spacer sequence set forth in SEQ ID NO:27, and a gRNA comprising the spacer sequence set forth in SEQ ID NO:37.

[0053] In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA, a second gRNA, a third gRNA and a fourth gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11, the second gRNA comprises the 6037971-sf22474-20029.40 spacer sequence set forth in SEQ ID NO:23, the third gRNA comprises the spacer sequence set forth in SEQ ID NO:27, and the fourth gRNA comprises the spacer sequence set forth in SEQ ID NO:37, optionally further comprising a fifth gRNA, wherein the fifth gRNA comprises the spacer sequence set forth in SEQ ID NO:25.

[0054] In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA and a second gRNA set forth by two gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence set forth in SEQ ID NO:42, a gRNA comprising the spacer sequence set forth in SEQ ID NO:50, a gRNA comprising the spacer sequence set forth in SEQ ID NO:56, and a gRNA comprising the spacer sequence set forth in SEQ ID NO:58, optionally wherein the two gRNAs are: (i) a gRNA comprising the spacer set forth in SEQ ID NO:42 and a gRNA comprising the spacer set forth in SEQ ID NO:50, (ii) a gRNA comprising the spacer set forth in SEQ ID NO:42 and a gRNA comprising the spacer set forth in SEQ ID NO:56; (iii) a gRNA comprising the spacer set forth in SEQ ID NO:42 and a gRNA comprising the spacer set forth in SEQ ID NO:58; (iv) a gRNA comprising the spacer set forth in SEQ ID NO:50 and a gRNA comprising the spacer set forth in SEQ ID NO:56; or (v) a gRNA comprising the spacer set forth in SEQ ID NO:50 and a gRNA comprising the spacer set forth in SEQ ID NO:58. In some of any of the provided embodiments, the DNA-targeting system comprises a first gRNA, a second gRNA and a third gRNA set forth by three gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence set forth in SEQ ID NO:42, a gRNA comprising the spacer sequence set forth in SEQ ID NO:50, a gRNA comprising the spacer sequence set forth in SEQ ID NO:56, and a gRNA comprising the spacer sequence set forth in SEQ ID NO:58, optionally wherein the three gRNAs are: (i) a gRNA comprising the spacer set forth in SEQ ID NO:42, a gRNA comprising the spacer set forth in SEQ ID NO:50 and a gRNA comprising the spacer set forth in SEQ ID NO:56; or (ii) a gRNA comprising the spacer set forth in SEQ ID NO:42, a gRNA comprising the spacer set forth in SEQ ID NO:50 and a gRNA comprising the spacer set forth in SEQ ID NO:58.

[0055] In some of any of the provided embodiments, the DNA-targeting system further comprises a fusion protein comprising a DNA-binding domain that is a zinc finger protein (ZFP), or variant thereof, and at least one transcriptional activator effector domain, wherein the ZFP targets a target site in IL-2 comprising the sequence set forth in any one of SEQ ID NOs: 186-288. 6037971-sf22474-20029.40

[0056] In some of any of the provided embodiments, each transcriptional activator effector domain is a NCOA3 domain, a FOXO3 domain, a NCOA3-FOXO3-NCOA3 domain, 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 wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing. In some of any of the provided embodiments, each transcriptional activator effector domain is p300.

[0057] In some of any of the provided embodiments, each transcriptional activator effector domain comprises at least one VP16 domain or a variant or portion thereof that exhibits transcriptional activation activity. In some of any of the provided embodiments, each transcriptional activator effector domain comprises a VP16 tetramer (VP64) domain or a variant or portion thereof that exhibits transcriptional activation activity. In some of any of the provided embodiments, each transcriptional activator effector domain is a VP64 domain.

[0058] In some of any of the provided embodiments, each transcriptional activator effector domain comprises a NCOA3 domain or a variant or portion thereof that exhibits transcriptional activation activity. In some of any of the provided embodiments, each transcriptional activator effector domain comprises a FOXO3 domain or a variant or portion thereof that exhibits transcriptional activation activity. In some of any of the provided embodiments, each transcriptional activator effector domain comprises a NCOA3-FOXO3-NCOA3 domain. In some of any of the provided embodiments, each transcriptional activator effector domain is a NCOA3-FOXO3-NCOA3 domain. In some of any of the provided embodiments, each transcriptional activator effector domain further comprises a VP16 tetramer (VP64) domain.

[0059] In some of any of the provided embodiments, the at least one transcriptional activator effector domain comprises the sequence set forth in SEQ ID NO: 66, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some of any of the provided embodiments, the at least one transcriptional activator effector domain comprises the sequence set forth in SEQ ID NO: 66.

[0060] In some of any of the provided embodiments, the at least one transcriptional activator effector domain comprises the sequence set forth in SEQ ID NO: 181, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some of any of the provided embodiments, the 6037971-sf22474-20029.40 at least one transcriptional activator effector domain comprises the sequence set forth in SEQ ID NO: 181.

[0061] In some of any of the provided embodiments, the at least one transcriptional activator effector domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C- terminus, of the DNA-binding domain.

[0062] In some of any of the provided embodiments, the fusion protein further comprises one or more nuclear localization signals (NLS). In some of any of the provided embodiments, the fusion protein further comprises one or more linkers connecting two or more of: the DNA- binding domain, the at least one effector domain, and the one or more nuclear localization signals. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:100 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:100.

[0063] In some of any of the provided embodiments, the fusion protein comprises any one of the sequences set forth in SEQ ID NOs: 5, 61, 182, and 213-215 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the fusion protein comprises any one of the sequences set forth in SEQ ID NOs: 5, 61, 182, and 213-215. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 5. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 61. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 182. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 213. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 214. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 215.

[0064] In some of any of the provided embodiments, transient delivery of the epigenetic- modifying DNA-targeting system to a lymphoid cell promotes increased IL-2 expression, optionally increased compared to a lymphoid cell that has not been delivered the epigenetic- modifying DNA-targeting system. In some of any of the provided embodiments, the lymphoid cell is a T cell. In some of any of the provided embodiments, the lymphoid cell is a natural killer (NK) cell. In some of any of the provided embodiments, the lymphoid cell is derived from a 6037971-sf22474-20029.40 primary cell. In some of any of the provided embodiments, the lymphoid cell is derived from a T or NK cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell. In some of any of the provided embodiments, the modified lymphoid cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor.

[0065] In some of any of the provided embodiments, transient delivery of the epigenetic- modifying DNA-targeting system to a T cell promotes increased IL-2 expression upon T cell stimulation, optionally increased compared to a T cell that has not been delivered the epigenetic- modifying DNA-targeting system. In some of any of the provided embodiments, the DNA- targeting system increases expression of IL-2 by a log2 fold-change of at or greater than 1.0 in the lymphoid cell contacted with the DNA-targeting system. In some of any of the provided embodiments, the DNA-targeting system increases expression of IL-2 by a log2 fold-change of at or greater than 2.0 in the lymphoid cell contacted with the DNA-targeting system. In some of any of the provided embodiments, the DNA-targeting system increases expression of IL-2 by a log2 fold-change of at or greater than 2.5 in the lymphoid cell contacted with the DNA-targeting system. In some of any of the provided embodiments, the DNA-targeting system increases expression of IL-2 by a log2 fold-change of at or greater than 2.75 in the lymphoid cell contacted with the DNA-targeting system.

[0066] In some of any of the provided embodiments, the T cell stimulation is with an anti- CD3 and anti-CD28 activation reagent. In some of any of the provided embodiments, the T cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor or a T cell receptor (eTCR). In some of any of the provided embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR) directed against an antigen and the T cell stimulation is an antigen-specific stimulation of the CAR or eTCR, optionally wherein the T cell stimulation is with antigen-expressing target cells. In some of any of the provided embodiments, the T cell expresses a chimeric antigen receptor (CAR) directed against an antigen and the T cell stimulation is an antigen-specific stimulation of the CAR, optionally wherein the T cell stimulation is with antigen-expressing target cells. In some of any of the provided embodiments, the T cell stimulation is a restimulation after at least one prior T cell stimulation of the T cells.

[0067] In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:8. In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:41. 6037971-sf22474-20029.40

[0068] In some of any of the provided embodiments, wherein the DNA-targeting system does not introduce a genetic disruption or a DNA break.

[0069] In some aspects, provided herein is a guide RNA (gRNA) that targets a target site of the interleukin (IL-2) gene, wherein the target site is selected from a target site comprising the sequence set forth in any one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, a portion of any of the foregoing comprising at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0070] In some of any of the provided embodiments, the target site is set forth in any one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt.

[0071] In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:11 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:11. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:23 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:23. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:25 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:25. In some of any of 6037971-sf22474-20029.40 the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:27 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:27. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:37 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:37.

[0072] In some of any of the provided embodiments, the gRNA comprises a spacer sequence between 14 nt and 24 nt. In some of any of the provided embodiments, the gRNA comprises a spacer sequence between 16 nt and 22 nt in length. In some of any of the provided embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0073] In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:8.

[0074] In some aspects, provided herein is a guide RNA (gRNA) that targets a target site of the interleukin (IL-2) gene, wherein the target site is selected from a target site comprising the sequence set forth in any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59, a portion of any of the foregoing comprising at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site is set forth in any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59, or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt.

[0075] In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:42. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence 6037971-sf22474-20029.40 comprising the sequence set forth in SEQ ID NO:50 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:50.

[0076] In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:56 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:56. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:58 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:58.

[0077] In some of any of the provided embodiments, the gRNA comprises a spacer sequence between 14 nt and 24 nt. In some of any of the provided embodiments, the gRNA comprises a spacer sequence between 16 nt and 22 nt in length. In some of any of the provided embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0078] In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:41.

[0079] In some aspects, provided herein is a combination of gRNAs comprising two or more gRNAs, each selected from a gRNA provided herein. In some aspects, provided herein is a combination of gRNAs comprising two or more gRNAs, each selected from a gRNA provided herein.

[0080] In some aspects, provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof from Streptococcus pyogenes; and (b) at least one gRNA provided herein. In some of any of the provided embodiments, the Cas protein or variant thereof is a deactivated (dSpCas9) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO:62. In some of any of the provided embodiments, the dSpCas9 comprises the sequence set forth in SEQ ID NO:63, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 is set forth in SEQ ID NO:63.

[0081] In some aspects, provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof from Staphylococcus aureus; and (b) at least one gRNA provided 6037971-sf22474-20029.40 herein. In some of any of the provided embodiments, the Cas protein or variant thereof is a deactivated (dSaCas9) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dSaCas9 protein comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:64. In some of any of the provided embodiments, the dSaCas9 comprises the sequence set forth in SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 is set forth in SEQ ID NO:65.

[0082] In some aspects, provided herein is a polynucleotide encoding an epigenetic- modifying DNA-targeting system provided herein. In some aspects, provided herein is a polynucleotide encoding at least one DNA-targeting module of an epigenetic-modifying DNA- targeting system provided herein. In some aspects, provided herein is a polynucleotide encoding the fusion protein and the at least one gRNA of an epigenetic-modifying DNA-targeting system provided herein.

[0083] In some aspects, provided herein is a polynucleotide encoding a gRNA provided herein. In some aspects, provided herein is a polynucleotide encoding a combination of gRNAs provided herein. In some aspects, provided herein is a polynucleotide encoding a Cas-gRNA combination provided herein. In some aspects, provided herein is a polynucleotide encoding the fusion protein of an epigenetic-modifying DNA-targeting system provided herein, and one or more gRNAs provided herein. In some of any of the provided embodiments, the polynucleotide encoding the fusion protein is mRNA.

[0084] In some aspects, provided herein is a vector comprising a polynucleotide provided herein. In some of any of the provided embodiments, the vector is a viral vector. In some of any of the provided embodiments, the vector is an adeno-associated virus (AAV) vector. In some of any of the provided embodiments, the vector is selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

[0085] In some of any of the provided embodiments, the vector is a non-viral vector. In some of any of the provided embodiments, the non-viral vector is selected from: a lipid nanoparticle, a liposome, an exosome, or a cell penetrating peptide. In some of any of the provided embodiments, the non-viral vector is a lipid nanoparticle. In some of any of the provided embodiments, the vector exhibits immune cell tropism, optionally wherein the vector exhibits T-cell tropism. 6037971-sf22474-20029.40

[0086] In some aspects, provided herein is a modified lymphoid cell comprising an epigenetic-modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, or a polynucleotide provided herein.

[0087] In some aspects, provided herein is a modified lymphoid cell comprising an epigenetic or phenotypic modification resulting from being contacted by an epigenetic- modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, or a polynucleotide provided herein.

[0088] In some of any of the provided embodiments, the modified lymphoid cell is a modified T cell. In some of any of the provided embodiments, the modified lymphoid cell is a modified natural killer (NK) cell. In some of any of the provided embodiments, the modified lymphoid cell is derived from a primary cell. In some of any of the provided embodiments, the modified lymphoid cell is derived from a T or NK cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell. In some of any of the provided embodiments, the modified lymphoid cell further comprises a chimeric antigen receptor (CAR).

[0089] In some aspects, provided herein is a modified T cell comprising an epigenetic- modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, or a polynucleotide provided herein. In some aspects, provided herein is a modified T cell comprising an epigenetic or phenotypic modification resulting from being contacted by an epigenetic-modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, or a polynucleotide provided herein. In some of any of the provided embodiments, the modified T cell is derived from a cell from a subject. In some of any of the provided embodiments, the modified T cell is derived from a primary T cell. In some of any of the provided embodiments, the modified T cell is derived from a T cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell. In some of any of the provided embodiments, the T cell is a tumor infiltrating lymphocyte (TIL). In some of any of the provided embodiments, the modified T cell further comprises an engineered T cell receptor (eTCR) or chimeric antigen receptor (CAR).

[0090] In some aspects, provided herein is a method of increasing the transcription of IL-2 in a lymphoid cell, the method comprising introducing into the lymphoid cell an epigenetic- 6037971-sf22474-20029.40 modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, a polynucleotide provided herein, or a vector provided herein.

[0091] In some aspects, provided herein is a method of increasing the production of IL-2 in or by a lymphoid cell, the method comprising introducing into the lymphoid cell an epigenetic- modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, a polynucleotide provided herein, or a vector provided herein. In some of any of the provided embodiments, the lymphoid cell is a T cell. In some of any of the provided embodiments, the lymphoid cell is a natural killer (NK) cell. In some of any of the provided embodiments, the lymphoid cell is derived from a primary cell. In some of any of the provided embodiments, the lymphoid cell is derived from a T or NK cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell. In some of any of the provided embodiments, the lymphoid cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor (CAR).

[0092] In some aspects, provided herein is a method of increasing the transcription of IL-2 in a T cell, the method comprising introducing into a T cell an epigenetic-modifying DNA- targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, a polynucleotide provided herein, or a vector provided herein. In some aspects, provided herein is a method of increasing the production of IL-2 in or by a T cell, the method comprising introducing into a T cell an epigenetic-modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, a polynucleotide provided herein, or a vector provided herein. In some of any of the provided embodiments, the T cell is a tumor infiltrating lymphocyte (TIL). In some of any of the provided embodiments, the T cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor or a T cell receptor (eTCR).

[0093] In some aspects, provided herein is a method of promoting persistence of an immune cell upon repeat stimulations, the method comprising the method comprising introducing into a T cell an epigenetic-modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, a polynucleotide provided herein, or a vector provided herein, wherein after the introducing the T cell is subjected to a plurality of repeat stimulations that initiate a T cell 6037971-sf22474-20029.40 activating signal. In some of any of the provided embodiments, the stimulation is with an anti- CD3 and anti-CD28 activation reagent. In some of any of the provided embodiments, the T cell is a tumor infiltrating lymphocyte (TIL). In some of any of the provided embodiments, the T cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor or a T cell receptor (eTCR). In some of any of the provided embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR) directed against an antigen and the T cell stimulation is an antigen-specific stimulation of the CAR or eTCR, optionally wherein the T cell stimulation is with antigen-expressing target cells. In some of any of the provided embodiments, the T cell expresses a chimeric antigen receptor (CAR) directed against an antigen and the T cell stimulation is an antigen-specific stimulation of the CAR, optionally wherein the T cell stimulation is with antigen-expressing target cells. In some of any of the provided embodiments, the T cell stimulation is a restimulation after at least one prior T cell stimulation of the T cells. In some of any of the provided embodiments, the T cell is a T cell in a subject and the method is carried out in vivo.

[0094] In some of any of the provided embodiments, the T cell is a T cell from a subject, or derived from a cell from the subject, and the method is carried out ex vivo. In some of any of the provided embodiments, the method is carried out in vitro.

[0095] In some of any of the provided embodiments, T cell is a primary T cell. In some of any of the provided embodiments, the T cell is derived from a T cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell.

[0096] In some of any of the provided embodiments, the introducing is by transient delivery into the T cell. In some of any of the provided embodiments, the introducing is by electroporation, transfection, or transduction.

[0097] In some aspects, provided herein is modified lymphoid cell produced by a method provided herein.

[0098] In some aspects, provided herein is a pharmaceutical composition comprising a plurality of modified lymphoid cells provided herein. In some aspects, provided herein is a pharmaceutical composition comprising a plurality of modified T cells provided herein. In some of any of the provided embodiments, a pharmaceutical composition provided herein comprises a pharmaceutically acceptable excipient.

[0099] In some aspects, provided herein is a method of treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising a 6037971-sf22474-20029.40 modified lymphoid cell provided herein or a pharmaceutical composition provided herein. In some aspects, provided herein is a method of treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising a modified T cell provided herein or a pharmaceutical composition provided herein.

[0100] In some of any of the provided embodiments, the T cells or modified T cells are an adoptive T cell therapy for treating a disease or condition in the subject. In some of any of the provided embodiments, the T cells or modified T cells are tumor infiltrating lymphocytes (TILs). In some of any of the provided embodiments, the modified T cells express a recombinant receptor specific for a target antigen associated with the disease or condition.

[0101] In some aspects, provided herein is a method of treating a disease or condition in a subject, the method comprising administering to a subject: an adoptive T cell therapy for treating a disease or condition in the subject; and an epigenetic-modifying DNA-targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, a polynucleotide provided herein, or a vector provided herein.

[0102] 2 In some of any of the provided embodiments, the T cells are tumor infiltrating lymphocytes (TILs). In some of any of the provided embodiments, the T cells express a recombinant receptor specific for a target antigen associated with the disease or condition. In some of any of the provided embodiments, the recombinant receptor is an engineered T cell receptor (eTCR) or chimeric antigen receptor (CAR).

[0103] In some of any of the provided embodiments, the target antigen is a tumor antigen.

[0104] In some of any of the provided embodiments, the disease or condition is a cancer. In some of any of the provided embodiments, the cancer is a hematological cancer or is a solid tumor.

[0105] In some of any of the provided embodiments, the disease or condition is an autoimmune condition and / or an inflammatory condition.

[0106] In some of any of the provided embodiments, the administering increases transcription of IL-2 in lymphoid cells.

[0107] In some of any of the provided embodiments, the administering increases transcription of IL-2 in T cells. Brief Description of the Drawings 6037971-sf22474-20029.40

[0108] FIG.1A shows IL-2 locus within human genome assembly GRCh38 (hg38) genomic coordinates chr4:122,451,261-122,593,946, with 7 annotated distinct regions. FIG.1B shows a log 2 fold change (log2fc) to log2 fold change plot between two different donors. Large black dots, which have been boxed, represents verified hits. FIG.1C shows the distribution of target site hits across various regions within the IL-2 locus.

[0109] FIG.2A shows the percent IL-2 expression following a first stimulation of Her2 CAR T cells that were delivered mRNA encoding a dSpCas9-2xVP64 effector fusion protein and various SpCas9 IL-2-targeting gRNA. FIG.2B shows percent IL-2 expression following a second stimulation of the same Her2 CAR T cells shown in FIG.2A. FIG.2C shows percent IL-2 expression following a third stimulation of the same Her2 CAR T cells shown in FIG.2A. The solid line demarcates the percent IL-2 expression when using the control guide RNA IL-2 gRNA-1. The dashed line demarcates the percent IL-2 expression of Her2 CAR T cells (“CAR”).

[0110] FIG.3A shows the change in percent IL-2 expression between the first stimulation and third stimulation for gRNA IL-2_H, gRNA IL-2 gRNA-1 and gRNA SpNT as well as other gRNAs. FIG.3B shows the fold IL-2+ CAR T cells over the non-targeting gRNA SpNT between the first stimulation and third stimulation for gRNA IL-2_H, gRNA IL-2 gRNA-1 and other gRNAs. Data points representative of gRNA IL-2_H and gRNA IL-2 gRNA-1 are denoted. The open circle data points represent data from using the gRNA SpNT.

[0111] FIG.4 shows the mean fluorescence intensity (MFI) of Her2 CAR T cells that transiently expressed dCas9 effector fusion proteins for activation of IL-2 targeted by guide RNA IL-2_H compared to other guides. Data points of gRNA IL-2_H and gRNA IL-2 gRNA-1 are denoted. The open circle data points represent data from using the gRNA SpNT.

[0112] FIG.5A shows a schematic depicting the experimental design of testing the functionality of induced pluripotent stem cell (iPSC)-derived natural killer cells for increased functionality and cytokine secretion following delivery of a DNA-targeting system targeting IL- 2 by differentiating iPSCs into immune effector cells (induced Natural Killer, or iNK, cells) then delivering CAR lentivirus and mRNA encoding a dSpCas9-2xVP64 fusion protein and a gRNA targeting IL-2.

[0113] FIG.5B shows the tumor cell count over time, normalized to the tumor cell count at the onset of the experiment (T0), following incubation with either induced Natural Killer cells 6037971-sf22474-20029.40 (iNK), CAR+ iNK cells (iNK+CAR), or CAR+ iNK cells that were delivered mRNA encoding a dSpCas9-2xVP64 fusion protein and a gRNA targeting IL-2 (iNK+CAR+IL-2).

[0114] FIG.5C shows the secretion of IL-2 in induced Natural Killer (iNK) cells or CAR-T cells, as measured in pg / mL. FIG.5D shows the secretion of interferon gamma (IFNγ) in induced Natural Killer (iNK) cells or CAR-T cells, as measured in pg / mL. Different conditions of iNK cells were tested, including those that were not transduced with a CAR lentivirus nor electroporated with an IL-2-targeted DNA-targeting system (-), iNK cells only transduced with a CAR lentivirus (CAR+), and iNK cells both transduced with a CAR lentivirus and electroporated with an IL-2-targeted DNA-targeting system (+CAR+IL-2)

[0115] FIG.6A shows the fold increase in IL-2+ cells (which is the ratio of the absolute count of edited CAR+ IL-2+ cells to the absolute count of NT control, CAR+ IL-2+ cells at the third round of serial killing) against the IL-2 activation durability in an exemplary donor. FIG. 6B and FIG.6C similarly show the fold increase in IL-2+ cells against the IL-2 activation durability in two additional exemplary donors. Select data points representing select combinations of gRNAs are highlighted for each exemplary donor.

[0116] FIG.7A shows percent IL-2 expression following a first stimulation of Her2 CAR T cells that were delivered mRNA encoding a dSpCas9-2xVP64 effector fusion protein and various SpCas9 IL-2-targeting gRNA or a dSaCas9-2xVP64 effector fusion protein and various SaCas9 IL-2-targeting gRNA. FIG.7B shows percent IL-2 expression following a second stimulation of the same Her2 CAR T cells shown in FIG.7A.

[0117] FIG.8 shows normalized tumor target cell count over two stimulations of T cells derived from two donors (Donor 1 and Donor 2) that were delivered a chimeric antigen receptor (CAR) mRNA and mRNA encoding a dSpCas9-2xVP64 effector fusion protein and either an IL-2 targeting gRNA (bottom left subplot) or a non-targeting (NT) gRNA (top right subplot). As negative controls, T cells also received no mRNA (mock; bottom right subplot) or only a CAR mRNA (top left subplot).

[0118] FIG.9 depicts two exemplary dSaCas9 fusion proteins for transcriptional activation: dSaCas9 covalently linked to effector domains NCOA3-FOXO3-NCOA3 (NFN) and VP64 (dSaCas9-NFN-VP64) on the left and dSaCas9 covalently linked to 2 VP64 domains (dSaCas9- 2xVP64) on the right.

[0119] FIG.10 shows the intracellular expression of IL-2 after first stimulation using an average of two donors as %IL-2 positive cells in the left panel and mean fluorescent levels 6037971-sf22474-20029.40 (MFI; corresponding to average expression levels) in the right panel, in cells that were delivered mRNA encoding dSaCas9-2xVP64, dSaCas9-VP64-NFN, or dSpCas9-2xVP64 mRNA with corresponding IL-2 targeting gRNA(s). No delivery of exemplary fusion protein (CAR only) was used as a negative control.

[0120] FIGS.11-13 show alignments of a subset of designed engineered zinc finger protein (ZFP) target sites for IL-2 gene region 4 (FIG.11), IL-2 region 5 (FIG.12), and IL-2 transcription start site (TSS; FIG.13). Each solid rectangle represents a given target site and top-performing ZFPs (see Table E4) and guide RNAs (gRNAs) are annotated using a box.

[0121] FIG.14A shows the expression of IL-2 following delivery of the top-performing IL- 2-targeting ZFP fusion proteins set forth in Table E4, dSpCas9-2xVP64 and gRNA IL2_H (SpCas9), or no delivery (CAR alone). IL-2 expression was represented as % IL-2 positivity following a first stimulation 72 hours post-delivery (top) or cell counts of IL-2-positive CAR T- cells following a second stimulation (bottom).

[0122] FIG.14B show overall cell count 72 hours post-delivery of top-performing IL-2- targeting ZFP fusion proteins as set forth in Table E4. As controls, cells were delivered a dSpCas9 fusion protein and a corresponding IL-2 gRNA (SpCas9) or did not receive any fusion proteins (CAR alone). Detailed Description

[0123] Provided herein is an epigenetic-modifying DNA-targeting system in which the DNA-targeting system comprises a plurality of DNA-targeting modules for increasing transcription of the interleukin (IL-2) gene, wherein each of the DNA- targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain for targeting to a target site of the IL-2 gene; and (b) at least one transcriptional activator effector domain. Further provided herein is an epigenetic-modifying DNA-targeting system in which the DNA-targeting system comprises at least one DNA-targeting module composed of a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site in the interleukin-2 (IL-2) gene or regulatory DNA element thereof in a lymphoid cell (e.g., T or natural killer cell, also referred to as NK cell); and (b) at least one transcriptional activator effector domain capable of activating transcription of the IL-2 gene or regulatory element thereof. In some embodiments, the target site is in the IL-2 gene or regulatory region thereof found herein to be a positive regulator of lymphoid cell (e.g., T or NK cell) function after transient transcriptional modulation of the IL-2 6037971-sf22474-20029.40 gene. In some such embodiments, the at least one effector domain is a transcriptional activator domain, such as VP64. In some embodiments, the target site is within 1000 base pairs of a transcriptional start site (TSS) of the IL-2 gene. The target site may be within a regulatory region, such as a promoter or enhancer of the IL-2 gene. In some embodiments, the target site is within 50-150 kb upstream of the IL-2 gene.

[0124] In some embodiments, the epigenetic-modifying DNA-targeting systems are synthetic transcription factors that are able to increase (or upregulate) transcription of the IL-2 gene in a targeted manner. Provided herein is a epigenetic-modifying DNA-targeting system in which the DNA-binding domain of each fusion protein comprises: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I- SceI enzyme or a variant thereof, optionally wherein the DNA- binding domain comprises a catalytically inactive variant of any of the foregoing, wherein when the DNA-binding domain of each fusion protein comprises a Cas protein, the DNA-targeting system further comprises at least two gRNAs, each capable of targeting the Cas protein to a target site.

[0125] In some embodiments, the epigenetic-modifying DNA-binding domain of the DNA- targeting system is a nuclease-inactive Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein (e.g., a dCas protein) or variant thereof, which is complexed with a guide RNA (gRNA). Also provided are gRNAs for targeting to a target site in the IL-2 gene or a regulatory DNA element thereof in a lymphoid cell (e.g., T or NK cell), in which it is found that transient epigenetic modulation of transcription of the gene promotes lymphoid cell (e.g., T or NK cell) function. Also provided are CRISPR-Cas / gRNA combinations composed of the gRNA and a nuclease inactivated Cas, such as a dCas9. Also provided herein are polynucleotides encoding the DNA-targeting system or the fusion protein of the DNA-targeting system, and vectors and cells containing the same. Also provided herein are methods of using the epigenetic-modifying DNA-targeting system for modulating transcription or phenotype or function of lymphoid cells (e.g., T or NK cells) and the resulting modified cells. Also provided herein are methods of using the epigenetic-modifying DNA-targeting system for increasing transcription of the IL-2 gene or regulatory element thereof. Also provided herein are cells, such as lymphoid cells (e.g., T or NK cells), which are modified using any of the compositions and / or methods provided herein.

[0126] In some embodiments, the epigenetic-modifying DNA-targeting system contains at 6037971-sf22474-20029.40 least one DNA-targeting module, where each DNA-targeting module of the system is a component of the DNA-targeting system that is independently capable of targeting one target site for the IL-2 gene or regulatory element thereof as provided. In some embodiments, each DNA-targeting module includes (a) a DNA-binding domain capable of being targeted to a target site for the IL-2 gene or regulatory element and (b) an effector domain capable of increasing (e.g. activating) transcription of the gene. In some embodiments, the epigenetic-modifying DNA-targeting system contains at a plurality of DNA-targeting modules. In some embodiments, the plurality of DNA-targeting modules is 2-6 DNA targeting modules. In particular embodiments, the plurality of DNA-targeting modules is 2 DNA targeting modules. In particular embodiments, the plurality of DNA-targeting modules is 3 DNA targeting modules. In particular embodiments, the plurality of DNA-targeting modules is 4 DNA targeting modules. In particular embodiments, the plurality of DNA-targeting modules is 5 DNA targeting modules.

[0127] In some embodiments, the DNA-targeting system includes a single DNA-targeting module for targeting activation or increased expression of IL-2. In some embodiments, the DNA-targeting module includes (a) a DNA-binding domain capable of being targeted to a target site of the IL-2 gene or regulatory element, and (b) an effector domain capable of activating transcription of the gene.

[0128] In some embodiments, the DNA-targeting system includes a plurality of DNA- targeting modules, in which each DNA-targeting module is for targeting activation or increased expression of different target sites of the IL-2 gene or regulatory element thereof. In some embodiments, the DNA-targeting systems are multiplexed DNA-targeting systems, i.e. targeted to multiple target sites for the IL-2 gene or regulatory element thereof. Hence, the terms DNA- targeting system may include a multiplexed epigenetic-modifying DNA targeting system that includes more than one DNA-targeting module. A multiplexed epigenetic-modifying DNA targeting system provided herein can comprise 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 multiplexed epigenetic-modifying DNA targeting system comprises between 2-6 DNA targeting modules. In some embodiments, the multiplexed epigenetic- modifying target system comprises 2 DNA targeting modules. In some embodiments, the multiplexed epigenetic-modifying target system comprises 3 DNA targeting modules. In some embodiments, the multiplexed epigenetic-modifying target system comprises 4 DNA targeting modules. In some embodiments, the multiplexed epigenetic-modifying target system comprises 6037971-sf22474-20029.40 5 DNA targeting modules. In some embodiments, the multiplexed epigenetic-modifying target system comprises 6 DNA targeting modules. In some embodiments, the plurality of DNA- targeting modules target a plurality of (i.e. multiple) target sites for the IL-2 gene or regulatory element thereof.

[0129] In some embodiments, any two DNA-targeting modules of a DNA-targeting system comprise separate (i.e. non-overlapping) components. In some embodiments, different DNA- targeting modules of a DNA-targeting system comprise separate (i.e. non-overlapping) components. For example, a 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.

[0130] In some embodiments, any two DNA-targeting modules of a DNA-targeting system may comprise shared (i.e. overlapping) components. In some embodiments, different DNA- targeting modules of a DNA-targeting system comprise shared (i.e. overlapping) components. For example, in one aspect, a DNA-targeting system may comprise a first DNA-targeting module comprising (a) a fusion protein comprising a Cas protein and a transcriptional effector (e.g. activator) domain, and (b) a first gRNA that complexes with the Cas protein and targets a first target site, and a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes 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 be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g. SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs. Thus, it is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA- targeting modules.

[0131] The provided embodiments relate to compositions and methods for promoting lymphoid cell (e.g., T or NK cell) function, such as one or more lymphoid cell (e.g., T or NK cell) effector functions, by epigenetically modifying target sites in the IL-2 gene or regulatory element thereof. In some embodiments, the methods can be used in connection with lymphoid cell (e.g., T cell) therapies, such as in connection with adoptive T cell therapies. In some embodiments, increasing transcription of the IL-2 gene or regulatory element thereof increases 6037971-sf22474-20029.40 or improves one or more lymphoid cell (e.g., T or NK cell) phenotype or function. In some embodiments, the lymphoid cell (e.g., T or NK cell) effector function of increasing the ability to produce IL-2 is increased. In some embodiments, a lymphoid cell (e.g., T or NK cell) effector function is increased, such as the ability to produce cytokines, for example IL-2 or IFN-gamma (IFNg), the ability of lymphoid cell (e.g., T or NK cell)s to proliferate, the ability of lymphoid cell (e.g., T or NK cell)s to kill target cells, or the ability of lymphoid cell (e.g., T or NK cell)s to exhibit a persistent immune response. In particular embodiments, activation of the IL-2 gene or regulatory element thereof improves lymphoid cell (e.g., T or NK cell) effector functions after or upon lymphoid cell (e.g., T or NK cell) stimulation, including following serial stimulation that mimic conditions of repeated antigen encounter as occurs in vivo.

[0132] The administration of T cells targeting a specific antigen, 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 the transferred T cells can significantly differ between different T cell subsets and among T cells from different patients. Recent clinical trials for ACT suggest that the ability to persist long term in the circulation is dependent on the differentiation stage of the T cell, including the ability to retain a network of transcription factors and metabolic regulators (Pilipow K., et.al., Journal of Clinical Investigation Insight 2018;3(18):e122299). The T cells transferred into the patient are often terminally differentiated and therefore fail to persist in the long term, ultimately limiting effective anti-tumor response. For instance, while the first CAR-T cell therapy was FDA- approved as a cell & gene therapy in 2017, patients whose cancer relapse or do not respond to treatment often suffer from lack of CAR T cell persistence (Mueller et al, Blood (2018)). Moreover, no durable benefit has yet been observed for CAR T cell therapies in solid tumors.

[0133] Strategies to mitigate these challenges and enhance the persistence, expansion, and anti-tumor activity of chimeric antigen receptor (CAR) engineered T cells have been tested in preclinical and clinical settings. For instance, strategies for optimizing ex vivo T cell culture conditions, including the addition of cytokines during manufacturing (Besser M.J., Cytotherapy 2009;11(2):206-17), expression of cytokines and / receptors by the CAR T cells (Krenciute G., Cancer Immunol Res.201707;5(7):571-581), use of pharmacological inhibitors during expansion to inhibit signaling pathways such as AKT (Urak R.et.al., Journal of Immunotherapy Cancer 2017 Mar 21;5:26) or PI3K (Peterson C.T et.al., Blood Advances 2018 Feb 13;2(3):210- 6037971-sf22474-20029.40 223), immune-depletion and checkpoint blockade (Cherkassky L. et.al., Journal of clinical investigation 2016 Aug 1;126(8):3130-44) have been so far explored. However, existing strategies have not been entirely satisfactory. In some cases, concerns regarding cytokine- induced toxicity or the emergence of lymphoproliferative diseases as a result of the above- mentioned strategies have raised questions for alternative approaches.

[0134] The provided embodiments relate to identification of genomic locations in the IL-2 gene or regulatory element thereof that are epigenetically modified in a lymphoid cell (e.g., T or NK cell) to impact or promote lymphoid cell (e.g., T or NK cell) effector functions, such as upon stimulation (e.g., T cell stimulation), including those induced in a TCR and / or CAR- induced or dependent manner, such as demonstrated by assessment for cells producing IL-2. The provided embodiments relate to identification of genomic locations that are epigenetically modified in a lymphoid cell (e.g., T or NK cell) to impact or promote lymphoid cell (e.g., T or NK cell) effector functions, including upon stimulation (e.g., T cell stimulation), including those induced in a TCR and / or CAR-induced or dependent manner, such as demonstrated by assessment for cells producing IL-2, having the ability to proliferate, or having the ability to kill target cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. In some embodiments, the one or more agents are PMA and ionomycin. In some embodiments, the stimulation (e.g., T cell stimulation) is an antigen-specific stimulation, in which the cells ate stimulated with an agent providing an antigen or epitope thereof that is specific to, or recognized by, an antigen receptor (e.g. CAR) expressed on the lymphoid cell (e.g., T or NK cell). For instance, the stimulating agent may include antigen-expressing target cells. In particular embodiments, the phenotype is or includes the production or secretion of a cytokine, such as IL-2 or IFN-g, in response to a stimulation (e.g., T cell stimulation). The production and / or the secretion of cytokines contributes to immune responses, and is involved in different processes including the induction of anti-viral proteins and the induction of lymphoid cell (e.g., T or NK cell) proliferation. Cytokines are not pre-formed factors but are rapidly produced and secreted in response to cellular activation. The production or secretion of cytokines may be measured, detected, and / or 6037971-sf22474-20029.40 quantified by any suitable technique known in the art.

[0135] In certain embodiments, the lymphoid cell (e.g., T or NK 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 (ICS) by flow cytometry is a technique well-suited for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the cell (such as within the endoplasmic reticulum) after cell stimulation, allowing for the identification of cell populations that are positive or negative for production of a particular cytokine or for the separation of high producing and low producing cells based on a threshold. ICS can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or with MHC multimers to access cytokine production in a particular subgroup 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 lymphoid cell (e.g., T or NK cell) cloning.

[0136] In some aspects, certain target sites of the present disclosure were identified by a screening method involving transient delivery, in which the DNA-binding domain-effector fusion proteins (also called “epi-editors”) were delivered to the lymphoid cell (e.g., T or NK cell) transiently (i.e. delivered by a method that results in transient expression and / or presence of the fusion protein in the lymphoid cell (e.g., T or NK cell)) followed by primary or serial stimulation of the cells to assess impact on functional lymphoid cell (e.g., T or NK cell) cytokines. It was found herein that the transient delivery of the epigenetic modifying DNA- targeting systems allowed identification of genomic targets whose modulation substantially impacts lymphoid cell (e.g., T or NK cell) function, but without requiring permanent presence of the epigenetic modifying DNA-targeting systems, and / or stable knock down or knockout of the IL-2 gene or regulatory element This approach is advantageous because it permits identification of target sites that provide a better safety profile as their modulation is not reliant on a permanent editor integration, such as by lentiviral transduction. Moreover, the transient screening strategies allow for identification of target sites therein in which there is a durability of the effect of the epigenetic modifying DNA-targeting system that is not masked as a result of permanent integration into the genome and expression therefrom. This is in contrast to other screening approaches in which lentiviral delivery of DNA-systems has been employed 6037971-sf22474-20029.40 (Schmidt et al.2022 Science, 375, DOI: 10.1126 / science.abj4008; Freimer et al.2022 Nature Genetics, 54:1133-1144).

[0137] The provided embodiments can be used to target the IL-2 gene or regulatory element thereof that when transcriptionally altered by epigenetic modification, can vastly facilitate or promote lymphoid cell (e.g., T or NK cell) function, including effector activities required for lymphoid cell (e.g., T or NK cell) persistence and function. Such a lymphoid cell (e.g., T or NK cell) profile is expected to produce durable effector functions, such as the ability to produce IL-2 upon TCR or antigen stimulation. Such a lymphoid cell (e.g., T or NK cell) profile is expected to produce durable effector functions, have better fitness / proliferation benefit, and have the ability to produce pro-proliferation cytokines (e.g. IL-2) and / or cytotoxic cytokines (e.g. IFNg) upon TCR or antigen stimulation. In particular, the provided embodiments provide for epigenetic-modifying DNA-targeting systems (i.e. “epi-editing systems”) and methods that can provide for long-lasting effector function with better fitness. This approach offers substantial clinical solutions to circumvent the problems with lymphoid cell (e.g., T or NK cell) persistence, suboptimal functionality, and / or exhaustion. Moreover, the epigenetic modification of the cell does not modify DNA at the sequence level, thereby avoiding safety concerns with gene editing approaches. The ability to epigenetically control the differentiation fate of lymphoid cells (e.g., T or NK cells) provides an advantageous approach for increasing the percentage or number of lymphoid cells (e.g., T or NK cells) in a population of lymphoid cells (e.g., T or NK cells).

[0138] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0139] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. DNA-TARGETING SYSTEMS

[0140] Provided herein is an epigenetic-modifying DNA-targeting system comprising a plurality of DNA-targeting modules for increasing transcription of the interleukin (IL-2) gene, wherein each of the DNA- targeting modules comprises a fusion protein comprising: (a) a DNA- 6037971-sf22474-20029.40 binding domain for targeting to a target site of the IL-2 gene; and (b) at least one transcriptional activator effector domain. In some embodiments, provided are DNA-targeting systems capable of specifically targeting a target site for the IL-2 gene or regulatory element thereof, and activating transcription of the IL-2 gene or regulatory element thereof. In some embodiments, the DNA-targeting system targets one or more target sites for the IL-2 gene or regulatory element thereof and increases (e.g. activates) transcription of the gene. In some embodiments, the IL-2 gene is in a lymphoid cell, such as a T cell. In some embodiments, the IL-2 gene is in a lymphoid cell, such as a NK cell. In some embodiments, the target site for the IL-2 gene is a target site in the IL-2 gene or a regulatory DNA element thereof. In some embodiments, the transcription modulation is increased transcription of the IL-2 gene or regulatory element thereof. In provided embodiments, for the IL-2 gene or regulatory element thereof that is targeted, the DNA-targeting system includes a fusion protein that comprises a DNA-binding domain that binds to the target site for the gene, and an effector domain for increasing transcription of the IL-2 gene or regulatory element thereof. In some embodiments, the provided DNA-targeting systems are able to modulate, such increase, transcription of the IL-2 gene or regulatory element thereof in the cell. In some embodiments, transcriptional modulation of gene expression by the DNA-targeting systems provided herein can promote or improve function of the lymphoid cells. In particular embodiments, the provided DNA-targeting systems promote lymphoid cell (e.g., T or NK cell) function, such as one or more lymphoid cell (e.g., T or NK cell) effector functions, by epigenetically modifying target sites in the IL-2 gene or regulatory element thereof.

[0141] In some embodiments, the at least one effector domain is a transcriptional activator effector domain for increasing transcription of the IL-2 gene or regulatory element thereof (e.g. activates or increases transcription of the gene as compared to transcription of the gene in the absence of the DNA-targeting system), such as any effector domain for transcriptional activation. In some embodiments, the effector domain is a transcriptional activator effector domain.

[0142] In some embodiments, the effector domain directly or indirectly leads to increased transcription of the IL-2 gene or regulatory element thereof. In some embodiments, the effector domain induces, catalyzes or leads to transcription activation. In some embodiments, the effector domain induces transcription activation. In some aspects, the effector domain comprises: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP 6037971-sf22474-20029.40 domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing. In some embodiments, the effector domain is VP64.

[0143] In some embodiments, the DNA-targeting system includes a fusion protein comprising (a) at least one DNA-binding domain capable of being targeted to the target site; and (b) at least one effector domain capable of increasing transcription of the IL-2 gene or regulatory element thereof. In some embodiments, the at least one effector domain is a transcription activator effector domain. The fusion protein can be any suitable fusion protein, for example as described in Section I.F.

[0144] 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), meganuclease, homing endonuclease, I-SceI enzyme, or variants thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive (e.g. nuclease- inactive or nuclease-inactivated) variant of any of the foregoing. In some embodiments, the DNA-binding domain comprises a deactivated Cas9 (dCas9) protein or variant thereof that is a catalytically inactivated so that it is inactive for nuclease activity and is not able to cleave the DNA. The DNA-binding domain can be any suitable DNA-binding domain, for example as described in Sections I.C and I.D.

[0145] In some embodiments, the DNA-binding domain comprises or is derived from a Cas protein or variant thereof, such as a nuclease-inactive Cas or dCas (e.g. dCas9, and the DNA- targeting system comprises one or more guide RNAs (gRNAs), such as a combination of gRNAs (e.g. two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence that is capable of targeting and / or hybridizing to the target site. In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some aspects, the gRNA directs or recruits the Cas protein or variant thereof to the target site. The gRNA can be any suitable gRNA, for example as described in section I.C.2.

[0146] In some embodiments, the DNA-targeting system is for increasing transcription of IL-2 gene or regulatory element thereof, and the fusion protein of a DNA-targeting module thereof is a dCas9-VP64 fusion protein, such as a dCas9-2xVP64 fusion protein. In some embodiments, the fusion protein is any as described herein, for example in Section I.F.

[0147] Exemplary components and features of the DNA-targeting systems are provided below in the following subsections. 6037971-sf22474-20029.40 A. DNA-Targeting Modules and Multiplexed DNA-Targeting Systems

[0148] In some embodiments, the epigenetic-modifying DNA-targeting system contains at least one DNA-targeting module, where each DNA-targeting module of the system is a component of the DNA-targeting system that is independently capable of targeting one target site for the IL-2 gene or regulatory element thereof. In some embodiments, each DNA-targeting module includes (a) a DNA-binding domain capable of being targeted to the target site, and (b) an effector domain for increasing transcription of the IL-2 gene or regulatory element thereof. In some embodiments, the DNA-binding domain of the at least one DNA-targeting module comprises: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-SceI enzyme or a variant thereof, optionally wherein the DNA- binding domain comprises a catalytically inactive variant of any of the foregoing, wherein when the DNA-binding domain of each fusion protein comprises a Cas protein, the DNA-targeting system further comprises at least two gRNAs, each capable of targeting the Cas protein to a target site.

[0149] In some embodiments, a DNA-targeting module is a CRISPR / Cas-based DNA- targeting module. In some embodiments, in a CRISPR / Cas-based DNA-targeting module, the DNA-binding domain of the fusion protein is a Cas protein or variant thereof (e.g. a dCas protein, such as dCas9) and the DNA-targeting module further comprises a gRNA for targeting the DNA-binding domain to the target site.

[0150] In some embodiments, a 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).

[0151] In some embodiments, a 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.

[0152] In some embodiments, the DNA-targeting system includes a plurality of DNA- targeting modules, in which each DNA-targeting module targets a different target site in the IL-2 gene or regulatory element thereof. In some embodiments, the DNA-targeting system includes a plurality of DNA-targeting modules, in which each DNA-targeting module targets the same target site in the IL-2 gene or regulatory element thereof. In some embodiments, the DNA- targeting system is a multiplexed DNA-targeting system. In some embodiments, the 6037971-sf22474-20029.40 multiplexed DNA-targeting system targets different target sites for the IL-2 gene or regulatory element thereof. In some embodiments, the multiplexed DNA-targeting system targets same target sites for the IL-2 gene or regulatory element thereof. Hence, the term DNA-targeting system may include a multiplexed epigenetic-modifying DNA targeting system that includes more than one DNA-targeting module. In some embodiments, a multiplexed epigenetic- modifying 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 epigenetic-modifying DNA-targeting system contains at a plurality of DNA-targeting modules. In some embodiments, the plurality of DNA-targeting modules is 2-6 DNA targeting modules. In particular embodiments, the plurality of DNA- targeting modules is 2 DNA targeting modules. In particular embodiments, the plurality of DNA-targeting modules is 3 DNA targeting modules. In particular embodiments, the plurality of DNA-targeting modules is 4 DNA targeting modules. In particular embodiments, the plurality of DNA-targeting modules is 5 DNA targeting modules.

[0153] 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 may 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.

[0154] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise shared (i.e. overlapping) components. For example, a DNA-targeting system may comprise: 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 complexes with the Cas protein and targets a first target site, and ii) a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes 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 be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g. SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, as described herein. Thus, it 6037971-sf22474-20029.40 is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA-targeting modules.

[0155] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising a plurality of DNA-targeting modules for increasing transcription of the IL-2 gene or regulatory element thereof. In some embodiments, the plurality of DNA-targeting modules comprises a first DNA-targeting module for increasing transcription of the IL-2 gene or regulatory element thereof by targeting a first target site, and a second DNA-targeting module for increasing transcription of the IL-2 gene or regulatory element thereof by targeting a second target site. In some embodiments, each DNA-targeting module comprises a fusion protein comprising: (a) a DNA-binding domain for targeting a first target site of IL-2 gene or regulatory element thereof for the DNA-targeting module, and (b) at least one effector domain. In some embodiments, each DNA-targeting module comprises a transcriptional activator effector domain for increasing transcription of the IL-2 gene or regulatory element thereof B. IL-2 Gene Target Sites For Promoting Lymphocyte (e.g. T or NK cell) Activation and Function

[0156] In some aspects, provided herein are target sites for the IL-2 gene or regulatory element thereof in which increasing transcription of the IL-2 gene or regulatory element thereof promotes lymphoid cell (e.g., T cell or NK cell) activation or function. In some embodiments, the target site is targeted using any of the provided DNA-targeting systems.

[0157] In some embodiments, the target site is in the IL-2 gene or regulatory element thereof in which increased expression of the gene promotes lymphoid cell activation or function. In some embodiments, the target site is in the IL-2 gene or regulatory element thereof in which increased expression of the gene promotes lymphoid cell (e.g., T or NK cell) activation or function. In some embodiments, the target site is in the IL-2 gene or regulatory element thereof in which increased expression of the gene promotes natural killer cell activation or function.

[0158] In some embodiments, the target site is targeted by a DNA-targeting system, such as by a DNA-targeting module of the DNA-targeting system, such as any described herein. In some embodiments, the target site is a target site for the IL-2 gene or regulatory element thereof. In some embodiments, the target site for the IL-2 gene is in the IL-2 gene body or is a regulatory DNA element thereof. In some embodiments, the target site is a target site in the IL-2 gene. In some aspects, the gene is a IL-2 gene or regulatory element thereof. In some embodiments, the gene is a IL-2 gene or regulatory element thereof in a cell. In some embodiments, the cell is an 6037971-sf22474-20029.40 immune cell, such as a T cell or a NK cell. In some embodiments, provided herein are multiplexed epigenetic-modifying DNA-targeting systems that target a combination of at least two target sites of the IL-2 gene or regulatory element thereof described herein.

[0159] In some embodiments, the DNA-targeting system targets to or binds to a target site in the IL-2 gene or regulatory element thereof, such as any described herein. In some embodiments, the target site is located in the IL-2 gene or regulatory element thereof. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of a gene. In some embodiments, the regulatory DNA element is a distal regulatory element of a gene. In some embodiments, the regulatory DNA element is a proximal regulatory DNA element of a gene. In some embodiments, the regulatory DNA element is a upstream regulatory element of a gene. In some embodiments, the gene is IL-2. 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 to begin transcription of the gene. In some embodiments, a promoter is a nucleotide sequence located within about 100bp, about 500bp, about 1000bp, or more, of a transcriptional start site of the gene. In some embodiments, a promoter is within 500bp of a transcriptional start site of the gene. In some embodiments the target site is located within a sequence of unknown or known function that is suspected of being able to control expression of a gene. In some embodiments, the target site is located within 50 to 150 kb, inclusive, upstream of the IL-2 gene. 1. Lymphoid Cells and Modulated Effector Functions

[0160] In some embodiments, the provided DNA-targeting systems and / or a DNA-targeting provide for transcriptional activation to increase expression of the IL-2 gene or regulatory element thereof. In some embodiments, the IL-2 gene is a gene for which expression of the gene regulates a cellular phenotype. In some embodiments, the IL-2 gene is capable of regulating a phenotype in a lymphoid cell (e.g., T or NK cell). In some embodiments, increased expression of the IL-2 gene or regulatory element thereof, regulates the phenotype. In some embodiments, modulated expression of the IL-2 gene promotes increased lymphoid cell function. In some embodiments, modulated expression of the IL-2 gene promotes increased T cell effector 6037971-sf22474-20029.40 function upon T cell stimulation. In some embodiments, modulated expression of the IL-2 gene promotes increased NK cell effector function.

[0161] In some embodiments, the increased lymphoid cell effector function is increased compared to a lymphoid cell in which expression of the IL-2 gene has not been increased with a provided DNA-targeting system. In some embodiments, the increased T cell effector function is increased compared to a T cell in which expression of the IL-2 gene has not been increased with a provided DNA-targeting system. In some embodiments, the increased NK cell effector function is increased compared to a NK cell in which expression of the IL-2 gene has not been increased with a provided DNA-targeting system. Methods for modulating T cell function or functions of other lymphoid cells by provided DNA-targeting systems are further described below and in Section IV.

[0162] In some embodiments, the IL-2 gene is increased by a DNA-targeting system, such as any DNA-targeting system provided herein. In some embodiments, the DNA-targeting system is transiently delivered to the cell. In some embodiments, delivery of the DNA-targeting system, for example by transient delivery, promotes increased modified lymphoid cell effector function. In some embodiments, the lymphoid cell effector function is increased in comparison to a comparable lymphoid cell to which the DNA-targeting system has not been delivered. In some embodiments, delivery of the DNA-targeting system, for example by transient delivery, promotes increased T cell effector function upon T cell stimulation. In some embodiments, the T cell effector function is increased in comparison to a comparable T cell to which the DNA- targeting system has not been delivered. In some embodiments, delivery of the DNA-targeting system, for example by transient delivery, promotes increased natural killer cell effector function. In some embodiments, the NK cell effector function is increased in comparison to a comparable NK cell to which the DNA-targeting system has not been delivered.

[0163] In some aspects, transient delivery refers to any method of delivery that results in expression and / or presence of one or more components of the DNA-targeting system in the cell for a limited duration. For example, delivery of mRNA (such as by electroporation) encoding the fusion protein of the DNA-targeting system to a cell can result in transient expression of the fusion protein in the cell, for example until the mRNA is degraded. In other examples, the DNA-targeting system can be expressed from one or more nucleic acids encoding the DNA- targeting system, wherein the nucleic acids encoding the DNA-targeting system are not incorporated into the genome of the cell, and are eventually degraded and / or removed from the 6037971-sf22474-20029.40 cell such that expression of the DNA-targeting system does not persist. In other examples, one or more components of the DNA-targeting system, such as a fusion protein and optionally a gRNA can be synthesized in vitro and delivered directly to the cell (e.g. by electroporation) without the need for an expression vector, resulting in transient presence of the DNA-targeting system, for example until the fusion protein and / or gRNA are degraded. In some aspects, transient delivery differs from non-transient methods of delivery that result in stable expression, such as methods involving incorporation of an expression vector for a DNA-targeting system or component thereof into the genome of the cell.

[0164] In some embodiments, delivery of the DNA-targeting system to the cell (e.g. lymphoid cell, such as T cell or NK cell), such as by transient delivery, promotes a phenotype in the cell (e.g. lymphoid cell, such as T cell or NK cell). In some embodiments, the phenotype is increased activation or function in the cell (e.g. lymphoid cell, such as T cell or NK cell). In some embodiments, delivery of the DNA-targeting system to the cell (e.g. modified lymphoid cell, such as T cell or NK cell), such as by transient delivery, promotes increased activation or function in the cell (e.g., lymphoid cell, such as T cell or NK cell). In some embodiments, the phenotype is modified lymphoid cell function upon modified lymphoid cell stimulation. In some embodiments, the phenotype is increased T cell effector function upon T cell stimulation. In some embodiments, the phenotype is increased NK cell effector function upon NK cell stimulation. In some embodiments, the lymphoid cell function is increased compared to a lymphoid cell that has not been delivered the epigenetic-modifying DNA-targeting system. In some embodiments, the T cell effector function is increased compared to a T cell that has not been delivered the epigenetic-modifying DNA-targeting system. In some embodiments, the NK cell effector function is increased compared to a NK cell that has not been delivered the epigenetic-modifying DNA-targeting system. In some embodiments, increased expression (e.g. transcription) of the IL-2 gene or regulatory element thereof, leads to increased T cell effector function upon T cell stimulation. In some embodiments, increased expression (e.g. transcription) of the IL-2 gene or regulatory element thereof, leads to increased NK cell effector function. In some embodiments, the T cell effector function is characterized by IL-2 production. 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. In some embodiments, the NK cell effector function is characterized by IL-2 production. In some embodiments, the NK cell 6037971-sf22474-20029.40 effector function is characterized by an activity selected from the group consisting of IL-2 production, IFN-gamma production, or a combination of any of the foregoing.

[0165] In some embodiments, provided DNA-targeting systems promote or increase an improved T cell effector function as may occur after stimulation in vitro, ex vivo or in vivo. In some embodiments, the T cell stimulation is a polyclonal T cell stimulation. In some embodiments, the T cell stimulation is with an anti-CD3 and anti-CD28 activation reagent. In some embodiments, the T cell stimulation is an antigen-specific activity that is mediated or induced by specific binding of an antigen to an antigen receptor on the surface of the T cell. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR) directed against an antigen and the T cell stimulation is an 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 cell contacts a cell expressing the antigen. In some embodiments, the T cell stimulation is a restimulation after at least one prior T cell stimulation of the T cells. In some embodiments, the T cells are stimulated and then are transiently delivered a provided DNA-targeting system prior to assessment of a T cell effector function or phenotype.

[0166] In certain embodiments, the cell composition that contains T cells is stimulated with an anti-CD3 / anti-CD28 activation reagent for an amount of time, and an effector function is measured at one or more time points during or after the incubation. In some embodiments, such an activation reagent has anti-CD3 / anti-CD28 coated on a support, such as magnetic beads or other matrix. Exemplary activation reagent Dynabeads™ or T cell TransAct™. In some embodiments, the T cells are incubated with the activation reagent, for 3 hours to 72 hours, such as 12 hours to 48 hours, for example, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours, or any value between any of the foregoing. In some embodiments, cells can be assessed directly for an effector function, such as production of cytokines or ability to proliferate. In some embodiments, the supernatant of the culture can be collected and the amount of a soluble factor, e.g., a cytokine is detected. In some embodiments, the T cells can be collected and re-exposed to the activation reagent to monitor cytolytic activity. In some embodiments, cells can be restimulated one or more time, such as by serial stimulation methods, and serially assessed for effector functions after each stimulation.

[0167] In certain embodiments, the antigen-specific activity is measured by incubating the cell composition that contains lymphoid cells (e.g., T cells or NK cells) expressing the antigen 6037971-sf22474-20029.40 receptor, e.g., a CAR, with antigen-expressing cells for an amount of time, and an effector function is measured at one or more time points during or after the incubation. In some embodiments, the lymphoid cells (e.g., T or NK cells) are incubated with the antigen specific agent, such as antigen-expressing cells, for 3 hours to 96 hours, such as 12 hours to 72 hours, for example, 12 hours, 24 hours, 48 hours, 72 hours or any value between any of the foregoing. In some embodiments, cells can be assessed directly for an effector function, such as production of cytokines or ability to proliferate. In some embodiments, the supernatant of the culture can be collected and the amount of a soluble factor, e.g., a cytokine is detected. In some embodiments, the lymphoid cells (e.g., T or NK cells) can be collected and re-exposed to antigen-expressing target cells to monitor cell killing (cytolytic activity) of target cells. In some embodiments, cells can be restimulated one or more time, such as by serial stimulation methods, and serially assessed for effector functions after each stimulation. In some embodiments, the lymphoid cells (e.g., T or NK cells) with the engineered antigen receptor (e.g., a CAR) are incubated with a constant number of the antigen-expressing cells, such as at an effector to target (E:T) ratio of 1:4 to 4:1, such as at a ratio of 1:4, 1:3, 1:2 or 1:1.

[0168] In some embodiments, the lymphoid cells (e.g. T cell or NK cell) exhibits increased cytokine production. In some embodiments, the increased cytokine production occurs upon T cell stimulation. In some embodiments, T cell effector function is characterized by cytokine production. In some embodiments, natural killer cell effector function is characterized by cytokine production. In some embodiments, the 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 greater, in comparison to a cell that has not been delivered the epigenetic-modifying DNA- targeting system. In some embodiments, the cytokine production is production of IL-2. In some embodiments, the cytokine production is production of IL-2, IFN-gamma, TNF-alpha, or a combination thereof. In some embodiments, the T cell effector function is characterized by IL-2 production. In some embodiments, the cell (e.g. T cell) exhibits increased IL-2 production. In some embodiments, the T cell effector function is characterized by IFN-gamma production. In some embodiments, the cell (e.g. T cell) exhibits increased IFN-gamma production. In some embodiments, the T cell effector function is characterized by IL-2 production and IFN-gamma production. In some embodiments, the cell (e.g. T cell) exhibits increased IL-2 production and increased IFN-gamma production. In some embodiments, the T cell effector function is characterized by polyfunctional production of IL-2, IFN-gamma and TNF-alpha. In some 6037971-sf22474-20029.40 embodiments, the cell (e.g. T cell) exhibits increased IL-2, IFN-gamma and TNF-alpha production.

[0169] In some embodiments, the NK cell effector function is characterized by IL-2 production. In some embodiments, the cell (e.g. NK cell) exhibits increased IL-2 production. In some embodiments, the natural killer cell effector function is characterized by IFN-gamma production. In some embodiments, the cell (e.g. NK cell) exhibits increased IFN-gamma production. In some embodiments, the NK cell effector function is characterized by IL-2 production and IFN-gamma production. In some embodiments, the cell (e.g. NK cell) exhibits increased IL-2 production and increased IFN-gamma production. In some embodiments, the NK cell effector function is characterized by polyfunctional production of IL-2, IFN-gamma and TNF-alpha. In some embodiments, the cell (e.g. NK cell) exhibits increased IL-2, IFN-gamma and TNF-alpha production.

[0170] Suitable techniques for the measurement of the production or secretion of a soluble factor, such as a cytokine, are known in the art. Production and / or secretion of a soluble factor can be measured by determining the concentration or amount of the extracellular amount of the factor, or determining the amount of transcriptional activity of the gene that encodes the factor. Suitable techniques include, but are not limited to assays such as an immunoassay, an aptamer- based assay, a histological or cytological assay, an mRNA expression level assay, an enzyme linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibition assay or avidity assay, protein microarrays, high-performance liquid chromatography (HPLC), Meso Scale Discovery (MSD) electrochemiluminescence and bead based multiplex immunoassays (MIA). In some embodiments, the suitable technique may employ a detectable binding reagent that specifically binds the soluble factor.

[0171] In some embodiments, the cytokine production is measured as a percentage of cells being positive for the cytokine, for example as measured by intracellular cytokine staining (ICS) and flow cytometry. Intracellular cytokine staining (ICS) by flow cytometry is a technique well- suited for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing for the identification of cell populations that are positive or negative for production of a particular cytokine or for the separation of high producing and low producing cells based on a threshold. 6037971-sf22474-20029.40 ICS can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or with MHC multimers to access cytokine production in a particular subgroup of cells, making it an extremely 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.

[0172] In some embodiments, the cytokine production is measured as the amount of cytokine secreted from the cell, for example as measured by ELISA (enzyme-linked immunosorbent assay). ELISA is a plate-based assay technique designed for detecting and quantifying substances such as peptides, cytokines, antibodies and hormones. In an ELISA, the soluble factor, such as a cytokine, must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a detectable signal.

[0173] In some embodiments, the lymphoid cell (e.g., T or NK cell) effector function is characterized by activity that further comprises lymphoid cell (e.g., T or NK cell) proliferation. In some embodiments, the cell (e.g. T or NK cell) exhibits increased proliferation. In some embodiments, the increased proliferation occurs upon T cell stimulation. In some embodiments, the 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 greater, in comparison to a cell that has not been delivered the epigenetic-modifying DNA-targeting system. In some embodiments, the proliferation is measured as the increase in cell numbers before and after stimulation. In some embodiments, the increased proliferation is measured as the number of cells after stimulation in a cell population delivered with the epigenetic-modifying DNA-targeting system compared to the number of cells after stimulation in a cell population not delivered with the epigenetic- modifying DNA-targeting system. In some embodiments, the cell (e.g. T or NK cell) does not exhibit increased proliferation.

[0174] In some embodiments, the lymphoid cell (e.g., T or NK cell) effector function is characterized by activity that further comprises killing of target cells. In some embodiments, the cell (e.g. T or NK cell) exhibits increased killing of target cells. In some embodiments, the increased killing of target cells occurs upon T cell stimulation. In some embodiments, the stimulation is performed by contacting the cells (e.g. T or NK cells) with target cells. In some embodiments, lymphoid cell (e.g., T or NK cell)s are incubated with antigen-expressing target cells at ratios between 4:1 and 1:4, inclusive, such as at ratios of 1:4, 1:3, 1:2 or 1:1. In some 6037971-sf22474-20029.40 embodiments, the killing of target cells 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 greater, in comparison to a cell that has not been delivered the epigenetic-modifying DNA-targeting system. In some embodiments, the killing is measured as the ability of the cells to kill target cells when contacted by the target cells.

[0175] In some embodiments, the natural killer cell effector function is characterized by activity that further comprises killing of target cells. In some embodiments, the cell (e.g. natural killer cell) exhibits increased killing of target cells. In some embodiments, the increased killing of target cells occurs upon natural killer cell stimulation. In some embodiments, the stimulation is performed by contacting the cells (e.g. natural killer cells) with target cells. In some embodiments, natural killer cells are incubated with antigen-expressing target cells at ratios between 4:1 and 1:4, inclusive, such as at ratios of 1:4, 1:3, 1:2 or 1:1. In some embodiments, the killing of target cells 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 greater, in comparison to a cell that has not been delivered the epigenetic-modifying DNA-targeting system. In some embodiments, the killing is measured as the ability of the cells to kill target cells when contacted by the target cells.

[0176] Killing of target cells can be measured by any suitable assay, for example as described herein in the Examples. In some embodiments, the killing is measured in an in vitro assay, wherein cells delivered with the epigenetic-modifying DNA-targeting system are co- cultured with the target cells, and the number of target cells are measured over time. In some embodiments, reduced numbers and / or proliferation of the target cells are indicative of target cell killing. The cytolytic activity can be measured by directly or indirectly measuring the target cell number over time. For example, the target cells may be incubated with a detectable marker prior to being incubated with antigen receptor (e.g. CAR)-expressing cells, such a marker that is detectable then the target cell is lysed, or a detectable marker that is detectable in viable target cells. These readouts provide direct or indirect of target cell number and / or target cell death, and can be measured at different time points during the assay. A reduction of target cell number and / or an increase of target cell death indicate 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, flow cytometric assays that use fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE), PKH-2, and PKH-26. 6037971-sf22474-20029.40

[0177] In some embodiments, the lymphoid cell (e.g., T or NK cell) effector function is characterized by activity that further comprises lymphoid cell (e.g., T or NK cell) persistence. In some embodiments, the cell (e.g. T or NK cell) exhibits increased persistence (e.g. T or NK cell persistence). In some embodiments, persistence relates to the ability of cells to remain present and / or maintain an immune response in the presence of target cells. In some embodiments, persistence can be measured in vitro or in vivo, for example after administration of cells to a subject. Persistence can be measured by any suitable method, for example as described in Section IV.

[0178] In certain embodiments, the ability of lymphoid cell (e.g., T or NK cell)s to persist can be measured as a pharmacokinetic property of the cell composition following its administration to a subject. In some embodiments, the pharmacokinetic parameter can include the exposure, number, concentration, persistence and proliferation. In some cases, pharmacokinetics can be assessed by measuring such parameters as the maximum (peak) plasma concentration (Cmax), the peak time (i.e. when maximum plasma concentration (Cmax) occurs; Tmax), the minimum plasma concentration (i.e. the minimum plasma concentration between doses of a therapeutic agent, e.g., CAR+ T cells; Cmin), the elimination half-life (T1 / 2) and area under the curve (i.e. the area under the curve generated by plotting time versus plasma concentration of the therapeutic agent CAR+ T cells; AUC), following administration. The parameters of the administered engineered lymphoid cells (e.g., NK or T cells) can be measured in samples of blood from the subject. For example, nucleic acid-based methods, such as quantitative PCR (qPCR) or flow cytometry-based methods, or other assays, such as an immunoassay, ELISA, or chromatography / mass spectrometry-based assays can be used.

[0179] In some aspects, nucleic acid-based methods, such as quantitative PCR (qPCR), is used to assess the quantity of cells expressing the antigen receptor (e.g., CAR-expressing cells administered for T cell based therapy) in the blood or serum or organ or tissue sample (e.g., disease site, e.g., tumor sample) of the subject. In some aspects, persistence is quantified as copies of 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 the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or lymphoid cell (e.g., T or NK cell)s per microliter of the 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 nucleic acids encoding the antigen receptor, 6037971-sf22474-20029.40 and / or other components or elements of the plasmid and / or vector, including regulatory elements, e.g., promoters, transcriptional and / or post-transcriptional regulatory elements or response elements, or markers, e.g., surrogate markers. In some embodiments, the primers can be specific for regulatory elements, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0180] In some embodiments, any of the phenotypes described herein, such as increased IL- 2 production, are observed after stimulation (e.g. T cell stimulation). In some embodiments, any of the phenotypes described herein, such as increased IL-2 production, increased IFN-gamma production, increased IL-2 production and increased IFN-gamma production, increased IL-2, IFN-gamma and TNF-alpha production, increased proliferation or proliferation that is not increased, increased killing of target cells, and / or increased persistence, are observed after stimulation (e.g. T cell stimulation).

[0181] In some embodiments, the phenotype, such as any phenotype described herein, including increased lymphoid cell (e.g., T or NK cell) effector function, occurs 48 hours or more after the transient delivery of the epigenetic-modifying DNA-targeting system to the lymphoid cell (e.g., T or NK cell). In some embodiments, the phenotype, such as increased lymphoid cell (e.g., T or NK cell) effector function, occurs up to 6 days, up to 9 days, up to 12 days, up to 15 days, up to 21 days, up to 28 days, up to 35 days, up to 42 days, up to 49 days, up to 56 days, up to 63 days, up to 71 days or more after the transient delivery of the epigenetic-modifying DNA- targeting system to the lymphoid cell (e.g., T or NK cell).

[0182] In some aspects, the phenotype is one that is characterized by a cell surface phenotype of the cells. In some embodiments, the phenotype comprises expression of IL-2+. In some embodiments, the phenotype comprises expression of one or more cell-surface markers selected from IL-2+, TNFa+, IFNg+, or any combination thereof. In some embodiments, the phenotype is a phenotype in a lymphoid cell (e.g., T or NK cell), such as a CD3+ T cell, which may be a CD4+ T-cell or CD8+ T cell. Thus, in some embodiments, the phenotype comprises expression of one or more cell-surface markers selected from CD3+, CD4+, CD8+, IL-2+, TNFa+, IFNg+, or any combination thereof. In some aspects, the phenotype comprises expression of IL-2+. In some embodiments, the phenotype comprises expression of IL-2- and IFNg+.

[0183] It is understood that embodiments of the provided epigenetic-modifying DNA- targeting systems are not limited to increasing (e.g., activating) expression of the IL-2 gene or 6037971-sf22474-20029.40 regulatory element thereof and promoting phenotypes in T cells. In addition to T cells, lymphoid cells can include NK cells, NKT cells, any cells that have been differentiated from stem cells into such lymphoid cells and / or have been differentiated from progenitor cells, such as common lymphoid progenitors (CLPs). In some embodiments, the lymphoid cells are differentiated from stem cells, such as hematopoietic stem or progenitor cells, or progenitor cells. In some embodiments, the lymphoid cells are trans-differentiated from a non-pluripotent cell of non-hematopoietic lineage.

[0184] In some embodiments, the lymphoid cell for modulation is an isolated or enriched population of lymphoid immune cells, such as a population isolated or enriched in T, NK and / or NKT cells. In some embodiments, the cells for modulation are isolated or enriched T cells. In some embodiments, the cells for modulation are isolated or enriched NK cells. In some embodiments, the cells for modulation are isolated or enriched NK T cells. In some embodiments, isolated or enriched populations or subpopulations of immune cells comprising T, NK, and / or NKT cells for modulation can be obtained from a unit of blood using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one embodiment, T, NK or NKT cells from the circulating blood of an individual are obtained by apheresis and separated from other nucleated white blood cells, 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 from a subject, such as a human subject.

[0185] In some embodiments, the lymphoid cells for modulation are differentiated in vitro from a stem cell or progenitor cell. In some embodiments, the lymphoid cells, such as T, NK or NKT cells or lineages thereof, can be differentiated from a stem cell, a hematopoietic stem or progenitor cell (HSC), or a progenitor cell. The progenitor cell can be a CD34+ hemogenic endothelium cell, a multipotent progenitor cell, a T cell progenitor, an NK cell progenitor, or an NKT cell progenitor. In some embodiments, the progenitor cells is a lymphoid progenitor cells such as a common lymphoid progenitor cell, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cell, T progenitor cell, NK progenitor cell or NKT progenitor cell. The stem cell can be a pluripotent stem cell, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). The iPSC is a non-naturally occurring reprogrammed pluripotent cell. Once the cells of a subject have been reprogrammed to a pluripotent state, the cells can then be programmed or differentiated to a desired cell type or subtypes, such as T, NK, or NKT 6037971-sf22474-20029.40 cells.

[0186] In some embodiments, the iPSC is differentiated to a T, NK or NKT cells by a multi- stage differentiation platform wherein cells from various stages of development can be induced to assume a hematopoietic phenotype, ranging from mesodermal stem cells, to fully differentiated T, NK or NKT cells (See e.g. U.S. Pat. No.10,626,372).

[0187] In some embodiments, the population or subpopulation of lymphoid cells is trans- differentiated in vitro from a non-pluripotent cell of non-hematopoietic fate to a hematopoietic lineage cell or from a non-pluripotent cell of a first hematopoietic cell type to a different hematopoietic cell type, which can be a T, NK, or NKT progenitor cell or a fully differentiated specific type of immune cell, such as T, NK, or NKT cell (See e.g. U.S. Pat. No.9,376,664 and U.S. application Ser. No.15 / 072,769, the disclosure of which is incorporated herein in their entirety). In some embodiments, the non-pluripotent cell of non-hematopoietic fate is a somatic cell, such as a skin fibroblast, an adipose tissue-derived cell and a human umbilical vein endothelial cell (HUVEC). Somatic cells useful for trans-differentiation may be immortalized somatic cells.

[0188] Various strategies are being pursued to induce pluripotency, or increase potency, 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 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., 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 hereby incorporated by reference in their entireties.

[0189] It is understood that a cell that is positive (+) for a particular cell surface marker is a cell that expresses the marker on its surface at a level that is detectable. Likewise, it is understood that a cell that is negative (-) for a particular cell surface marker is a cell that expresses the marker on its surface at a level that is not detectable. Antibodies and other binding entities can be used to detect expression levels of marker proteins to identify or detect a given 6037971-sf22474-20029.40 cell surface marker. Suitable antibodies may include polyclonal, monoclonal, fragments (such as Fab fragments), single chain antibodies and other forms of specific binding molecules. Antibody reagents for cell surface markers above are readily known to a skilled artisan. A number of well-known methods for assessing expression level of surface markers or proteins may be used, such as detection by affinity-based methods, e.g., immunoaffinity-based methods, e.g., in the context of surface markers, such as by flow cytometry. In some embodiments, the label is a fluorophore and the method for detection or identification of cell surface markers on cells (e.g. T or NK cells) is by flow cytometry. In some embodiments, different labels are used for each of the different markers by 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 the binding of the antibody to the marker.

[0190] In some embodiments, a cell (e.g. T or NK cell) is positive (pos or +) for a particular marker if there is detectable presence on or in the cell of a particular marker, which can be an intracellular marker or a surface marker. In some embodiments, surface expression is positive if staining by flow cytometry is detectable at a level substantially above the staining detected carrying out the same procedures with an isotype-matched control under otherwise identical conditions and / or at a level substantially similar to, or in some cases higher than, a cell known to be positive for the marker and / or at a level higher than that for a cell known to be negative for the marker.

[0191] In some embodiments, a cell (e.g. T or NK cell) is negative (neg or -) for a particular marker if there is an absence of detectable presence on or in the cell of a particular marker, which can be an intracellular marker or a surface marker. In some embodiments, surface expression is negative if staining is not detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedures with an isotype-matched control under otherwise identical conditions and / or at a level substantially lower than a cell known to be positive for the marker and / or at a level substantially similar to a cell known to be negative for the marker.

[0192] In some aspects, the phenotype can be characterized by one or more functions of the cells. In some aspects, the phenotype is characterized by polyfunctional activity of the lymphoid cells (e.g., T or NK cells) to produce more than one lymphoid cell (e.g., T or NK cell) stimulatory cytokine, such as determined in a polyfunctional cytokine secretion assay following stimulation of the lymphoid cells with a stimulatory agent. In some embodiments, the lymphoid 6037971-sf22474-20029.40 cell (e.g., T or NK cell) is polyfunctional for producing two or more cytokines. In some embodiments, a lymphoid cell (e.g., T or NK cell) is polyfunctional for producing two or more cytokines selected from among interferon-gamma (IFN-gamma), interleukin 2 (IL-2) and TNF- alpha. In some embodiments, a polyfunctional lymphoid cell (e.g., T or NK cell) produces IFN- gamma, IL-2, and TNF-alpha. In some embodiments, the stimulatory agent is a non-specific or non-antigen-dependent lymphoid cell stimulatory agent. In some embodiments, the non-specific or non-antigen dependent lymphoid 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 lymphoid cell stimulatory agent contains PMA / ionomycin.

[0193] In particular embodiments, the production of one or more cytokines is measured, detected, and / or quantified by intracellular cytokine staining. Intracellular cytokine staining (ICS) by flow cytometry is a technique well-suited for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing for the identification of cell populations that are positive or negative for production of a particular cytokine or for the separation of high producing and low producing cells based on a threshold. In some embodiments, as described above, the stimulation can be performed using nonspecific stimulation, e.g., is not an antigen- specific stimulation. For example, PMA / ionomycin can be used for nonspecific cell stimulation. ICS can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or with MHC multimers to access cytokine production in a particular subgroup of cells, making it an extremely 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, the assays to assay polyfunctional cytokine secretion of multiple cytokines, can include multiplexed assays or other assays to assess polyfunctionality (see, e.g., Xue et al., (2017) Journal for ImmunoTherapy of Cancer 5:85). 2. Target sites for increasing transcription of IL-2

[0194] In some embodiments, delivery of the DNA-targeting system increases transcription of IL-2 gene or regulatory element thereof. In some embodiments, provided herein are target sites for IL-2, for which increased transcription promotes a phenotype in a cell. In some 6037971-sf22474-20029.40 embodiments, the target sites can be located on the IL-2 gene body or be a regulatory DNA element of IL-2 described herein. In some embodiments, provided herein are target sites for the IL-2 gene or regulatory DNA element thereof described herein for which increased transcription promotes increased lymphoid cell (e.g., T or NK cell) effector function. In some embodiments, the increased transcription promotes increased lymphoid cell (e.g., T or NK cell) effector function upon cell stimulation (e.g., T cell stimulation).

[0195] In some embodiments, each of the target site of the epigenetic-modifying DNA- targeting system is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4:122,451,261-122,593,946. In some embodiments, each target site is in a putative regulatory region of the IL-2 gene, wherein the putative regulatory region is characterized by having one or more of an epigenetic mark, a regulatory feature or a transcription factor motif. In some embodiments, the putative regulatory region is a promoter or an enhancer. In some embodiments, the target site is in a promoter or enhancer.

[0196] 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 target at least a first target site of the IL-2 gene or regulatory element thereof and a second target site of the IL-2 gene or regulatory element thereof. In some embodiments, the second target site is different from the first target site. In some embodiments, the second target site is the same as the first target site.

[0197] In some embodiments, the plurality of DNA-targeting modules target at least a first target site, a second target site, and a third target site of the IL-2 gene or regulatory element thereof. In some embodiments, the first target site, the second target site and the third target site are all different from each other.

[0198] In some embodiments, the DNA-targeting system targets a combination of target sites for the IL-2 gene or a regulatory element described herein for transcriptional activation. In some embodiments, the target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4:122,451,261-122,593,946. In some embodiments, the target site is located in the gene and / or a regulatory DNA element of the gene. In some embodiments, the target site is located on the IL-2 gene body. In some embodiments, the target site is located on the IL-2 locus. In some embodiments, the target site is a regulatory element located on the IL-2 locus. In some embodiments, a regulatory element is a sequence to which a gene regulatory protein may bind and affect transcription of the gene. In some embodiments, the regulatory 6037971-sf22474-20029.40 DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of a gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of an IL-2 gene.

[0199] In some embodiments, within the IL-2 locus, there are seven identified regions of interest. In some embodiments, a region of interest exhibits characteristics indicative of regulatory regions. In particular embodiments, the characteristic can be epigenetic marks. In particular embodiments, the characteristics can be regulatory features. In particular embodiments, the characteristics can be relevant transcription factor (TF) motifs.

[0200] In some embodiments, the target site is located on the IL-2 gene body. In some embodiments, the target site is within 100 kb of the IL-2 transcription start site (TSS). In some embodiments, the target site is 50 to 150 kilobases (kb) upstream of the IL-2 gene and the IL-2 transcription start site (TSS). In some embodiments, the target site is a regulatory DNA element of the IL-2 gene and is 50 to 150 kb upstream of the IL-2 gene and the IL-2 TSS.

[0201] In some embodiments, each target site of the epigenetic-modifying DNA-targeting system is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410- 122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some embodiments, each target site of the epigenetic-modifying DNA-targeting system is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300- 122,544,050.

[0202] In some embodiments, the target site is located on the IL-2 gene body. In some embodiments, the target site is within 100 kb of the IL-2 transcription start site (TSS). In some embodiments, the target site is located within GRCh38 (hg38) chr4: 122,451,000-122,460,000. In some embodiments, the target site is located in a region of interest identified as Region 1. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 12, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 6037971-sf22474-20029.40 some embodiments, the target site is a contiguous portion of SEQ ID NO: 12 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:12. In some embodiments the target site is set forth in SEQ ID NO:12. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 14, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 14 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:14. In some embodiments the target site is set forth in SEQ ID NO:14. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 16, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 16 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:16. In some embodiments the target site is set forth in SEQ ID NO:16. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 18, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 18 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:18. In some embodiments the target site is set forth in SEQ ID NO:18. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 20, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 20 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:20. In some embodiments, the target site is set 6037971-sf22474-20029.40 forth in SEQ ID NO:20. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 43, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 43 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:43. In some embodiments the target site is set forth in SEQ ID NO:43. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 45, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 45 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:45. In some embodiments the target site is set forth in SEQ ID NO:45. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 47, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 47 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:47. In some embodiments the target site is set forth in SEQ ID NO:47.

[0203] In some embodiments, the target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,465,000-122,472,000. In some embodiments, the target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,479,410-122,482,750. In some embodiments, the target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,488,840-122,491,890.

[0204] In some embodiments, the target site is 50 to 150 kilobases (KB) upstream of the IL- 2 gene and the IL-2 transcription start site (TSS). In some embodiments, the target site is a regulatory DNA element of the IL-2 gene and is 50 to 150 kb upstream of the IL-2 gene and the 6037971-sf22474-20029.40 IL-2 TSS. In some embodiments, the regulatory DNA element of is a distal regulatory element of a IL-2 gene. In some embodiments, the regulatory DNA element is an upstream regulatory element of the IL-2 gene. In some embodiments, the target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,507,000-122,508,985. In some embodiments, the target site is located in a region of interest identified as Region 4. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 22, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 22 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:22. In some embodiments the target site is set forth in SEQ ID NO:22. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 24, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 24 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:24. In some embodiments the target site is set forth in SEQ ID NO:24. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 26, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 26 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:26. In some embodiments the target site is set forth in SEQ ID NO:26. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 49, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 49 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In 6037971-sf22474-20029.40 some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:49. In some embodiments the target site is set forth in SEQ ID NO:49. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 51, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 51 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:51. In some embodiments the target site is set forth in SEQ ID NO:51. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 53, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 53 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:53. In some embodiments the target site is set forth in SEQ ID NO:53. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 55, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 55 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:55. In some embodiments the target site is set forth in SEQ ID NO:55. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 186, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 186 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is within a sequence that comprises the sequence set forth in SEQ ID NO: 186. In some embodiments, the target site is set forth in SEQ ID NO: 186. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 187, a contiguous 6037971-sf22474-20029.40 portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 187 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is within a sequence that comprises the sequence set forth in SEQ ID NO: 187. In some embodiments, the target site is set forth in SEQ ID NO: 187. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 188, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 188 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is within a sequence that comprises the sequence set forth in SEQ ID NO: 188. In some embodiments, the target site is set forth in SEQ ID NO: 188.

[0205] In some embodiments, the target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,539,300-122,544,050. In some embodiments, the target site is located in a region of interest identified as Region 5. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 28, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 28 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:28. In some embodiments the target site is set forth in SEQ ID NO:28. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 30, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 30 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:30. In some embodiments the target site is set forth in SEQ ID NO:30. In some embodiments, the target sites comprises a sequence set forth in 6037971-sf22474-20029.40 SEQ ID NO: 32, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 32 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:32. In some embodiments the target site is set forth in SEQ ID NO:32. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 57, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 57 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:57. In some embodiments the target site is set forth in SEQ ID NO:57. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 59, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 59 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:59. In some embodiments the target site is set forth in SEQ ID NO:59.

[0206] In some embodiments, the target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,576,890-122,579,315. In some embodiments, the target site is located in a region of interest identified as Region 6. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 34, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 34 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:34. In some embodiments the target site is set forth in SEQ 6037971-sf22474-20029.40 ID NO:34. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 36, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 36 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:36. In some embodiments the target site is set forth in SEQ ID NO:36. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 38, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 38 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:38. In some embodiments the target site is set forth in SEQ ID NO:38. In some embodiments, the target sites comprises a sequence set forth in SEQ ID NO: 40, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 40 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments the target site is within a sequence that comprises the sequence set forth in SEQ ID NO:40. In some embodiments, the target site is set forth in SEQ ID NO:40.

[0207] Exemplary target sites for IL-2 are shown in Table 1. Table 1. Regions of Interest and Target Sites6037971-sf22474-20029.40

[0208] In some embodiments, at least two of the plurality of DNA targeting modules of the epigenetic-modifying DNA-targeting system targets the same target site. In some embodiments, the at least two same target sites of the epigenetic-modifying DNA-targeting system are in the same target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840- 122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some embodiments, the at least two same target sites of the epigenetic-modifying DNA-targeting system are in the same target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0209] In some embodiments, the DNA-targeting system targets a first target site for IL-2 and a second target site for IL-2, wherein the first and second target sites are the same. In particular embodiments, each of the same target site for IL-2 comprises the sequence set forth in SEQ ID NO:12. In particular embodiments, each of the same target site for IL-2 comprises the sequence set forth in SEQ ID NO: 24. In particular embodiments, each of the same target for IL-2 site comprises the sequence set forth in SEQ ID NO: 26.

[0210] In some embodiments, at least two of the plurality of DNA targeting modules of the epigenetic-modifying DNA-targeting system targets a different target site. In some 6037971-sf22474-20029.40 embodiments, the at least two different target sites of the epigenetic-modifying DNA-targeting system are in two different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000- 122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some embodiments, the at least two different target sites of the epigenetic-modifying DNA-targeting system are in two different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0211] In some embodiments, the DNA-targeting system targets a first target site and a second target site, wherein the first and second target sites are different. In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO 12 and a second target site for IL-2 comprises the sequence set forth in SEQ ID NO:24. In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO 12 and a second target site for IL-2 comprises the sequence set forth in SEQ ID NO:26. In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO 12 and a second target site for IL-2 comprises the sequence set forth in SEQ ID NO:28. In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO 12 and a second target site for IL-2 comprises the sequence set forth in SEQ ID NO:38. In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO 24 and a second target site for IL-2 comprises the sequence set forth in SEQ ID NO:26. In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO 12 and a second target site for IL-2 comprises the sequence set forth in SEQ ID NO:28. In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO 12 and a second target site for IL-2 comprises the sequence set forth in SEQ ID NO:38.

[0212] Exemplary paired combinations of target sites are shown in Table 2. Table 2. Exemplary Paired Combinations of Target Sites6037971-sf22474-20029.40

[0213] In some embodiments, the DNA-targeting system targets a first target site, a second target site and a third target site, wherein all three target sites are different. In some embodiments, at least three of the plurality of DNA targeting modules of the epigenetic- modifying DNA-targeting system targets a different target site. In some embodiments, the at least three different target sites of the epigenetic-modifying DNA-targeting system are in three different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840- 122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some embodiments, the at least three different target sites of the epigenetic-modifying DNA-targeting system are in three different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0214] In some embodiments, the DNA-targeting system targets a first target site, a second target site, a third target site and a fourth target site, wherein all four target sites are different. In some embodiments, at least four of the plurality of DNA targeting modules of the epigenetic- modifying DNA-targeting system targets a different target site. In some embodiments, the at least four different target sites of the epigenetic-modifying DNA-targeting system are in four different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840- 122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some embodiments, the at least four different target sites of the epigenetic-modifying DNA-targeting system are in four different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group 6037971-sf22474-20029.40 consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0215] In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO: 12 (GACTTAGTGCAATGCAAGAC), a second target site for IL-2 comprises the sequence set forth in SEQ ID NO: 24 (CTCTCTCTGCAGACAGGGCA), a third target site for IL-2 comprises the sequence set forth in SEQ ID NO: 28 (GGCAGGGTAGAGAAGTAGAG) and a fourth target site for IL-2 comprises the sequence set forth in SEQ ID NO:38 (GGAAATGACATGCTTGAAGT).

[0216] In some embodiments, the DNA-targeting system targets a first target site, a second target site, a third target site, a fourth target site and a fifth target site, wherein all five target sites are different. In some embodiments, at least five of the plurality of DNA targeting modules of the epigenetic-modifying DNA-targeting system targets a different target site. In some embodiments, the at least five different target sites of the epigenetic-modifying DNA-targeting system are in five different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000- 122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315. In some embodiments, the at least five different target sites of the epigenetic-modifying DNA-targeting system are in five different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

[0217] In particular embodiments, a first target site for IL-2 comprises the sequence set forth in SEQ ID NO: 12 (GACTTAGTGCAATGCAAGAC), a second target site for IL-2 comprises the sequence set forth in SEQ ID NO: 24 (CTCTCTCTGCAGACAGGGCA), a third target site for IL-2 comprises the sequence set forth in SEQ ID NO: 26 (GGCAGGGTAGAGAAGTAGAG) , a fourth target site for IL-2 comprises the sequence set forth in SEQ ID NO: 28 (GGCAGGGTAGAGAAGTAGAG) and a fifth target site for IL-2 comprises the sequence set forth in SEQ ID NO:38 (GGAAATGACATGCTTGAAGT).

[0218] In some embodiments, delivery of the DNA-targeting system increases expression (e.g. transcription) of the IL-2 gene or regulatory element thereof. In some embodiments, the increase in gene expression in a cell (e.g. T or NK cell) is about a log2 fold change of greater 6037971-sf22474-20029.40 than 1.0. In some embodiments, the increase in IL-2 expression in a cell (e.g. T or NK cell) is about a log2 fold change of greater than 1.0. For instance, the log2 fold change is greater than at or about 1.5, at or about 2.0, at or about 2.5, at or about 3.0, at or about 4.0, at or about 5.0, at or about 6.0, at or about 7.0, at or about 8.0, at or about 9.0, at or about 10.0 or any value between any of the foregoing compared to the level of expression of the IL-2 gene in a control cell. C. CRISPR / Cas-Based DNA-Targeting Systems and DNA-Binding Domains

[0219] Provided herein are multiplexed epigenetic-targeting DNA-targeting systems based on CRISPR / Cas systems, i.e. CRISPR / Cas-based DNA-targeting systems, that are able to bind to a target site for the IL-2 gene or regulatory element thereof, or to a combination of target sites, e.g. for a combination of target sites within the IL-2 gene or regulatory element thereof. Exemplary target sites include any, or any combination, as described in above in Section I.B. In some embodiments, the CRISPR / Cas DNA-binding domain is nuclease inactive, such as includes a dCas (e.g. dCas9) so that the system binds to the target site for the IL-2 gene or regulatory element thereof without mediating nucleic acid cleavage at the target site. The CRISPR / Cas-based DNA-targeting systems may be used to activate or increase expression of the IL-2 gene or regulatory element thereof in a cell, such as a lymphoid cell (e.g., T or NK cell). In some embodiments, the CRISPR / Cas-based DNA-targeting system can include any known Cas enzyme, and generally a nuclease-inactive or dCas. In some embodiments, the CRISPR / Cas-based DNA-targeting system includes a fusion protein of a nuclease-inactive Cas protein or a variant thereof and an effector domain, and at least one gRNA. In some embodiments, the effector domain increases transcription of the IL-2 gene or regulatory element thereof (e.g. the effector domain is a transcriptional activator, such as any described in Section I.E.1).

[0220] The CRISPR system (also known as CRISPR / Cas system, or CRISPR-Cas system) refers to a conserved microbial nuclease system, found in the genomes of bacteria and archaea, that provides a form of acquired immunity against invading phages and plasmids. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), refers to loci containing multiple repeating DNA elements that are separated by non-repeating DNA sequences called spacers. Spacers are short sequences of foreign DNA that are incorporated into the genome between CRISPR repeats, serving as a “memory” of past exposures. Spacers encode the DNA-targeting portion of RNA molecules that confer specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes, 6037971-sf22474-20029.40 which can act as RNA-guided nucleases for mediating the cleavage, as well as non-protein coding DNA elements that encode RNA molecules capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0221] In Type II CRISPR / Cas systems with 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) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The trans- activating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas / RNA RNP complex.

[0222] Naturally occurring CRISPR / Cas systems, such as those with Cas9, have been engineered to allow efficient programming of Cas / RNA RNPs to target desired sequences in cells of interest, both for gene-editing and modulation of gene expression. The tracrRNA and crRNA have been engineered to form a single chimeric guide RNA molecule, commonly referred to as a guide RNA (gRNA), for example as described in WO 2013 / 176772, WO 2014 / 093661, WO 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 chosen by a user to target the Cas / gRNA RNP complex to a desired locus, e.g. a desired target site in the target gene.

[0223] Cas proteins have also been engineered to be catalytically inactivated or nuclease inactive to allow targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as deactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. An exemplary deactivated Cas9 (dCas9) derived from S. pyogenes contains silencing mutations of the RuvC and HNH nuclease domains (D10A and H840A), for example as described in WO 2013 / 176772, WO 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 variants derived from the Cas12 system (i.e. Cpf1) are described, for example in WO 2017 / 189308 and Zetsche, B. et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologues, and can be mutated to produce inactive variants, for example as described in Zetsche, B. et al. Cell 163(3):759-71 (2015). 6037971-sf22474-20029.40

[0224] dCas-fusion proteins with transcriptional and / or epigenetic regulators have been used as a versatile platform for ectopically regulating gene expression in target cells. These include fusion of a Cas with an effector domain, such as a transcriptional activator. 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 the Herpes simplex virus) can result in robust induction of gene expression. A variety of dCas-fusion proteins with effector domains can be engineered for regulation of gene expression, for example as described 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. Nat. Biotechnol.31, 833–838 (2013), Maeder, M. L. et al. Nat. Methods 10, 977–979 (2013), Gilbert, L. A. et al. Cell 154(2):442-451 (2013), and Nuñez, J.K. et al. Cell 184(9):2503-2519 (2021).

[0225] In some aspects, provided is a DNA-targeting system comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or variant thereof, and an effector domain for increasing transcription or inducing transcriptional activation (i.e. a transcriptional activator) when targeted to the IL-2 gene or regulatory element in a cell (e.g. a lymphoid cell (e.g., T or NK cell)). In some aspects, provided is a DNA-targeting system comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or variant thereof, and an effector domain for increasing transcription or inducing transcriptional activation (i.e. a transcriptional activator) when targeted to the IL-2 gene or regulatory element thereof in a cell (e.g. a lymphoid cell (e.g., T or NK cell)). In some embodiments, the dCas protein is any suitable dCas protein, such as any described in Section I.C.1. In some embodiments, the dCas protein is a dCas9 protein, such as a dSpCas9 or dSaCas9. In some embodiments, the at least one effector domain is any suitable transcriptional activator effector domain, such as any described in Section I.E.1, such as VP64. In some embodiments, the epigenetic mark includes Histone H3K27 acetylation. In some embodiments, the effector domain catalyzes acetylation of histone H3 lysine 27 at the target site or is able to recruit an enzyme that catalyzes acetylation of histone H3 lysine 27 at the target site. In some embodiments, the enzyme that catalyzes the acetylation is an acetyltransferase.

[0226] In some embodiments, the at least one effector domain is VP64. In some embodiments, the fusion protein is a dCas9-VP64 fusion protein, for example as described in Section I.F. In such embodiments, the DNA-targeting system also includes one or more gRNAs 6037971-sf22474-20029.40 (e.g. as described in Section I.C.2), provided in combination or as a complex with the dCas protein or variant thereof, for targeting of the DNA-targeting system to the target site of the IL-2 gene or regulatory element thereof. In some embodiments, the fusion protein is guided to a specific target site sequence of the IL-2 gene or regulatory element thereof by the guide RNA, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of the IL-2 gene or regulatory element thereof. In some embodiments, a combination of gRNAs guides the fusion protein to a combination of target site sequences in a combination of target sites, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of the combination of target sites found on the IL-2 gene or regulatory element thereof. Any of a variety of effector domains that increase or activate transcription can be used as described further below. 1. CRISPR / Cas-Based DNA-Binding Domains

[0227] In some aspects, the DNA-binding domain comprises a CRISPR-associated (Cas) protein or variant thereof, or is derived from a Cas protein or variant thereof. In particular embodiments herein, the Cas protein is nuclease-inactive (i.e. is a dCas protein).

[0228] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e. 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. single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Type V CRISPR system. In some embodiments, the Cas protein is derived from a Cas12 protein (i.e. Cpf1) or variant thereof, for example as described 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 derived from a Cas9 protein or variant thereof, for example as described in 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. Nat. Biotechnol.31, 833–838 (2013). Various CRISPR / Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S.B. et al. Exp. Mol. Med.51, 1–11 (2019), Zhang, F. Q. Rev. Biophys.52, E6 (2019), and Makarova K.S. et al. Methods Mol. Biol.1311:47-75 (2015). 6037971-sf22474-20029.40

[0229] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule, or 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 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.

[0230] Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to: Cas proteins identified in 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 degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree; Clostridium botulinum Ba4 str.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 subsp. bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. 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 sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737). 6037971-sf22474-20029.40

[0231] In some aspects, the Cas protein is a variant that lacks nuclease activity (i.e. is a dCas protein). In some embodiments, the Cas protein is mutated so that nuclease activity is reduced or eliminated. Such Cas proteins are referred to as deactivated Cas or dead Cas (dCas) or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or that is a deactivated Cas9 (dCas9, or iCas9) protein.

[0232] In some embodiments, the Cas9 protein or a variant thereof is derived from a Staphylococcus aureus Cas9 (SaCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) that comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:64. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0233] In some embodiments, the Cas9 protein or variant thereof is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein that comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO:62. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:63, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. 2. Guide RNAs (gRNAs)

[0234] In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (also known as a spacer sequence or a guide sequence) that is capable of hybridizing to the target site or is complementary to the target site, such as any target site described herein, for example, any target site in a genome. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein. In some embodiments, a gRNA specific to a target locus of interest (e.g. a regulatory DNA element of a IL-2locus) is used to recruit an RNA- guided protein (e.g. a Cas protein) or variant thereof or a fusion protein comprising such RNA- guided protein (e.g., a Cas polypeptide), to the target site.

[0235] In some embodiments, the Cas protein (e.g. dCas9) is provided in combination or as a complex with one or more guide RNA (gRNA). In some aspects, the gRNA is a nucleic acid 6037971-sf22474-20029.40 that promotes the specific targeting or homing of the gRNA / Cas RNP complex to the target site of the IL-2 gene or regulatory element thereof, such as any described above in Section I.B. In some embodiments, a target site of a gRNA may be referred to as a protospacer.

[0236] Provided herein are gRNAs, such as gRNAs that target or bind to a target site for IL- 2 gene or regulatory element thereof, such as any described herein, for example in Section I.B. Provided herein are gRNAs, such as gRNAs that target or can bind to a regulatory DNA element of a IL-2 locus. In some embodiments, the gRNAs bind to a target site that is located in the IL-2 gene and / or a regulatory DNA element of the IL-2 gene. In some embodiments, the gRNAs bind to a target site that is located in the IL-2 gene. In some embodiments, the gRNAs bind to a target site that is located in a regulatory DNA element of the IL-2 gene.

[0237] In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e. a spacer sequence or a guide sequence) that is capable of hybridizing to the target site, or that is complementary to the target site, such as any target site described herein. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein.

[0238] In some aspects, a “gRNA molecule” is a nucleic acid that promotes the specific targeting or homing of a gRNA molecule / Cas9 molecule complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. In general, a spacer sequence of the guide RNA, is any polynucleotide sequences comprising at least a sequence portion that has sufficient complementarity with a target polynucleotide sequence, such as the at the IL-2 locus in humans, to hybridize with the target sequence at the target site and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, in the context of formation of a CRISPR complex, “target sequence” is to a sequence to which a spacer sequence is designed to have complementarity, where hybridization between the target sequence and a spacer sequence of the guide RNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. Generally, a spacer sequence is selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0239] In some embodiments, a guide RNA (gRNA) specific to a target locus of interest (e.g. at the IL-2 locus in humans) is used with RNA-guided nucleases or variants thereof, e.g., 6037971-sf22474-20029.40 nuclease-inactive Cas variants, to target the provided DNA-targeting system to the target site or target position. Methods for designing gRNAs and exemplary spacer sequences are known. Exemplary gRNA structures that can be associated with particular RNA-guided nucleases or variants thereof, e.g., nuclease-inactive Cas variants, with particular domains and scaffold regions, are also known. In some aspects, gRNA molecules comprise a scaffold sequence, e.g., sequences that can be complexed with the Cas protein. In some aspects, the scaffold sequence is specific for the Cas protein.

[0240] In some embodiments, the gRNAs provided herein are chimeric gRNAs. In general, gRNAs can be unimolecular (i.e. composed of a single RNA molecule), or modular (comprising more than one, and typically two, separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, wherein sequences from the separate modular RNA molecules are comprised 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, for example as described in WO 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, wherein the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein. Exemplary types of CRISPR / Cas systems and associated gRNA structures include those described in, for example, Moon et al. Exp. Mol. Med. 51, 1–11 (2019), Zhang, F. Q. Rev. Biophys.52, E6 (2019), Makarova et al. Methods Mol. Biol. 1311:47-75 (2015), WO 2013 / 176772, or Jinek, M. et al. Science 337(6096):816-21 (2012).

[0241] A guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V guide RNA (gRNA), the crRNA forms a duplex. In both systems, the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. The gRNA can provide target specificity to the complex by virtue of its association with the site-directed polypeptide. The gRNA thus can direct the activity of the site- directed polypeptide.

[0242] In some aspects, the spacer sequence of a gRNA is a polynucleotide sequence comprising at least a portion that has sufficient complementarity with the target site to hybridize 6037971-sf22474-20029.40 with the target site in the target gene and direct sequence-specific binding of a Cas / gRNA (or a CRISPR) complex to the sequence of the target site. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. In some embodiments, the gRNA comprises a spacer sequence that is complementary, e.g., at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., fully complementary), to the target site. The strand of the target nucleic acid comprising the target site sequence may be referred to as the “complementary strand” of the target nucleic acid. In some aspects, the spacer sequence is a user-defined sequence. Guidance on the selection of spacer sequences can be found, e.g., in Fu et al., Nat Biotechnol 201432:279–284 and Sternberg et al., Nature 2014507:62-67.

[0243] In some aspects, a gRNA targets a target site in double-stranded DNA. Thus, in some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer hybridizes to, or by the sequence complementary to the sequence that the gRNA spacer hybridizes to. In some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer displaces in order to hybridize to the DNA. In some embodiments, the sequence of the target site is the sequence that the gRNA hybridizes to.

[0244] In some embodiments, the gRNA spacer sequence is between about 14 nucleotides (nt) and about 26 nt, or between 16 nt and 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 18 nt in length. In some embodiments, the gRNA spacer sequence is 19 nt in length. In some embodiments, the gRNA spacer sequence is 20 nt in length. In some embodiments, the gRNA spacer sequence is 21 nt in length. In some embodiments, the gRNA spacer sequence is 22 nt in length.

[0245] In some embodiments the gRNA is a concatenation of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence. The gRNA may target a desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II CRISPR / Cas system (e.g., Cas9). This duplex, which may include, for example, a 42-nucleotide crRNA and a 75- nucleotide tracrRNA, acts as a guide for the Cas9 protein to cleave the target nucleic acid. The “target region”, “target sequence” or “protospacer” as used interchangeably herein refers to a region of the IL-2 gene or regulatory element to which the CRISPR / Cas9-based system targets. 6037971-sf22474-20029.40 The CRISPR / Cas9-based system may include two or more gRNAs, wherein the two or more gRNAs target different DNA sequences. The target DNA sequences may be overlapping or non-overlapping. The target DNA sequences may be located within or near the same gene or different genes. The target sequence or protospacer is followed by a PAM sequence at the 3′ end of the protospacer. Different Type II systems have differing PAM requirements. For example, the Streptococcus pyogenes Type II system uses an “NGG” sequence, where “N” can be any nucleotide.

[0246] A target site of a 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: 68), where N is any nucleotide. In some embodiments, the PAM of a gRNA for complexing with a Type V CRISPR / Cas system, such as with Cas12a (also known as Cpf1) or variant thereof uses TTTV (SEQ ID NO: 67), where V is A, C, or G. In some embodiments, S. aureus Cas9 uses the PAM 5’- NNGRRT-3’ (SEQ ID NO: 69), 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: 70), where N is any nucleotide. In some embodiments, C. jejuni Cas9 uses the PAM 5′-NNNNRYAC-3′ (SEQ ID NO: 71), where N is any nucleotide, R is G or A, and Y is C or T. In some embodiments, S. thermophilus uses the PAM 5’-NNAGAAW-3’ (SEQ ID NO: 72), where N is any nucleotide and W is A or T. In some embodiments, F. Novicida Cas9 uses the PAM 5’-NGG-3’ (SEQ ID NO: 68), where N is any nucleotide. In some embodiments, T. denticola Cas9 uses the PAM 5’-NAAAAC-3’ (SEQ ID NO: 73), 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: 74). In some embodiments, Cas proteins may use or be engineered to use different PAMs from those listed above. For example, mutated SpCas9 proteins may use the PAMs 5’-NGG-3’ (SEQ ID NO: 68), 5’-NGAN-3’ (SEQ ID NO: 75), 5’-NGNG-3’ (SEQ ID NO: 76), 5’-NGAG-3’ (SEQ ID NO: 77), or 5’-NGCG-3’ (SEQ ID NO: 78), where N is any nucleotide. In some embodiments, the protospacer-adjacent motif (PAM) of a gRNA for complexing with S. pyogenes Cas9 or variant thereof is NGG, as set forth in SEQ ID NO: 68. In some embodiments, the PAM of a gRNA for complexing with S. aureus Cas9 or variant thereof is NNGRRT, as set forth in SEQ ID NO: 69. Methods for designing or identifying gRNA spacer 6037971-sf22474-20029.40 sequences and / or protospacer sequences in a particular region, are known. gRNA spacer sequences and / or protospacer sequences can be determined based on the type of Cas protein used and the associated PAM sequence.

[0247] A 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.

[0248] In some embodiments, the gRNA (including the guide sequence) will comprise the base uracil (U), whereas DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in some embodiments, it is believed that the complementarity of the guide sequence with the target sequence contributes to specificity of the interaction of the gRNA molecule / Cas molecule complex with a target nucleic acid. It is understood that in a guide sequence and target sequence pair, the uracil bases in the guide sequence will pair with the adenine bases in the target sequence. A gRNA spacer sequence herein may be defined by the DNA sequence encoding the gRNA spacer, and / or the RNA sequence of the spacer.

[0249] In some embodiments, the gRNA comprises modified nucleotides, e.g. for increased stability. In some embodiments, one, more than one, or all of the nucleotides of a gRNA can have a modification, e.g., to render the gRNA less susceptible to degradation and / or improve bio-compatibility. By way of non-limiting example, the backbone of the gRNA can be modified with a phosphorothioate, or other modification(s). In some cases, a nucleotide of the gRNA can comprise a 2’ modification, e.g., a 2-acetylation, e.g., a 2’ methylation, or other modification(s).

[0250] Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., International PCT Pub. Nos. WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, WO 2015 / 089427, WO 2016 / 049258, WO 2016 / 123578, WO 2021 / 076744, WO 2014 / 191128, WO 2015 / 161276, WO 2017 / 193107, and WO 2017 / 093969.

[0251] In some aspects, the gRNA comprises scaffold sequences. In some aspects, the scaffold sequence (in some cases including a crRNA sequence and / or a tracrRNA sequence) will be different depending on the Cas protein. In some aspects, different CRISPR / Cas systems have different gRNA scaffold sequences for associating with Cas protein. In some embodiments, an exemplary scaffold sequence for S. aureus Cas9 comprises a sequence set forth in SEQ ID NO:41, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 6037971-sf22474-20029.40 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:41. In some embodiments, an exemplary scaffold sequence for S. aureus Cas9 comprises a sequence set forth in SEQ ID NO:41. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:8, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:8. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:8.

[0252] In some embodiments, an exemplary scaffold sequence for Acidaminococcus sp. Cas12a comprises a sequence set forth in SEQ ID NO:123, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:123. In some embodiments, an exemplary scaffold sequence for CasPhi-2 comprises a sequence set forth in SEQ ID NO:124, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:124. In some embodiments, an exemplary scaffold sequence for Un1Cas12f1 comprises a sequence set forth in SEQ ID NO:125, the sequence “GGAATGAAC” (SEQ ID NO: 126), or the sequence “TTTTATTTT” (SEQ ID NO: 127), or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:125, 126 or 127. In some embodiments, an exemplary scaffold sequence for Un1Cas12f1 comprises a sequence set forth in SEQ ID NO:213, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:213. In some embodiments, an exemplary scaffold sequence for Un1Cas12f1 comprises a sequence set forth in SEQ ID NO:126, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:126. In some embodiments, an exemplary scaffold sequence for Un1Cas12f1 comprises a sequence set forth in SEQ ID NO:127, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:127. In some embodiments, an exemplary scaffold sequence for C. jejuni Cas9 comprises a sequence set forth in SEQ ID NO:128, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:128. In some embodiments, an exemplary scaffold sequence for Cas12k comprises a sequence set forth in SEQ ID NO:129, or a sequence having at or at 6037971-sf22474-20029.40 least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:129. In some embodiments, an exemplary scaffold sequence for CasMini comprises a sequence set forth in SEQ ID NO:130, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:130.

[0253] In some aspects, the gRNA can target the DNA-targeting system to direct the activities of an associated polypeptide (e.g., fusion protein, DNA-targeting system, effector domain, etc.) to a specific target site within a target nucleic acid (e.g., regulatory DNA element of a IL-2 locus). a. gRNAs for transcriptional activation

[0254] In some embodiments, a gRNA provided herein targets a target site for the IL-2 gene or regulatory element thereof for transcriptional activation. In some embodiments, a gRNA provided herein targets a target site for the IL-2 gene or regulatory element thereof for transcriptional activation. In some embodiments, the target site is located on the IL-2 gene. In some embodiments, the target site is located in a regulatory DNA element of the gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of the gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of the IL-2 gene. Exemplary target sites and combinations of target sites for gRNA of the multiplexed DNA- binding systems include any described in Section I.B.

[0255] In some embodiments, any of the provided gRNA sequences is complexed with or is provided in combination with a fusion protein comprising Cas9. In some embodiments, the Cas9 is a dCas9. In some embodiments, the dCas9 is a dSpCas9, such as a dSpCas9 with a sequence “DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF 6037971-sf22474-20029.40 EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK PAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL SQLGGD” as set forth in SEQ ID NO:63.

[0256] In some embodiments, the gRNA targets a target site that comprises a sequence selected from any one of SEQ ID NOS: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40, as shown in Table 1, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 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: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

[0257] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:12, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 the sequence set forth in SEQ ID NO:12 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:12. 6037971-sf22474-20029.40

[0258] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:24, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 the sequence set forth in SEQ ID NO:24 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:24.

[0259] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:26, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 the sequence set forth in SEQ ID NO:26 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:26.

[0260] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:28, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 the sequence set forth in SEQ ID NO:28 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:28.

[0261] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:38, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 the sequence set forth in SEQ ID NO:38 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:38.

[0262] In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:8 (GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC), or a sequence having at or at least 6037971-sf22474-20029.40 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a portion thereof. In some embodiments, the scaffold sequence is set forth in SEQ ID NO:8.

[0263] In some embodiments, any of the provided gRNA sequences is complexed with or is provided in combination with a fusion protein comprising Cas9. In some embodiments, the Cas9 is a dCas9. In some embodiments, the dCas9 is a dSpCas9, such as a dSpCas9 set forth in SEQ ID NO:63.

[0264] In some embodiments, provided herein is a multiplexed epigenetic-modifying DNA- targeting system comprising the combination of gRNAs.

[0265] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOS:9, 11, 13, 15, 17, 19, 21, 23,25,27, 29, 31, 33, 35, 37 and 39, as shown in Table 3, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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: 9, 11, 13, 15, 17, 19, 21, 23,25,27, 29, 31, 33, 35, 37 and 39 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: 9, 11, 13, 15, 17, 19, 21, 23,25,27, 29, 31, 33, 35, 37 and 39.

[0266] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:11, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:11 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:11.

[0267] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:23, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:23 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:23.

[0268] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:25, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 6037971-sf22474-20029.40 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:25 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:25.

[0269] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:27, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:27 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:27.

[0270] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:37, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:37 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:37. Table 3. Exemplary Guide RNAs (gRNAs) for Combination with Cas (e.g., SpCas9) for transcriptional activation of IL-26037971-sf22474-20029.40

[0271] In some embodiments, provided herein is a combination of gRNAs that each target a target site for IL-2 gene or regulatory element thereof for transcriptional activation. In some embodiments, provided herein is a multiplexed epigenetic-modifying DNA-targeting system comprising the combination of gRNAs.

[0272] In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting at least two different target sites for transcriptional activation of IL-2. In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting two same target sites for transcription activation. In some embodiments, the gRNAs target a combination of target sites selected from the combinations of target sites listed in Table 1. In some embodiments, the gRNAs target a combination of target sites listed in Table 2.

[0273] In some embodiments, each gRNA of the combination of gRNAs is selected from any of the gRNAs described herein for targeted transcriptional activation. Exemplary paired combinations of gRNAs are listed in Table 4. Table 4. Exemplary Paired Combinations of SpCas9 gRNAs

[0274] In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting the same target site In some embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site and a second gRNA targeted to the same first target site. In some embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site and a second gRNA targeted to a second target site, wherein the target sites are the same.

[0275] In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:11, and a second 6037971-sf22474-20029.40 gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:11. In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:23, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:23. In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:25, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:25.

[0276] In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting at least two target sites. In some embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site and a second gRNA targeted to a second target site, wherein the target sites are different.

[0277] In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:11, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:23. In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:11, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:25. In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:11, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:27. In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:11, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:37. In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:23, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:25. In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:23, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:27. In some embodiments, the combination of gRNAs comprises a first gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:23, and a second gRNA that targets a target site for IL-2 comprising the sequence set forth in SEQ ID NO:37. 6037971-sf22474-20029.40

[0278] In some embodiments, the combination of gRNAs comprises at least three gRNAs targeting at least three target sites. In particular embodiments, the at least three target sites are different target sites. In particular embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site, a second gRNA targeted to a second target site and a third gRNA targeted to a third target site. In some embodiments, at least two of the three target sites can be the same. In some embodiments, the combination of gRNAs comprises at least three gRNAs targeting at least two target sites.

[0279] In some embodiments, the combination of gRNAs comprises at least four gRNAs targeting at least four target sites. In particular embodiments, the at least four target sites are different target sites. In particular embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site, a second gRNA targeted to a second target site, a third gRNA targeted to a third target site and a fourth gRNA targeted to a fourth target site. In some embodiments, at least two of the four target sites can be the same. In some embodiments, the combination of gRNAs comprises at least four gRNAs targeting at least three target sites.

[0280] In particular embodiments, a first gRNA comprises the spacer sequence set forth in SEQ ID NO:11(GACUUAGUGCAAUGCAAGAC), a second gRNA comprises the spacer sequence set forth in SEQ ID NO:23 (CUCUCUCUGCAGACAGGGCA), a third gRNA comprises the spacer sequence set forth in SEQ ID NO:27 (AGAGGGAAGUGUCACAUAAU) and a fourth gRNA comprises the spacer sequence set forth in SEQ ID NO:37 (GGAAAUGACAUGCUUGAAGU).

[0281] In some embodiments, the combination of gRNAs comprises at least five gRNAs targeting at least five target sites. In particular embodiments, the at least five target sites are different target sites. In particular embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site, a second gRNA targeted to a second target site, a third gRNA targeted to a third target site, a fourth gRNA targeted to a fourth target site and a fifth target gRNA targeted to a fifth target site. In some embodiments, at least two of the five target sites can be the same. In some embodiments, the combination of gRNAs comprises at least five gRNAs targeting at least four target sites.

[0282] In particular embodiments, a first gRNA comprises the spacer sequence set forth in SEQ ID NO:11(GACUUAGUGCAAUGCAAGAC), a second gRNA comprises the spacer sequence set forth in SEQ ID NO:23 (CUCUCUCUGCAGACAGGGCA), a third gRNA comprises the spacer sequence set forth in SEQ ID NO: 25 6037971-sf22474-20029.40 (GGCAGGGUAGAGAAGUAGAG), a fourth gRNA comprises the spacer sequence set forth in SEQ ID NO:27 (AGAGGGAAGUGUCACAUAAU) and a fifth gRNA comprises the spacer sequence set forth in SEQ ID NO:37 (GGAAAUGACAUGCUUGAAGU).

[0283] In some embodiments, the combination of gRNAs targets a combination of target sites for a combination of target sites for transcriptional activation, for example as shown in Table 2.

[0284] Exemplary paired SpCas9 guide RNAs (gRNAs) and target site combinations are shown in Table 5. Table 5. Exemplary Paired SpCas9 Guide RNA (gRNA) and Target Site Combinations for Transcriptional Activation6037971-sf22474-20029.40

[0285] In some embodiments, any of the provided gRNA sequences is complexed with or is provided in combination with a fusion protein comprising Cas9. In some embodiments, the Cas9 is a dCas9. In some embodiments, the dCas9 is a dSaCas9, such as a dSaCas9 set forth in SEQ ID NO:65.

[0286] In some embodiments, the gRNA targets a target site that comprises a sequence selected from any one of SEQ ID NOS: 43, 45, 47, 49, 51, 53, 55, 57 and 59 as shown in Table 1, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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: 43, 45, 47, 49, 51, 53, 55, 57 and 59 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: 43, 45, 47, 49, 51, 53, 55, 57 and 59.

[0287] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:43, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 the sequence set forth in SEQ ID NO:43 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:43.

[0288] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:51, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 6037971-sf22474-20029.40 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 the sequence set forth in SEQ ID NO:51 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:51.

[0289] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:57, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 the sequence set forth in SEQ ID NO:57 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:57.

[0290] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO:59, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at 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 the sequence set forth in SEQ ID NO:59 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site sequence is set forth in SEQ ID NO:59.

[0291] In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:41 (GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUU AUCUCGUCAACUUGUUGGCGAGAUUUU), or a sequence having at 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 portion thereof. In some embodiments, the scaffold sequence is set forth in SEQ ID NO:41.

[0292] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOS:42, 44, 46, 48, 50, 52, 54, 56 and 58, as shown in Table 6, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:42, 44, 46, 48, 50, 52, 54, 56 and 58 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 6037971-sf22474-20029.40 any one of SEQ ID NOS:42, 44, 46, 48, 50, 52, 54, 56 and 58.

[0293] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:42, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:42 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:42.

[0294] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:50, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:50 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:50.

[0295] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:56, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:56 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:56.

[0296] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt, or a sequence having at 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:58 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:58. Table 6. Exemplary SaCas9 Guide RNAs (gRNAs) for transcriptional activation6037971-sf22474-20029.40

[0297] In some embodiments, provided herein is a combination of gRNAs that each target a target site for the IL-2 gene or regulatory element thereof for transcriptional activation. In some embodiments, provided herein is a multiplexed epigenetic-modifying DNA-targeting system comprising the combination of gRNAs.

[0298] In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting at least two different target sites for transcriptional activation. In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting two same target sites for transcription activation. In some embodiments, the gRNAs target a combination of target sites selected from combining the target sites listed in Table 1. In some embodiments, the gRNAs target a combination of target sites selected from the group consisting of SEQ ID NOS: 43, 45, 47, 49, 51, 53, 55, 57 and 59.

[0299] In some embodiments, each gRNA of the combination of gRNAs is selected from any of the gRNAs described herein for targeted transcriptional activation. In some embodiments, the combinations of gRNAs are selected from the gRNAs listed in Table 6. In some embodiments, the gRNAs have spacer sequences selected from the group consisting of SEQ ID NOS: 42, 44, 46, 48, 50, 52, 54, 56 and 58.

[0300] In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting the same target site. In some embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site and a second gRNA targeted to the same first target site. In some embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site and a second gRNA targeted to a second target site, wherein the target sites are the same.

[0301] In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting at least two target sites. In some embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site and a second gRNA targeted to a second target site, wherein the target sites are different.

[0302] In some embodiments, the combination of gRNAs comprises at least three gRNAs targeting at least three target sites. In particular embodiments, the at least three target sites are 6037971-sf22474-20029.40 different target sites. In particular embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site, a second gRNA targeted to a second target site and a third gRNA targeted to a third target site. In some embodiments, at least two of the three target sites can be the same. In some embodiments, the combination of gRNAs comprises at least three gRNAs targeting at least two target sites.

[0303] In some embodiments, the combination of gRNAs comprises at least four gRNAs targeting at least four target sites. In particular embodiments, the at least four target sites are different target sites. In particular embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site, a second gRNA targeted to a second target site, a third gRNA targeted to a third target site and a fourth gRNA targeted to a fourth target site. In some embodiments, at least two of the four target sites can be the same. In some embodiments, the combination of gRNAs comprises at least four gRNAs targeting at least three target sites.

[0304] In some embodiments, the combination of gRNAs comprises at least five gRNAs targeting at least five target sites. In particular embodiments, the at least five target sites are different target sites. In particular embodiments, the combination of gRNAs comprises a first gRNA targeted to a first target site, a second gRNA targeted to a second target site, a third gRNA targeted to a third target site, a fourth gRNA targeted to a fourth target site and a fifth target gRNA targeted to a fifth target site. In some embodiments, at least two of the five target sites can be the same. In some embodiments, the combination of gRNAs comprises at least five gRNAs targeting at least four target sites. D. Other DNA-Binding Domains and DNA-Targeting Systems

[0305] In some of any of the provided embodiments, the DNA-binding domain comprises a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-SceI enzyme or a variant thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing. In some embodiments, the fusion protein of the DNA-targeting system, or one or more DNA-targeting modules thereof, comprises a DNA-binding domain described herein, such as a DNA-binding domain that is an engineered zinc finger protein (eZFP) or a TALE.

[0306] In some embodiments, a ZFP, a zinc finger DNA binding protein, or zinc finger DNA binding domain, is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid 6037971-sf22474-20029.40 sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial, or engineered ZFPs (eZFPs), comprising ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers. ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (−1, 2, 3, and 6) on a zinc finger recognition helix. Thus, for example, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is an eZFP that is engineered to bind to a target site of choice.

[0307] In some embodiments, zinc fingers are custom-designed (i.e. designed by the user), or obtained from a commercial source. Various methods for designing zinc finger proteins are available. For example, methods for designing zinc finger proteins to bind to a target DNA sequence of interest are described, for example in Liu, Q. et al., PNAS, 94(11):5525-30 (1997); Wright, D.A. et al., Nat. Protoc., 1(3):1637-52 (2006); Gersbach, C.A. et al., Acc. Chem. Res., 47(8):2309-18 (2014); Bhakta M.S. et al., Methods Mol. Biol., 649:3-30 (2010); and Gaj et al., Trends Biotechnol, 31(7):397-405 (2013). In addition, various web-based tools for designing zinc finger proteins to bind to a DNA target sequence of interest are publicly available. See, for example, the Zinc Finger Tools design web site from Scripps available on the world wide web at scripps.edu / barbas / zfdesign / zfdesignhome.php. Various commercial services for designing zinc finger proteins to bind to a DNA target sequence of interest are also available. See, for example, the commercially available services or kits offered by Creative Biolabs (world wide web at creative-biolabs.com / Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium Modular Assembly Kit available from Addgene (world wide web at addgene.org / kits / zfc-modular-assembly / ), or the CompoZr Custom ZFN Service from Sigma Aldrich (world wide web at sigmaaldrich.com / life-science / zinc-finger-nuclease- technology / custom-zfn.html).

[0308] In some embodiments, the fusion protein of the DNA-targeting system comprises an eZFP DNA-binding domain and an effector domain.

[0309] Transcription activator-like effectors (TALEs), are proteins naturally found in Xanthomonas bacteria. TALEs comprise a plurality of repeated amino acid sequences, each 6037971-sf22474-20029.40 repeat having binding specificity for one base in a target sequence. Each repeat comprises a pair of variable residues in position 12 and 13 (repeat variable diresidue; RVD) that determine the nucleotide specificity of the repeat. In some embodiments, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A. In some embodiments, RVDs can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. Binding domains with similar modular base-per-base nucleic acid binding properties can also be derived from different bacterial species. These alternative modular proteins may exhibit more sequence variability than TALE repeats.

[0310] In some embodiments, a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains, each comprising a repeat variable diresidue (RVD), are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TALE proteins may be designed to bind to a target site using canonical or non-canonical RVDs within the repeat units. See, e.g., U.S. Pat. Nos. 8,586,526 and 9,458,205.

[0311] In some embodiments, the fusion protein of the DNA-targeting system comprises a TALE DNA-binding domain and an effector domain.

[0312] Zinc finger and TALE DNA-binding domains can be engineered to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger protein, by engineering of the amino acids in a TALE repeat involved in DNA binding (the repeat variable diresidue or RVD region), or by systematic ordering of modular DNA-binding domains, such as TALE repeats or ZFP domains. Therefore, engineered zinc finger proteins or TALE proteins are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering zinc finger proteins and TALEs are design and selection. A designed protein is a protein not occurring in nature whose design / composition results principally from rational criteria. Rational 6037971-sf22474-20029.40 criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Pat. Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. 1. Exemplary ZFPs

[0313] In some aspects, the DNA-binding domain comprises a zinc finger protein (ZFP). In some aspects, provided herein are exemplary ZFPs that are capable of binding to, or bind to, a target site, such as any provided herein in Section I.B. In some aspects, the exemplary ZFPs can facilitate specific targeting of effector domains for transcriptional activation of a target site provided in Section I, e.g., target sites in the IL-2 gene, for gene-specific transcriptional activation of IL-2. Thus, in some aspects, the exemplary ZFPs facilitates promotion of lymphoid activation and function.

[0314] In some embodiments, the target site for a ZFP provided herein is within the IL-2 gene. In some embodiments, the target site within the IL-2 gene is within region 4, region 5, or the region surrounding or including the transcriptional start site (TSS). In some embodiments, the region surrounding or including the TSS is 50 to 150 kilobases (KB) upstream of the IL-2 gene and the IL-2 transcription start site (TSS). In some embodiments, the target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,507,000- 122,508,985. In some embodiments, the target site within the IL-2 gene is within region 4 or region 5.

[0315] In some embodiments, the target site for a ZFP provided herein comprises the nucleotide sequence set forth in any one of SEQ ID NOS:186-188, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the target site for a ZFP provided herein comprises the nucleotide sequence set forth in any one of SEQ ID NOS:186-188. In some embodiments, the target site is comprised in double-stranded DNA, such as genomic DNA. In some embodiments, the target site is double-stranded DNA, such as genomic DNA. In some embodiments, the ZFP is capable of binding to the target site. In some embodiments, the ZFP binds to the target site. In some embodiments, the binding is target- specific. For example, in some embodiments, an ZFP binds to the target site, and not to other sites comprising different sequences. For example, in some embodiments, an individual ZFP disclosed herein binds to the target site set forth in SEQ ID NO:186, and does not bind to a 6037971-sf22474-20029.40 different target site, such as the target site set forth in SEQ ID NO:188. In some embodiments, the target site for an ZFP provided herein comprises a sequence set forth in Table 7. Table 7. ZFP target sequences

[0316] In some embodiments, the target site for an ZFP provided herein comprises the nucleotide sequence set forth in SEQ ID NO:186, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the target site for an ZFP provided herein comprises the sequence set forth in SEQ ID NO: 186.

[0317] In some embodiments, the target site for an ZFP provided herein comprises the nucleotide sequence set forth in SEQ ID NO:187, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the target site for an ZFP provided herein comprises the sequence set forth in SEQ ID NO: 187.

[0318] In some embodiments, the target site for an ZFP provided herein comprises the nucleotide sequence set forth in SEQ ID NO:188, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the target site for a ZFP provided herein comprises the sequence set forth in SEQ ID NO: 188.

[0319] In some embodiments, characteristics of ZFPs targeting specific target sites provided herein are shown in Table 8. In some embodiments, the ZFP comprises six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, each comprising a corresponding recognition region F1-F6, as shown in Table 8. In some embodiments, the recognition regions F1-F6 facilitate specific binding to the indicated target site sequence in Table 8. In some embodiments, the ZFP comprises an amino acid sequence comprising the recognition regions, as shown in Table 8. In some embodiments, the ZFP can be encoded by a DNA sequence as shown in Table 8. 6037971-sf22474-20029.40 Table 8. Exemplary ZFP engineered zinc finger proteins

[0320] In some embodiments, provided herein is an ZFP, such as IL2_R4_A as described herein. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO:186, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO:186. In some embodiments, the target site is 6037971-sf22474-20029.40 double-stranded DNA. In some embodiments, the ZFP comprises six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1 through F6, and the amino acid sequence of each zinc finger recognition region is as follows: F1: QNAHRKT (SEQ ID NO: 195), F2: RKYYLAK (SEQ ID NO: 196), F3: RSAHLSR (SEQ ID NO: 197), F4: QSGDLTR (SEQ ID NO: 198), F5: RSDHLTQ (SEQ ID NO: 199), and F6: DSANLSR (SEQ ID NO: 200). In some embodiments, the ZFP comprises the amino acid sequence set forth in SEQ ID NO:189, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the ZFP comprises the amino acid sequence set forth in SEQ ID NO:189. In some embodiments, the ZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:192, or a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the ZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:192.

[0321] In some embodiments, provided herein is an ZFP, such as IL2_R4_B as described herein. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO:187, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO:187. In some embodiments, the target site is double-stranded DNA. In some embodiments, the ZFP comprises six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1 through F6, and the amino acid sequence of each zinc finger recognition region is as follows: F1: DSSHLEL (SEQ ID NO: 201), F2: DRSNLTR (SEQ ID NO: 202), F3: RSDNLSE (SEQ ID NO: 203), F4: VRRALSS (SEQ ID NO: 204), F5: QSGALAR (SEQ ID NO: 205), and F6: RLDWLPM (SEQ ID NO: 206). In some embodiments, the ZFP comprises the amino acid sequence set forth in SEQ ID NO:190, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the ZFP comprises the amino acid sequence set forth in SEQ ID NO:190. In some embodiments, the ZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:191, or a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the ZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:191. 6037971-sf22474-20029.40

[0322] In some embodiments, provided herein is an ZFP, such as IL2_R5_A as described herein. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO:188, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO:188. In some embodiments, the target site is double-stranded DNA. In some embodiments, the ZFP comprises six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1 through F6, and the amino acid sequence of each zinc finger recognition region is as follows: F1: RSDNLSV (SEQ ID NO: 207), F2: RSAHLSR (SEQ ID NO: 208), F3: QNAHRKT (SEQ ID NO: 209), F4: LRHHLTR (SEQ ID NO: 210), F5: TSSNRKT (SEQ ID NO: 211), and F6: TSSNLSR (SEQ ID NO: 212). In some embodiments, the ZFP comprises the amino acid sequence set forth in SEQ ID NO:191, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the ZFP comprises the amino acid sequence set forth in SEQ ID NO:191. In some embodiments, the ZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:194, or a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the ZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:194. E. Effector Domains

[0323] In some aspects, the DNA-targeting systems provided herein further include one or more effector domains. In some embodiments, the one or more effector domains are transcriptional activator effector domains. In some embodiments, in a DNA-targeting system having a plurality of effector domains, each effector domain is a transcriptional activator. In some embodiments, provided herein is a DNA-targeting system comprising a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site in an IL-2 gene or regulatory DNA element thereof, such as any described above in Section I.C or Section I.D, and (b) at least one effector domain. 1. Effector Domains for Transcriptional Activation

[0324] In some aspects, the DNA-targeting systems provided herein further include one or more effector domains, such as a transcriptional activator effector domain. In some embodiments, provided herein is a DNA-targeting system comprising a fusion protein 6037971-sf22474-20029.40 comprising: (a) a DNA-binding domain capable of being targeted to a target site in the IL-2 gene or regulatory element thereof or regulatory DNA element thereof, such as any DNA-binding domain described above in Section I.D or Section I.E, and (b) at least one effector domain. In some aspects, the effector domain is capable of increasing transcription of IL-2. In some aspects, the effector domain comprises a transcription activation domain.

[0325] In some aspects, the effector domain activates, induces, catalyzes, or leads to increased transcription of a gene when ectopically recruited to the gene or DNA regulatory element thereof. In some embodiments, the effector domain activates, induces, catalyzes, or leads to: transcription activation, transcription co-activation, transcription elongation, transcription de-repression, transcription factor release, polymerization, histone modification, histone acetylation, histone deacetylation, nucleosome remodeling, chromatin remodeling, reversal of heterochromatin formation, proteolysis, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, DNA methylation, DNA demethylation, histone methylation, histone demethylation, or DNA base oxidation. In some embodiments, the effector domain activates, induces, catalyzes or leads to transcription activation, transcription co- activation, or transcription elongation. In some embodiments, the effector domain induces transcription activation. In some embodiments, the effector domain has one of the aforementioned activities itself (i.e. acts directly). In some embodiments, the effector domain recruits and / or interacts with a polypeptide domain that has one of the aforementioned activities (i.e. acts indirectly).

[0326] Gene expression of endogenous mammalian genes, such as human genes, can be achieved by targeting a fusion protein comprising a DNA-binding domain, such as a dCas9, and an effector domain, such as a transcription activation domain, to mammalian genes or regulatory DNA elements thereof (e.g. a promoter or enhancer) via one or more gRNAs. Any of a variety of effector domains for transcriptional activation (e.g. transcription activation domains) are known and can be used in accord with the provided embodiments. Transcription activation domains, as well as activation of target genes by Cas fusion proteins (with a variety of Cas molecules) and the transcription activation domains, are described, for example, in WO 2014 / 197748, WO 2016 / 130600 , WO 2017 / 180915, WO 2021 / 226555 , WO 2021 / 226077, WO 2013 / 176772 , WO 2014 / 152432, WO 2014 / 093661, WO 2024 / 015881, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973–976 (2013), Mali, P. et al. Nat. Biotechnol.31, 833–838 (2013), and Maeder, M. L. et al. Nat. Methods 10, 977–979 (2013). 6037971-sf22474-20029.40

[0327] In some embodiments, a transcriptional activation domain comprises a domain of a protein selected from among VP64, p65, Rta, p300, CBP, VPR, VPH, HSF1, a TET protein (e.g. TET1), a partially or fully functional fragment or domain thereof, or a combination of any of the foregoing. In some embodiments, a transcriptional activator domain further comprises at least one domain of a protein selected from among FOXO3 and NCOA3, that exhibits transcriptional activation, is capable of inducing or activating transcription from a gene, is a functional transcriptional activation domain, and / or has a function of transcription activation. In some embodiments, a transcriptional activator domain further comprises at least one domain selected from among FOXO3 and NCOA3.

[0328] In some embodiments, the epigenetic mark includes Histone H3K27 acetylation. In some embodiments, the effector domain catalyzes acetylation of histone H3 lysine 27 at the target site or is able to recruit an enzyme that catalyzes acetylation of histone H3 lysine 27 at the target site. In some embodiments, the enzyme that catalyzes the acetylation is an acetyltransferase. In some embodiments, the transcriptional activation domain comprises a VP64 domain. For example, dCas9-VP64 can be targeted to a target site by one or more gRNAs to activate a gene. VP64 is a polypeptide composed of four tandem copies of VP16, a 16 amino acid transactivation domain of the Herpes simplex virus. VP64 domains, including in dCas fusion proteins, have been described, for example, in WO 2014 / 197748, WO 2013 / 176772, WO 2014 / 152432, and WO 2014 / 093661. In some embodiments, the transcriptional activation domain comprises at least one VP16 domain, or a VP16 tetramer (“VP64”) or a variant thereof. An exemplary VP64 domain is set forth in SEQ ID NO:66. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:66, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:66, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:66.

[0329] In some embodiments, the transcriptional activation domain comprises a p65 activation domain (p65AD). p65AD is the principal transactivation domain of the 65kDa polypeptide of the nuclear form of the NF-KB transcription factor. An exemplary sequence of human transcription factor p65 is available at the Uniprot database under accession number Q04206. p65 domains, including in dCas fusion proteins, have been described, for example in WO 2017 / 180915 and Chavez, A. et al. Nat. Methods 12, 326–328 (2015). An exemplary p65 activation domain is set forth in SEQ ID NO:79. In some embodiments, the transcriptional 6037971-sf22474-20029.40 activation domain comprises SEQ ID NO:79, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:79, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:79.

[0330] In some embodiments, the transcriptional activation domain comprises an R transactivator (Rta) domain. Rta is an immediate-early protein of Epstein-Barr virus (EBV), and is a transcriptional activator that induces lytic gene expression and triggers virus reactivation. The Rta domain, including in dCas fusion proteins, has been described, for example in WO 2017 / 180915 and Chavez, A. et al. Nat. Methods 12, 326–328 (2015). An exemplary Rta domain is set forth in SEQ ID NO:80. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:80, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:80, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:80.

[0331] In some embodiments, the transcriptional activation domain comprises a CREB- binding protein (CBP) domain or a p300 domain. In some aspects, CBP refers to the CREB- binding protein encoded by the human CREBBP gene. CBP is a coactivator that interacts with cAMP-response element binding protein (CREB). In some aspects, p300 refers to the Histone acetyltransferase p300 protein encoded by the human EP300 gene, and is a coactivator closely related to CBP. CBP and p300 each interact with a variety of transcriptional activators to affect gene transcription (Gerritsen, M.E. et al. PNAS 94(7):2927-2932 (1997)). In some embodiments, the transcriptional activation domain comprises a p300 domain. p300 domains (such as the catalytic core of p300) including in dCas fusion proteins for gene activation, has been described, for example, in WO 2016 / 130600, WO 2017 / 180915, and Hilton, I.B. et al., Nat. Biotechnol.33(5):510-517 (2015). An exemplary human CBP sequence is set forth in SEQ ID NO:81. An exemplary human p300 sequence is set forth in SEQ ID NO:82. An exemplary p300 domain is set forth in SEQ ID NO:83. In some embodiments, the transcriptional activation domain comprises any one of SEQ ID NOS:81-83, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:81-83, or a portion thereof. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:83, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% 6037971-sf22474-20029.40 sequence identity to SEQ ID NO:83, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:83.

[0332] In some embodiments, the transcriptional activation domain comprises a HSF1 domain. In some aspects, HSF1 refers to the Heat shock factor protein 1 protein encoded by the human HSF1 gene. HSF1, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555, WO 2015 / 089427, and Konermann et al. Nature 517(7536):583-8 (2015). An exemplary human HSF1 sequence is set forth in SEQ ID NO:84. An exemplary HSF1 domain sequence is set forth in SEQ ID NO:84. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:84 or SEQ ID NO:177, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:84 or SEQ ID NO:1775, or a portion thereof. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:84, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:84, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:84.

[0333] In some embodiments, the transcriptional activation domain comprises the tripartite activator VP64-p65-Rta (also known as VPR). VPR comprises three transcription activation domains (VP64, p65, and Rta) fused by short amino acid linkers, and can effectively upregulate target gene expression. VPR, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555 and Chavez, A. et al. Nat. Methods 12, 326–328 (2015). An exemplary VPR polypeptide is set forth in SEQ ID NO:85. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:85, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:85, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:85.

[0334] In some embodiments, the transcriptional activation domain comprises VPH. VPH is a tripartite activator polypeptide comprising VP64, mouse p65, and HSF1. VPH, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555. An exemplary VPH polypeptide is set forth in SEQ ID NO:86. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:86, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% 6037971-sf22474-20029.40 sequence identity to SEQ ID NO:86, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:86.

[0335] In some embodiments, the transcriptional activation effector domain has demethylase activity. The effector domain may include an enzyme that remove methyl (CH3-) groups from nucleic acids, proteins (in particular histones), and other molecules. The effector domain may covert the methyl group to hydroxymethylcytosine in a mechanism for demethylating DNA. Alternatively, the transcriptional activation domain can convert the methyl group to hydroxymethylcytosine in a mechanism for demethylating DNA. The effector domain can catalyze this reaction. For example, the transcriptional activation domain that catalyzes this reaction may comprise a domain from a TET protein, for example TET1 (Ten-eleven translocation methylcytosine dioxygenase 1). In some aspects, TET1 refers to the Methylcytosine dioxygenase TET1 protein encoded by the human TET1 gene. TET1 catalyzes the conversion of the modified genomic base 5-methylcytosine (5mC) into 5- hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation. TET1, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555. An exemplary human TET1 sequence is set forth in SEQ ID NO:87. An exemplary TET1 catalytic domain is set forth in SEQ ID NO:88. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:87 or SEQ ID NO:88, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:87 or SEQ ID NO:88, or a portion thereof. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:88, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:88, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:88.

[0336] In some embodiments, the effector domain may comprise a SunTag domain. SunTag is a repeating peptide array, which can recruit multiple copies of an antibody-fusion protein that binds the repeating peptide. The antibody-fusion protein may comprise an additional effector domain, such as a transcription activation domain (e.g. VP64), to induce increased transcription of the target gene. SunTag, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2016 / 011070 and Tanenbaum, M. et al. Cell.159(3):635–646 (2014). An exemplary SunTag effector domain includes a repeating GCN4 peptide having the amino acid sequence LLPKNYHLENEVARLKKLVGER (SEQ ID NO:89) separated by linkers 6037971-sf22474-20029.40 having the amino acid sequence GGSGG (SEQ ID NO:90). In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO:89, a domain thereof, a portion thereof, or a variant thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some embodiments, the SunTag effector domain recruits an antibody-fusion protein that comprises a transcriptional activator effector domain (e.g. VP64) and binds the GCN4 peptide, thereby activating transcription at the target site and acting as a transcriptional activator effector domain.

[0337] In some embodiments, a transcriptional activation domain comprises a FOXO3 domain, i.e. a domain from FOXO3. In some aspects, FOXO3 refers to the Forkhead box protein O3 encoded by the human FOXO3 gene. FOXO3 functions as a transcriptional activator that recognizes and binds to specific DNA sequences. An exemplary human FOXO3 sequence is set forth in SEQ ID NO:219. An exemplary FOXO3 domain sequence is set forth in SEQ ID NO:220 and SEQ ID NO:221. In some embodiments, the transcriptional activation domain comprises a sequence set forth in any of SEQ ID NOS:219-221 or a domain or a portion thereof, such as a contiguous portion thereof of at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids, such as at least 20 amino acids, or a variant thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 219- 221 or a domain or a portion thereof, such as a contiguous portion thereof of at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids, such as at least 20 amino acids, or a variant thereof. In some embodiments, the transcriptional activation domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:219. In some embodiments, the transcriptional activation domain comprises a contiguous portion of SEQ ID NO:219 that is at least 80 amino acids in length. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:220. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:220. An exemplary nucleotide sequence encoding the transcriptional activation domain set forth in SEQ ID NO:220 is set forth in SEQ ID NO:222. In some embodiments, the transcriptional activation domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:221. In some embodiments, the transcriptional activation domain comprises a contiguous portion of SEQ ID NO:219 that is at least 42 amino acids in 6037971-sf22474-20029.40 length. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:221. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:221.

[0338] In some embodiments, a transcriptional activation domain comprises a NCOA3 domain, i.e. a domain from NCOA3. In some aspects, NCOA3 refers to the Nuclear receptor coactivator 3 protein encoded by the human NCOA3 gene. NCOA3 functions as a transcriptional coactivator for steroid receptors and nuclear receptors. An exemplary human NCOA3 sequence is set forth in SEQ ID NO:223. An exemplary NCOA3 domain sequence is set forth in SEQ ID NO:224 and SEQ ID NO:184. In some embodiments, the transcriptional activation domain comprises a sequence set forth in any of SEQ ID NOS:184, 223, and 224 or a domain or a portion thereof, such as a contiguous portion thereof of at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids, such as at least 20 amino acids, or a variant thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 184, 223, and 224 or a domain or a portion thereof, such as a contiguous portion thereof of at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids, such as at least 20 amino acids, or a variant thereof. In some embodiments, the transcriptional activation domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:224. In some embodiments, the transcriptional activation domain comprises a contiguous portion of SEQ ID NO:223 that is at least 80 amino acids in length. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:224. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:224. An exemplary nucleotide sequence encoding the transcriptional activation domain set forth in SEQ ID NO:224 is set forth in SEQ ID NO:185. In some embodiments, the transcriptional activation domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:184. In some embodiments, the transcriptional activation domain comprises a contiguous portion of SEQ ID NO:223 that is at least 49 amino acids in length. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:184. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:184.

[0339] In some embodiments, the transcriptional activation domain comprises a fusion of NCOA3 and FOXO3 domains as described herein, e.g. the NCOA3 domain set forth in SEQ ID 6037971-sf22474-20029.40 NO: 184 and the FOXO3 domain set forth in SEQ ID NO: 221. In some embodiments, the transcriptional activation domain comprises a fusion of two NCOA3 domains and one FOXO3. The fusion protein contains these domains can be arranged in any order. In some embodiments, the transcriptional activation domain is arranged, from N terminus to C terminus, as follows: a first NCOA3 domain, a FOXO3 domain, and a second NCOA3 domain, also referred to as a NCOA3-FOXO3-NCOA3 domain (NFN). In some embodiments, the NFN domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 181. In some embodiments, the NFN domain comprises SEQ ID NO: 181. In some embodiments, the NFN domain is SEQ ID NO: 181. In some embodiments, the domains are either directly linked to each other, or they are linked via a linker, such as a peptide linker.

[0340] In some embodiments, the fusion protein comprises a transcriptional activation domain that is a NFN domain. In some embodiments, the fusion protein further comprises an additional transcriptional activation domain that is a VP64 domain. In some embodiments, the NFN and VP64 domains are either directly linked to each other, or they are linked via a linker, or they are separated by a DNA-binding domain. In some embodiments, the NFN and VP64 domains are separated by a DNA-binding domain. F. Fusion Proteins

[0341] In some embodiments, the fusion protein comprises: (a) a DNA-binding domain capable of being targeted to multiple target sites for the IL-2 gene or regulatory element thereof, and (b) at least one transcriptional activator effector domain for increasing transcription of the IL-2 gene or regulatory element thereof.

[0342] In some embodiments, the fusion protein comprises at least one of any of the DNA- binding domains described herein in Section I.C or Section I.D, and at least one of any of the effector domains described herein in Section I.E. In some embodiments, the fusion protein contains a CRISPR / Cas-based DNA-binding domain, such as described in Section I.C, and at least one effector domain for transcriptional activation, as described in section I.E.1. In some embodiments, the fusion protein contains a ZFP DNA-binding domain, such as described in Section I.D or I.D.1, and at least one effector domain for transcriptional activation, as described in section I.E. In some aspects, the fusion protein is targeted to a target site in the IL-2 gene or regulatory element thereof, and leads to increased or activated transcription of the gene. In some 6037971-sf22474-20029.40 aspects, the fusion protein is targeted to target sites in a combination of target sites on the IL-2 gene or regulatory element thereof, and leads to increased or activated transcription of the IL-2 gene.

[0343] In some embodiments, the DNA-binding domain and effector domain of the fusion protein are heterologous, i.e. the domains are from different species, or at least one of the domains is not found in nature. In some aspects, the fusion protein is an engineered fusion protein, i.e. the fusion protein is not found in nature.

[0344] In some embodiments, the at least one effector domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain or a component thereof. The at least one effector domain may be fused to the DNA-binding domain directly, or via any intervening amino acid sequence, such as a linker sequence or a nuclear localization sequence (NLS).

[0345] In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises, from N- to C-terminal order: a transcriptional activator effector domain and a DNA-binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises, from N- to C-terminal order: a DNA-binding domain and a transcriptional activator effector domain.

[0346] In some embodiments, the at least one effector domain of the fusion protein includes more than one effector domain. In some embodiments, the fusion protein includes 2, 3 or 4 effector domains, or more than 4 effector domains. In some embodiments, at least two of the effector domains of the fusion protein are different. In some embodiments, each of the effector domains of the fusion protein are different. In some embodiments, the at least one effector domain includes two effector domains in which the two effector domains are different. In some embodiments, the effector domains and the DNA-binding domain can be arranged in any order.

[0347] In some aspects, each of the effector domains is a transcriptional activator effector domain.

[0348] In some embodiments, the at least one effector domain of the fusion protein includes two different effector domains. The two different effector domains and the DNA-binding domain can be arranged in any order. In some embodiments, each of the effector domains are N-terminal to the DNA-binding domain in which a first effector domain is fused to the N- terminus of the second effector domain and the second effector domain is fused to the N- terminus of the DNA-binding domain. In some embodiments, the fusion protein of a provided 6037971-sf22474-20029.40 DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a first effector domain, a second effector domain and the DNA binding domain. In some embodiments, each of the effector domains are C-terminal to the DNA-binding domain in which a first effector domain is fused to the C-terminus of the DNA-binding domain and the second effector domain is fused to the C-terminus of the first effector domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a DNA-binding domain, a first effector domain, and a second effector domain. In some embodiments, the DNA-binding domain is between the effector domains, in which one effector domain is fused to the N-terminus of the DNA-binding domain and the other effector domain is fused to the C-terminus of the DNA-binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA- targeting module thereof, comprises from N- to C-terminal order: a first effector domain, a DNA-binding domain, and a second effector domain. In some embodiments, one or more of the components may be fused to each other directly, or via any intervening amino acid sequence, such as via a linker sequence or a nuclear localization sequence (NLS).

[0349] In some embodiments, the fusion protein comprises one or more linkers. In some embodiments, the linker is a peptide linker. In some embodiments, the one or more linkers connect the DNA-binding domain or a component thereof to the at least one effector domain. A linker may be included anywhere in the polypeptide sequence of the fusion protein, for example, between the effector domain and the DNA-binding domain or a component thereof. A linker may be of any length and designed to promote or restrict the mobility of components in the fusion protein. A linker may comprise any amino acid sequence of about 2 to about 100, about 5 to about 80, about 10 to about 60, or about 20 to about 50 amino acids. A linker may comprise an amino acid sequence of at least about 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids. A linker may comprise an amino acid sequence of less than about 100, 90, 80, 70, 60, 50, or 40 amino acids. A skilled artisan can readily choose an appropriate linker for the connection of two domains. In some embodiments, the linker is a flexible linker. Flexible linkers are generally composed of small, non-polar or polar residues such as glycine, serine or threonine. A linker may include sequential or tandem repeats of an amino acid sequence that is 2 to 20 amino acids in length. Linkers may be rich in amino acids glycine (G), serine (S), and / or alanine (A). Linkers may include, for example, a GS linker. An exemplary GS linker is represented by the sequence GGGGS (SEQ ID NO:91). A linker may comprise repeats of a sequence, for example 6037971-sf22474-20029.40 as represented by the formula (GGGGS)n, wherein n is an integer that represents the number of times the GGGGS sequence is repeated (e.g. between 1 and 10 times). The number of times a linker sequence is repeated can be adjusted to optimize the linker length and achieve appropriate separation of the functional domains. For example, in some embodiments, the linker is the (GGGGS)nlinker, whereby n is an integer of 1 to 10. Other examples of linkers may include, for example, GGGGG (SEQ ID NO:92), GGAGG (SEQ ID NO:93), GGGGSSS (SEQ ID NO:94), or GGGGAAA (SEQ ID NO:95).

[0350] In some embodiments, artificial linker sequences can be used. In some embodiments, the linker is EASGSGRASPGIPGSTR (SEQ ID NO:96). In some embodiments, the linker is linker is GIHGVPAA (SEQ ID NO:97). In some embodiments, the linker is SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO:98). In some embodiments, the linker is KRPAATKKAGQAKKKKASDAKSLTAWS (SEQ ID NO:99).

[0351] In some embodiments, inclusion of a linker in the fusion protein leads to enhanced activation of the IL-2 gene or regulatory element thereof.

[0352] In some embodiments, the linker is an XTEN linker. In some aspects, an XTEN linker is a recombinant polypeptide (e.g., an unstructured recombinant peptide) lacking hydrophobic amino acid residues. Exemplary XTEN linkers are described in, for example, Schellenberger et al., Nature Biotechnology 27, 1186-1190 (2009) or WO 2021 / 247570. In some embodiments, a linker comprises the sequence set forth in SEQ ID NO:100, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:100. In some aspects, the linker comprises the sequence set forth in SEQ ID NO:77, or a contiguous portion of SEQ ID NO:100 of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 amino acids. In some aspects, the linker consists of the sequence set forth in SEQ ID NO:100, or a contiguous portion of SEQ ID NO:100 of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 amino acids. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:100. In some embodiments, the linker consists of the sequence set forth in SEQ ID NO:100. In some embodiments, a linker comprises the sequence set forth in SEQ ID NO:101, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some aspects, the linker comprises the sequence set forth in SEQ ID NO:101, or a contiguous portion of SEQ ID NO:101 of at least 5, 10, or15 amino acids. In some aspects, the linker consists of the sequence set forth in SEQ ID 6037971-sf22474-20029.40 NO:101, or a contiguous portion of SEQ ID NO:101 of at least 5, 10 or 15 amino acids. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:101. In some embodiments, the linker consists of the sequence set forth in SEQ ID NO:101. Appropriate linkers may be selected or designed based rational criteria known in the art, for example as described in Chen et al. Adv. Drug Deliv. Rev.65(10):1357-1369 (2013). In some embodiments, a linker comprises a linker described in WO 2021 / 247570.

[0353] In some embodiments, the fusion protein of the DNA-targeting system, or a DNA- targeting module thereof, comprises one or more nuclear localization signals (NLS). In some embodiments, a fusion protein described herein comprises one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO:102); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:103); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:104) or RQRRNELKRSP (SEQ ID NO:105); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:106); the sequen...

Claims

22474-20029.40 WHAT IS CLAIMED:

1. An epigenetic-modifying DNA-targeting system comprising a plurality of DNA- targeting modules for increasing transcription of the interleukin (IL-2) gene, wherein each of the DNA-targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain for targeting to a target site of the IL-2 gene; and (b) at least one transcriptional activator effector domain.

2. The epigenetic-modifying DNA-targeting system of claim 1, wherein the DNA- binding domain of each fusion protein comprises: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-SceI enzyme or a variant thereof, optionally wherein the DNA- binding domain comprises a catalytically inactive variant of any of the foregoing, wherein when the DNA-binding domain of each fusion protein comprises a Cas protein, the DNA-targeting system further comprises at least two gRNAs, each capable of targeting the Cas protein to a target site.

3. The epigenetic-modifying DNA-targeting system of claim 1 or claim 2, wherein the plurality of DNA-targeting modules is 2-6 DNA targeting modules.

4. The epigenetic-modifying DNA-targeting system of any of claims 1-3, wherein the plurality of DNA-targeting modules is 2 DNA-targeting modules.

5. The epigenetic-modifying DNA-targeting system of any of claims 1-3, wherein the plurality of DNA-targeting modules is 3 DNA-targeting modules.

6. The epigenetic-modifying DNA-targeting system of any of claim 1-3, wherein the plurality of DNA-targeting modules is 4 DNA-targeting modules or 5 DNA-targeting modules.

7. The epigenetic-modifying DNA-targeting system of any of claims 1-6, wherein each target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4:122,451,261-122,593,946.

8. The epigenetic-modifying DNA-targeting system of any one of claims 1-7, wherein each target site is in a putative regulatory region of the IL-2 gene, wherein the putative regulatory region is characterized by having one or more of an epigenetic mark, a regulatory feature or a transcription factor motif. 6037971-sf22474-20029.40 9. The epigenetic-modifying DNA-targeting system of claim 8, wherein the epigenetic mark includes Histone H3K27 acetylation.

10. The epigenetic-modifying DNA-targeting system of any of claims 1-9, wherein the at least one transcriptional activator effector domain catalyzes acetylation of histone H3 lysine 27 at the target site or is able to recruit an enzyme that catalyzes acetylation of histone H3 lysine 27 at the target site.

11. The epigenetic-modifying DNA-targeting system of claim 10, wherein the enzyme that catalyzes the acetylation is an acetyltransferase.

12. The epigenetic-modifying DNA-targeting system of claim 10 or claim 11, wherein the enzyme that catalyzes the acetylation is a histone acetyltransferase.

13. The epigenetic-modifying DNA-targeting system of any one of claims 8-12, wherein the putative regulatory region is a promoter or an enhancer.

14. The epigenetic-modifying DNA-targeting system of any of claims 1-13, wherein each target site is in a promoter or an enhancer.

15. The epigenetic-modifying DNA-targeting system of any one of claims 1-14, wherein each target site is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315.

16. The epigenetic-modifying DNA-targeting system of any one of claims 1-15, wherein each target site is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

17. The epigenetic-modifying DNA-targeting system of any one of claims 1-16, wherein at least two of the plurality of DNA-targeting modules target a different target site.

18. The epigenetic-modifying DNA-targeting system of any one of claims 1-15 and 17, wherein the at least two different target sites are in two different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000- 6037971-sf22474-20029.40 122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315.

19. The epigenetic-modifying DNA-targeting system of any one of claims 1-16, 17 and 18, wherein the at least two different targets site are in two different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

20. The epigenetic-modifying DNA-targeting system of any one of claims 1-15, wherein at least three of the plurality of DNA-targeting modules target a different target site.

21. The epigenetic-modifying DNA-targeting system of any one of claims 1-15 and 20, wherein the at least three different target sites are in three different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000- 122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315.

22. The epigenetic-modifying DNA-targeting system of any one of claims 1-16, 20 and 21, wherein the at least three different targets site are in three different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

23. The epigenetic-modifying DNA-targeting system of any one of claims 1-15, wherein at least four of the plurality of DNA-targeting modules target a different target site or at least five of the plurality of DNA-targeting modules target a different target site.

24. The epigenetic-modifying DNA-targeting system of any one of claims 1-15 and 23, wherein the at least four different target sites or the at least five different target sites are in four different target regions corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840- 122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315.

25. The epigenetic-modifying DNA-targeting system of any one of claims 1-16, 23 and 24, wherein the at least four different targets site or the at least five different target sites are in four different target regions corresponding to genomic coordinates human genome assembly 6037971-sf22474-20029.40 GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300- 122,544,050.

26. The epigenetic-modifying DNA-targeting system of any one of claim 1-15, wherein each of the plurality of DNA-targeting modules target a different target site.

27. The epigenetic-modifying DNA-targeting system of any one of claims 1-15 and 26, wherein each target site is in a different target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410- 122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, (7) chr4: 122,576,890-122,579,315.

28. The epigenetic-modifying DNA-targeting system of any one of claims 1-16, 26 and 27, wherein each target site is in a different target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000- 122,508,985, and (4) chr4: 122,539,300-122,544,050.

29. The epigenetic-modifying DNA-targeting system of any one of claims 1-25, wherein at least two of the DNA-targeting modules of the plurality of DNA-targeting modules target the same target region.

30. The epigenetic-modifying DNA-targeting system of any one of claims 1-29, wherein the DNA-binding domain is a zinc finger protein.

31. The epigenetic-modifying DNA-targeting system of any one of claims 1-30, wherein each fusion protein of the plurality of DNA-targeting modules is different.

32. The epigenetic-modifying DNA-targeting system of any of claims 1-29, wherein each of the DNA-targeting modules share the same fusion protein and each comprise a different guide nucleic acid that is complementary to a different target site.

33. The epigenetic-modifying DNA-targeting system of claim 32, wherein the guide nucleic acid is a guide RNA (gRNA).

34. The epigenetic-modifying DNA-targeting system of any one of claims 31-33, wherein the DNA-binding domain of the fusion protein is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof.

35. The DNA-targeting system of any one of claims 1-34, wherein at least one DNA- 6037971-sf22474-20029.40 targeting module targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,451,000-122,460,000.

36. The DNA-targeting system of any one of claims 1-34, wherein at least one DNA- targeting module targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,488,840-122,491,890.

37. The DNA-targeting system of any one of claims 1-34, wherein at least one DNA- targeting module targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,507,000-122,508,985.

38. The DNA-targeting system of any one of claims 1-34, wherein at least one DNA- targeting module targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,539,300-122,544,050.

39. An epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional activator effector domain; and (b) a plurality of guide RNAs (gRNAs) comprising at least two gRNAs that each target a target site of the interleukin-2 (IL-2) gene.

40. The epigenetic-modifying DNA-targeting system of claim 39, wherein the DNA- targeting system increases transcription of the interleukin (IL-2) gene.

41. The epigenetic-modifying DNA-targeting system of claim 39 or claim 40, wherein the plurality of gRNAs is 2-6 gRNAs.

42. The epigenetic-modifying DNA-targeting system of any of claims 39-41, wherein the plurality of gRNAs is 2 gRNAs.

43. The epigenetic-modifying DNA-targeting system of any of claims 39-41, wherein the plurality of gRNAs is 3 gRNAs.

44. The epigenetic-modifying DNA-targeting system of any of claim 39-41, wherein the plurality of gRNAs is 4 gRNAs or is 5 gRNAs.

45. The epigenetic-modifying DNA-targeting system of any of claims 39-44, wherein each target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr4:122,451,261-122,593,946.

46. The epigenetic-modifying DNA-targeting system of any one of claims 39-45, wherein each target site is in a putative regulatory region of the IL-2 gene, wherein the putative 6037971-sf22474-20029.40 regulatory region is characterized by having one or more of an epigenetic mark, a regulatory feature or a transcription factor motif.

47. The epigenetic-modifying DNA-targeting system of claim 46, wherein the epigenetic mark includes Histone H3K27 acetylation.

48. The epigenetic-modifying DNA-targeting system of any of claims 39-47, wherein the at least one transcriptional activator effector domain catalyzes acetylation of histone H3 lysine 27 at the target site or is able to recruit an enzyme that catalyzes acetylation of histone H3 lysine 27 at the target site.

49. The epigenetic-modifying DNA-targeting system of claim 48, wherein the enzyme that catalyzes the acetylation is an acetyltransferase.

50. The epigenetic-modifying DNA-targeting system of claim 48 or claim 49, wherein the enzyme that catalyzes the acetylation is a histone acetyltransferase.

51. The epigenetic-modifying DNA-targeting system of any one of claims 46-50, wherein the putative regulatory region is a promoter or an enhancer.

52. The epigenetic-modifying DNA-targeting system of any of claims 39-51, wherein each target site is in a promoter or an enhancer.

53. The epigenetic-modifying DNA-targeting system of any one of claims 39-52, wherein each target site is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,465,000-122,472,000, (3) chr4: 122,479,410-122,482,750, (4) chr4: 122,488,840-122,491,890, (5) 122,507,000-122,508,985, (6) chr4: 122,539,300-122,544,050, and (7) chr4: 122,576,890-122,579,315.

54. The epigenetic-modifying DNA-targeting system of any one of claims 39-53, wherein each target site is independently located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

55. The DNA-targeting system of any one of claims 39-54, wherein the DNA- targeting system targets at least two different target sites, optionally, 2, 3, 4 or 5 different target sites, wherein each different target site is located within a different target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,451,000-122,460,000, (2) chr4: 122,488,840-122,491,890, (3) 6037971-sf22474-20029.40 122,507,000-122,508,985, and (4) chr4: 122,539,300-122,544,050.

56. An epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional activator effector domain; and (b) at least one guide RNA (gRNA) that targets a target site of the interleukin-2 (IL-2) gene located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,465,000- 122,472,000, (2) chr4: 122,479,410-122,482,750, (3) chr4: 122,488,840-122,491,890, (4) 122,507,000-122,508,985, (4) chr4: 122,539,300-122,544,050, and (6) chr4: 122,576,890- 122,579,315.

57. The epigenetic-modifying DNA targeting system of claim 56, wherein the at least one gRNA targets a target site of the interleukin-2 (IL-2) gene located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,488,840-122,491,890, (2) 122,507,000-122,508,985, and (3) chr4: 122,539,300-122,544,050.

58. The epigenetic-modifying DNA-targeting system of claim 56 or claim 57, wherein the at least one gRNA is 1-6 gRNAs.

59. The epigenetic-modifying DNA-targeting system of any of claims 34-58, wherein the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein.

60. The epigenetic-modifying DNA-targeting system of claim 59, wherein the dCas protein lacks nuclease activity.

61. The epigenetic-modifying DNA-targeting system of claim 59 or claim 60, wherein the dCas protein is a dCas9 protein.

62. The epigenetic-modifying DNA-targeting system of claim 59 or claim 61, wherein the dCas protein is a dCas12 protein.

63. The epigenetic-modifying DNA-targeting system of any of claims 59-61, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

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

62. 6037971-sf22474-20029.40 65. The epigenetic-modifying DNA-targeting system of 63 or claim 64, wherein the dSpCas9 comprises the sequence set forth in SEQ ID NO:63, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

66. The epigenetic-modifying DNA-targeting system of any of claims 63-65, wherein the dSpCas9 is set forth in SEQ ID NO:

63.

67. The epigenetic-modifying DNA-targeting system of any of claims 59-61, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

68. The epigenetic-modifying DNA-targeting system of claim 67, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:

64.

69. The epigenetic-modifying DNA-targeting system of claim 67 or claim 68, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

70. The epigenetic-modifying DNA-targeting system of any of claims 67-69, wherein the dSaCas9 is set forth in SEQ ID NO:

65.

71. The epigenetic-modifying DNA-targeting system of any of claims 33-70, wherein each gRNA comprises a gRNA spacer sequence that is complementary to the target site of the respective gene.

72. The epigenetic-modifying DNA-targeting system of any one of claims 33-55 and 59-71, wherein each gRNA targets a target site in IL-2 comprising the sequence set forth in any one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, a portion of any of the foregoing comprising at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

73. The epigenetic-modifying DNA-targeting system of any one of claims 33-55 and 59-73, wherein each gRNA targets a target site in IL-2 set forth in any one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or a complementary sequence of any of the foregoing. 6037971-sf22474-20029.40 74. The epigenetic DNA-targeting system of any one of claims 33-55, 59-61, 63-66 and 71-73, wherein each gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt.

75. An epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a zinc finger protein (ZFP) or variant thereof, and at least one transcriptional activator effector domain; wherein the ZFP targets a target site of the interleukin-2 (IL-2) gene located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,465,000-122,472,000, (2) chr4: 122,479,410-122,482,750, (3) chr4: 122,488,840-122,491,890, (4) 122,507,000-122,508,985, (4) chr4: 122,539,300-122,544,050, and (6) chr4: 122,576,890-122,579,315.

76. The epigenetic-modifying DNA targeting system of claim 75, wherein the ZFP targets a target site of the interleukin-2 (IL-2) gene located within a target region corresponding to genomic coordinates human genome assembly GRCh38 (hg38) selected from the group consisting of: (1) chr4: 122,488,840-122,491,890, (2) 122,507,000-122,508,985, and (3) chr4: 122,539,300-122,544,050.

77. The epigenetic-modifying DNA-targeting system of claim 75 or claim 76, wherein the ZFP targets a target site in IL-2 comprising the sequence set forth in any one of SEQ ID NOs: 186-188.

78. The epigenetic-modifying DNA-targeting system of any one of claims 75-77, wherein the ZFP targets the target site set forth in SEQ ID NO:

186.

79. The epigenetic-modifying DNA-targeting system of claim 78, wherein the ZFP comprises a zinc finger recognition region comprising six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, selected from F1-F6 as follows: F1: QNAHRKT (SEQ ID NO: 195), F2: RKYYLAK (SEQ ID NO: 196), F3: RSAHLSR (SEQ ID NO: 197), F4: QSGDLTR (SEQ ID NO: 198), F5: RSDHLTQ (SEQ ID NO: 199), and F6: DSANLSR (SEQ ID NO: 200).

80. The epigenetic-modifying DNA-targeting system of claim 78 or 79, wherein the ZFP comprises the sequence set forth in SEQ ID NO: 189, or a portion thereof, or an amino acid 6037971-sf22474-20029.40 sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

81. The epigenetic-modifying DNA-targeting system of any one of claims 78-80, wherein the ZFP comprises the sequence set forth in SEQ ID NO:

189.

82. The epigenetic-modifying DNA-targeting system of any one of claims 78-81, wherein the ZFP is encoded by the sequence set forth in SEQ ID NO: 192 or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

83. The epigenetic-modifying DNA-targeting system of any one of claims 78-82, wherein the ZFP is encoded by the sequence set forth in SEQ ID NO:

192.

84. The epigenetic-modifying DNA-targeting system of any one of claims 75-77, wherein the ZFP targets the target site set forth in SEQ ID NO:

187.

85. The epigenetic-modifying DNA-targeting system of claim 84, wherein the ZFP comprises a zinc finger recognition region comprising six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, selected from F1-F6 as follows: F1: DSSHLEL (SEQ ID NO: 201), F2: DRSNLTR (SEQ ID NO: 202), F3: RSDNLSE (SEQ ID NO: 203), F4: VRRALSS (SEQ ID NO: 204), F5: QSGALAR (SEQ ID NO: 205), and F6: RLDWLPM (SEQ ID NO: 206).

86. The epigenetic-modifying DNA-targeting system of claim 84 or 85, wherein the ZFP comprises the sequence set forth in SEQ ID NO: 190, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

87. The epigenetic-modifying DNA-targeting system of any one of claims 84-86, wherein the ZFP comprises the sequence set forth in SEQ ID NO:

190.

88. The epigenetic-modifying DNA-targeting system of any one of claims 84-87, wherein the ZFP is encoded by the sequence set forth in SEQ ID NO: 193 or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

89. The epigenetic-modifying DNA-targeting system of any one of claims 84-88, wherein the ZFP is encoded by the sequence set forth in SEQ ID NO:

193.

90. The epigenetic-modifying DNA-targeting system of any one of claims 75-77, wherein the ZFP targets the target site set forth in SEQ ID NO:

188. 6037971-sf22474-20029.40 91. The epigenetic-modifying DNA-targeting system of claim 90, wherein the ZFP comprises a zinc finger recognition region comprising six zinc fingers denoted F1 through F6 in order from N-terminus to C-terminus, selected from F1-F6 as follows: F1: RSDNLSV (SEQ ID NO: 207), F2: RSAHLSR (SEQ ID NO: 208), F3: QNAHRKT (SEQ ID NO: 209), F4: LRHHLTR (SEQ ID NO: 210), F5: TSSNRKT (SEQ ID NO: 211), and F6: TSSNLSR (SEQ ID NO: 212).

92. The epigenetic-modifying DNA-targeting system of claim 90 or 91, wherein the ZFP comprises the sequence set forth in SEQ ID NO: 191, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

93. The epigenetic-modifying DNA-targeting system of any one of claims 90-92, wherein the ZFP comprises the sequence set forth in SEQ ID NO:

191.

94. The epigenetic-modifying DNA-targeting system of any one of claims 90-93, wherein the ZFP is encoded by the sequence set forth in SEQ ID NO: 194 or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

95. The epigenetic-modifying DNA-targeting system of any one of claims 90-94, wherein the ZFP is encoded by the sequence set forth in SEQ ID NO:

194.

96. An epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a deactivated Cas9 from Streptococcus pyogenes (dSpCas9) and at least one transcriptional activator effector domain; and (b) at least one guide RNA (gRNA) that targets a target site of the interleukin-2 (IL-2) gene, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt.

97. The epigenetic-modifying DNA-targeting system of claim 96, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO:

62. 6037971-sf22474-20029.40 98. The epigenetic-modifying DNA-targeting system of 96 or claim 97, wherein the dSpCas9 comprises the sequence set forth in SEQ ID NO:63, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

99. The epigenetic-modifying DNA-targeting system of any of claims 96-98, wherein the dSpCas9 is set forth in SEQ ID NO:

63.

100. The epigenetic-modifying DNA-targeting system of any one of claims 33-55 and 59-71, wherein each gRNA targets a target site in IL-2 comprising the sequence set forth in any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59, a portion of any of the foregoing comprising at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing..

101. The epigenetic-modifying DNA-targeting system of any one of claims 33-55, 59- 71 and 100, wherein each gRNA targets a target site in IL-2 set forth in any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59, or a complementary sequence of any of the foregoing.

102. The epigenetic DNA-targeting system of any one of claims 33-55, 59-61, 67-71, 100 and 101, wherein each gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt.

103. An epigenetic-modifying DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain that is a deactivated Cas9 from Staphylococcus aureus (dSaCas9) and at least one transcriptional activator effector domain; and (b) at least one guide RNA (gRNA) that targets a target site of the interleukin-2 (IL-2) gene, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt.

104. The epigenetic-modifying DNA-targeting system of claim 103, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:

64. 6037971-sf22474-20029.40 105. The epigenetic-modifying DNA-targeting system of claim 103 or claim 104, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

106. The epigenetic-modifying DNA-targeting system of any of claims 103-105, wherein the dSaCas9 is set forth in SEQ ID NO:

65.

107. The epigenetic-modifying DNA-targeting system of any of claims 33-74 and 96- 106, wherein each gRNA independently comprises a spacer sequence between 14 nt and 24 nt.

108. The epigenetic-modifying DNA-targeting system of any of claims 33-74 and 96- 107, wherein each gRNA independent comprises a spacer sequence between 16 nt and 22 nt in length.

109. The epigenetic-modifying DNA-targeting system of any of claims 33-74 and 96- 108, wherein each gRNA independently comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

110. The epigenetic DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96-99, and 107-109, wherein each gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt of any of the foregoing.

111. The epigenetic DNA-targeting system of any one of claims 33-55, 59-61, 67-71, and 100-109, wherein each gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt of any of the foregoing.

112. The epigenetic DNA-targeting system of any one of claims 33-74 and 96-111, wherein the DNA-targeting system comprises at least two gRNAs that target the same target site.

113. The epigenetic DNA-targeting system of claim 112, wherein the DNA-targeting system comprises at least two copies of the same gRNA.

114. The epigenetic DNA-targeting system of claim 113, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:

23. 6037971-sf22474-20029.40 115. The epigenetic DNA-targeting system of claim 113 or claim 114, wherein the gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:

23.

116. The epigenetic DNA-targeting system of any one of claims 33-74 and 96-111, wherein the DNA-targeting system comprises at least two gRNAs that target different target sites.

117. The epigenetic DNA-targeting system of any one of claims 33-74, 96-111, and 116, wherein each gRNA of the DNA-targeting system targets a different target site.

118. The DNA-targeting system of any one of claims 33-55 and 59-74, and 96-117, wherein at least one gRNA targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,451,000-122,460,000.

119. The DNA-targeting system of claim 118, wherein the at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or a contiguous portion thereof of at least 14 nt.

120. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, and 112-119, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:11, or a contiguous portion thereof of at least 14nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

11.

121. The DNA-targeting system of claim 118, wherein the least one gRNA comprises a gRNA spacer comprising the sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or a contiguous portion thereof of at least 14 nt.

122. The DNA-targeting system of any one of claims 33-55, 59-61, 67-71, 100-109, 111-113, 116-118, and 121, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

42.

123. The DNA-targeting system of any one of claims 33-74 and 96-122, wherein at least one gRNA targets a target site 50 to 150 kilobases (kb) upstream of the IL-2 transcriptional start site (TSS).

124. The DNA-targeting system of any one of claims 33-74 and 96-123, wherein at least one gRNA targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,488,840-122,491,890. 6037971-sf22474-20029.40 125. The DNA-targeting system of claim 124, wherein the at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or a contiguous portion thereof of at least 14 nt.

126. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 112-120, and 123-125, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:23, or a contiguous portion thereof of at least 14nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

23.

127. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 112-120, and 123-126, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:25 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

25.

128. The DNA-targeting system of claim 124, wherein the least one gRNA comprises a gRNA spacer comprising the sequence set forth in SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, or a contiguous portion thereof of at least 14 nt.

129. The DNA-targeting system of any one of claims 33-55, 59-61, 67-71, 100-109, 111-113, 116-118, 121-124, and 128, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:50, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

50.

130. The epigenetic DNA-targeting system of any one of claims 33-74 and 96-129, wherein at least one gRNA targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,507,000-122,508,985.

131. The DNA-targeting system of claim 130, wherein the at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or a contiguous portion thereof of at least 14 nt.

132. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 112-120, 123-127, 130, and 131, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:27, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

27.

133. The DNA-targeting system of claim 130, wherein the least one gRNA comprises a gRNA spacer comprising the sequence set forth in SEQ ID NO:56, SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt. 6037971-sf22474-20029.40 134. The DNA-targeting system of any one of claims 33-55, 59-61, 67-71, 100-109, 111-113, 116-118, 121-124, 128-130, and 133, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:56, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

56.

135. The DNA-targeting system of any one of claims 33-55, 59-61, 67-71, 100-109, 111-113, 116-118, 121-124, 128-130, 133, and 134, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

58.

136. The DNA-targeting system of any one of claims 33-74 and 96-135, wherein at least one gRNA targets a target site corresponding to genomic coordinates human genome assembly GRCh38 (hg38) chr4: 122,539,300-122,544,050.

137. The DNA-targeting system of claim 136, wherein the at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt.

138. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116-120, 123-127, 130-132, 136, and 137, wherein at least one gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:37, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

37.

139. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116-120, and 123-126, wherein the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:

23.

140. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116-120, and 123-126, wherein the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:

25.

141. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116-120, 123, and 130-132, wherein the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in 6037971-sf22474-20029.40 SEQ ID NO:11 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:

27.

142. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116-120, 123, and 136-138, wherein the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:

37.

143. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116, 117, 123-127, and 136-138, wherein the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:23 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:

37.

144. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116, 117, and 123-127, wherein the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:23 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:

25.

145. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116, 117, 123-127, and 130-132, wherein the DNA-targeting system comprises a first gRNA and a second gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:23 and the second gRNA comprises the spacer sequence set forth in SEQ ID NO:

27.

146. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116-120, 123-127, 130-132, and 136-138, wherein the DNA-targeting system comprises a first gRNA, a second gRNA, and a third gRNA set forth by three gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence set forth in SEQ ID NO:11, a gRNA comprising the spacer sequence set forth in SEQ ID NO:23, a gRNA comprising the spacer sequence set forth in SEQ ID NO:27, and a gRNA comprising the spacer sequence set forth in SEQ ID NO:

37.

147. The DNA-targeting system of any one of claims 33-55, 59-61, 63-66, 71-74, 96- 99, 107-110, 116-120, 123-127, 130-132, and 136-138, wherein the DNA-targeting system comprises a first gRNA, a second gRNA, a third gRNA and a fourth gRNA, wherein the first gRNA comprises the spacer sequence set forth in SEQ ID NO:11, the second gRNA comprises 6037971-sf22474-20029.40 the spacer sequence set forth in SEQ ID NO:23, the third gRNA comprises the spacer sequence set forth in SEQ ID NO:27, and the fourth gRNA comprises the spacer sequence set forth in SEQ ID NO:37, optionally further comprising a fifth gRNA, wherein the fifth gRNA comprises the spacer sequence set forth in SEQ ID NO:

25.

148. The DNA-targeting system of any one of claims 33-55, 59-61, 67-71, 100-109, 111-113, 116-118, 121-124, 128-130, 133, and 134, wherein the DNA-targeting system comprises a first gRNA and a second gRNA set forth by two gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence set forth in SEQ ID NO:42, a gRNA comprising the spacer sequence set forth in SEQ ID NO:50, a gRNA comprising the spacer sequence set forth in SEQ ID NO:56, and a gRNA comprising the spacer sequence set forth in SEQ ID NO:58, optionally wherein the two gRNAs are: (i) a gRNA comprising the spacer set forth in SEQ ID NO:42 and a gRNA comprising the spacer set forth in SEQ ID NO:50, (ii) a gRNA comprising the spacer set forth in SEQ ID NO:42 and a gRNA comprising the spacer set forth in SEQ ID NO:56; (iii) a gRNA comprising the spacer set forth in SEQ ID NO:42 and a gRNA comprising the spacer set forth in SEQ ID NO:58; (iv) a gRNA comprising the spacer set forth in SEQ ID NO:50 and a gRNA comprising the spacer set forth in SEQ ID NO:56; or (v) a gRNA comprising the spacer set forth in SEQ ID NO:50 and a gRNA comprising the spacer set forth in SEQ ID NO:

58.

149. The DNA-targeting system of any one of claims 33-55, 59-61, 67-71, 100-109, 111-113, 116-118, 121-124, 128-130133, and 134, wherein the DNA-targeting system comprises a first gRNA, a second gRNA and a third gRNA set forth by three gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence set forth in SEQ ID NO:42, a gRNA comprising the spacer sequence set forth in SEQ ID NO:50, a gRNA comprising the spacer sequence set forth in SEQ ID NO:56, and a gRNA comprising the spacer sequence set forth in SEQ ID NO:58, optionally wherein the three gRNAs are: (i) a gRNA comprising the spacer set forth in SEQ ID NO:42, a gRNA comprising the spacer set forth in SEQ ID NO:50 and a gRNA comprising the spacer set forth in SEQ ID NO:56; or 6037971-sf22474-20029.40 (ii) a gRNA comprising the spacer set forth in SEQ ID NO:42, a gRNA comprising the spacer set forth in SEQ ID NO:50 and a gRNA comprising the spacer set forth in SEQ ID NO:

58.

150. The epigenetic-modifying DNA-targeting system of any one of claims 96-149, wherein the DNA-targeting system further comprises a fusion protein comprising a DNA- binding domain that is a zinc finger protein (ZFP) or variant thereof, and at least one transcriptional activator effector domain; wherein the ZFP targets a target site in IL-2 comprising the sequence set forth in any one of SEQ ID NOs: 186-188.

151. The epigenetic-modifying DNA-targeting system of any one of claims 1-150, wherein each transcriptional activator effector domain is a NCOA3 domain, a FOXO3 domain, a NCOA3-FOXO3-NCOA3 domain, 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 wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing.

152. The epigenetic-modifying DNA-targeting system of any one of claims 1-151, wherein each transcriptional activator effector domain is p300.

153. The epigenetic-modifying DNA-targeting system of any one of claims 1-151, wherein each transcriptional activator effector domain comprises at least one VP16 domain or a variant or portion thereof that exhibits transcriptional activation activity.

154. The epigenetic-modifying DNA-targeting system of any one of claims 1-151 and 153, wherein each transcriptional activator effector domain comprises a VP16 tetramer (VP64) domain or a variant or portion thereof that exhibits transcriptional activation activity.

155. The epigenetic-modifying DNA-targeting system of any one of claims 1-151, 153, and 154, wherein each transcriptional activator effector domain is a VP64 domain.

156. The epigenetic-modifying DNA-targeting system of any one of claims 1-151, wherein each transcriptional activator effector domain comprises a NCOA3 domain or a variant or portion thereof that exhibits transcriptional activation activity.

157. The epigenetic-modifying DNA-targeting system of any one of claims 1-151, wherein each transcriptional activator effector domain comprises a FOXO3 domain or a variant or portion thereof that exhibits transcriptional activation activity. 6037971-sf22474-20029.40 158. The epigenetic-modifying DNA-targeting system of any one of claims 1-151, 156, and 157, wherein each transcriptional activator effector domain comprises a NCOA3- FOXO3-NCOA3 domain.

159. The epigenetic-modifying DNA-targeting system of any one of claims 1-151 and 156-158, wherein each transcriptional activator effector domain is a NCOA3-FOXO3-NCOA3 domain.

160. The epigenetic-modifying DNA-targeting system of claim 158, wherein each transcriptional activator effector domain further comprises a VP16 tetramer (VP64) domain.

161. The epigenetic-modifying DNA-targeting system of any one of claims 1-151 and 153-155, wherein the at least one transcriptional activator effector domain comprises the sequence set forth in SEQ ID NO: 66, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

162. The epigenetic-modifying DNA-targeting system of any one of claims 1-151 and 153-155, and 161, wherein the at least one transcriptional activator effector domain comprises the sequence set forth in SEQ ID NO:

66.

163. The epigenetic-modifying DNA-targeting system of any one of claims 1-151 and 158-160, wherein the at least one transcriptional activator effector domain comprises the sequence set forth in SEQ ID NO: 181, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

164. The epigenetic-modifying DNA-targeting system of any one of claims 1-151, 158-160, and 163, wherein the at least one transcriptional activator effector domain comprises the sequence set forth in SEQ ID NO:

181.

165. The epigenetic-modifying DNA-targeting system of any one of claims 1-164, wherein the at least one transcriptional activator effector domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain.

166. The epigenetic-modifying DNA-targeting system of any one of claims 1-165, wherein the fusion protein further comprises one or more nuclear localization signals (NLS).

167. The epigenetic-modifying DNA-targeting system of claim 166, wherein the fusion protein further comprises one or more linkers connecting two or more of: the DNA- 6037971-sf22474-20029.40 binding domain, the at least one effector domain, and the one or more nuclear localization signals.

168. The epigenetic-modifying DNA-targeting system of any one of claims 1-55, 59- 61, 63-66, 71-74, 96-99, 107-110, 116-120, 123-126, 130-132, 136-147, 151, and 153-167, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:100 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

169. The epigenetic-modifying DNA-targeting system of any one of claims 1-55, 59- 61, 63-66, 71-74, 96-99, 107-110, 116-120, 123-126, 130-132, 136-147, 151, and 153-168, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:

100.

170. The epigenetic-modifying DNA-targeting system of any one of claims 1-158 and 160-167, wherein the fusion protein comprises any one of the sequences set forth in SEQ ID NOs: 5, 61, 182, and 213-215 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

171. The epigenetic-modifying DNA-targeting system of any one of claims 1-158, 160-167, and 170, wherein the fusion protein comprises any one of the sequences set forth in SEQ ID NOs: 5, 61, 182, and 213-215.

172. The epigenetic-modifying DNA-targeting system of any one of claims 1-55, 59- 61, 63-66, 71-74, 96-99, 107-110, 116-120, 123-126, 130-132, 136-147, 151, 153-155, 161, 162, and 165-167, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:

5.

173. The epigenetic-modifying DNA-targeting system of any one of claims 1-55, 59- 61, 67-74, 96-99, 107-110, 116-120, 123-126, 130-132, 136-147, 151, 153-155, 161, 162, and 165-167, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:

61.

174. The epigenetic-modifying DNA-targeting system of any one of claims 1-55, 59- 61, 63-66, 71-74, 96-99, 107-110, 116-120, 123-126, 130-132, 136-147, 151, and 153-158, and 160-167, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:

182.

175. The epigenetic-modifying DNA-targeting system of any one of claims 75-83, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:

213.

176. The epigenetic-modifying DNA-targeting system of any one of claims 75-77 and 84-89, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:

214.

177. The epigenetic-modifying DNA-targeting system of any one of claims 75-77 and 90-95, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:

215. 6037971-sf22474-20029.40 178. The epigenetic-modifying DNA-targeting system of any one of claims 1-177, wherein transient delivery of the epigenetic-modifying DNA-targeting system to a lymphoid cell promotes increased IL-2 expression, optionally increased compared to a lymphoid cell that has not been delivered the epigenetic-modifying DNA-targeting system.

179. The epigenetic-modifying DNA-targeting system of claim 178, wherein the lymphoid cell is a T cell.

180. The epigenetic-modifying DNA-targeting system of claim 178, wherein the lymphoid cell is a natural killer (NK) cell.

181. The epigenetic-modifying DNA-targeting system of any one of claims 178-180, wherein the lymphoid cell is derived from a primary cell.

182. The epigenetic-modifying DNA-targeting system of any one of claims 178-181, wherein the lymphoid cell is derived from a T or NK cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell.

183. The epigenetic-modifying DNA-targeting system of any one of claims 178-182, wherein the lymphoid cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor.

184. The epigenetic-modifying DNA-targeting system of claim 179, wherein transient delivery of the epigenetic-modifying DNA-targeting system to the T cell promotes increased IL- 2 expression upon T cell stimulation, optionally increased compared to a T cell that has not been delivered the epigenetic-modifying DNA-targeting system.

185. The epigenetic-modifying DNA-targeting system of any one of claims 178-184, wherein the DNA-targeting system increases expression of IL-2 by a log2 fold-change of at or greater than 1.0 in the lymphoid cell contacted with the DNA-targeting system.

186. The epigenetic-modifying DNA-targeting system of any one of claims 178-185, wherein the DNA-targeting system increases expression of IL-2 by a log2 fold-change of at or greater than 2.0 in the lymphoid cell contacted with the DNA-targeting system.

187. The epigenetic-modifying DNA-targeting system of any one of claims 178-186, wherein the DNA-targeting system increases expression of IL-2 by a log2 fold-change of at or greater than 2.5 in the lymphoid cell contacted with the DNA-targeting system.

188. The epigenetic-modifying DNA-targeting system of any one of claims 178-187, wherein the DNA-targeting system increases expression of IL-2 by a log2 fold-change of at or greater than 2.75 in the lymphoid cell contacted with the DNA-targeting system. 6037971-sf22474-20029.40 189. The epigenetic-modifying DNA-targeting system of any of claims 184-188, wherein the T cell stimulation is with an anti-CD3 and anti-CD28 activation reagent.

190. The epigenetic-modifying DNA-targeting system of any of claims 184-189, wherein the T cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor or a T cell receptor (eTCR).

191. The epigenetic-modifying DNA-targeting system of claim 190, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR) directed against an antigen and the T cell stimulation is an antigen-specific stimulation of the CAR or eTCR, optionally wherein the T cell stimulation is with antigen-expressing target cells.

192. The epigenetic-modifying DNA-targeting system of claim 190, wherein the T cell expresses a chimeric antigen receptor (CAR) directed against an antigen and the T cell stimulation is an antigen-specific stimulation of the CAR, optionally wherein the T cell stimulation is with antigen-expressing target cells.

193. The epigenetic-modifying DNA-targeting system of any of claims 190-192, wherein the T cell stimulation is a restimulation after at least one prior T cell stimulation of the T cells.

194. The epigenetic-modifying DNA-targeting system of any of claims 33-55, 59-61, 63-66, 71-74, 96-99, 107-110, 116-120, 123-126, 130-132, 136-147, 151, 153-174, and 178-193, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:

8.

195. The epigenetic-modifying DNA-targeting system of any of claims 33-55, 59-61, 67-71, 100-109, 111-113, 116-118, 121-124, 128-130, 133-136, 148-167, and 178-193, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:

41.

196. The epigenetic-modifying DNA-targeting system of any of claims 1-195, wherein the DNA-targeting system does not introduce a genetic disruption or a DNA break.

197. A guide RNA (gRNA) that targets a target site of the interleukin (IL-2) gene, wherein the target site is selected from a target site comprising the sequence set forth in any one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, a portion of any of the foregoing comprising at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. 6037971-sf22474-20029.40 198. The gRNA of claim 197, wherein the target site is set forth in any one of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or a complementary sequence of any of the foregoing.

199. The gRNA of claim 197 or claim 198, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or a contiguous portion thereof of at least 14 nt.

200. The gRNA of any of claims 197-199, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:11 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

11.

201. The gRNA of any of claims 197-199, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:23 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

23.

202. The gRNA of any of claims 197-199, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:25 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

25.

203. The gRNA of any of claims 197-200, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:27 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

27. 6037971-sf22474-20029.40 204. The gRNA of any of claims 197-200, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:37 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

37.

205. The gRNA of any of claims 197-204, wherein the gRNA comprises a spacer sequence between 14 nt and 24 nt.

206. The gRNA of any of claims 197-205, wherein the gRNA comprises a spacer sequence between 16 nt and 22 nt in length.

207. The gRNA of any of claims 197-205, wherein the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

208. The gRNA of any one of claims 197-207, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:

8.

209. A guide RNA (gRNA) that targets a target site of the interleukin (IL-2) gene, wherein the target site is selected from a target site comprising the sequence set forth in any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59, a portion of any of the foregoing comprising at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

210. The gRNA of claim 209, wherein the target site is set forth in any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59, or a complementary sequence of any of the foregoing.

211. The gRNA of claim 209 or claim 210, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA comprises a gRNA spacer sequence set forth in SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56; or SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt.

212. The gRNA of any of claims 209-211, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:42 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

42. 6037971-sf22474-20029.40 213. The gRNA of any of claims 209-211, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:50 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

50.

214. The gRNA of any of claims 209-211, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:56 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

56.

215. The gRNA of any of claims 209-211, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:58 or a contiguous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:

58.

216. The gRNA of any of claims 209-215, wherein the gRNA comprises a spacer sequence between 14 nt and 24 nt.

217. The gRNA of any of claims 209-216, wherein the gRNA comprises a spacer sequence between 16 nt and 22 nt in length.

218. The gRNA of any of claims 209-217, wherein the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

219. The gRNA of any one of claims 209-218, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:

41.

220. A combination of gRNAs comprising two or more gRNAs, each selected from the gRNA of any of claims 197-208.

221. A combination of gRNAs comprising two or more gRNAs, each selected from the gRNA of any of claims 209-219.

222. A Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof from Streptococcus pyogenes; and (b) at least one gRNA of any of claims 197-208.

223. The Cas-gRNA combination of claim 222, wherein the Cas protein or variant thereof is a deactivated (dSpCas9) protein.

224. The Cas-gRNA combination of claim 223, wherein the dCas protein lacks nuclease activity. 6037971-sf22474-20029.40 225. The Cas-gRNA combination of claim 223 or claim 224, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO:

62.

226. The Cas-gRNA combination of any one of claims 223-225, wherein the dSpCas9 comprises the sequence set forth in SEQ ID NO:63, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

227. The Cas-gRNA combination of any of claims 223-225, wherein the dSpCas9 is set forth in SEQ ID NO:

63.

228. A Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof from Staphylococcus aureus; and (b) at least one gRNA of any of claims 209-219.

229. The Cas-gRNA combination of claim 228, wherein the Cas protein or variant thereof is a deactivated (dSaCas9) protein.

230. The Cas-gRNA combination of claim 229, wherein the dCas protein lacks nuclease activity.

231. The Cas-gRNA combination of claim 229 or claim 230, wherein the dSaCas9 protein comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:

64.

232. The Cas-gRNA combination of any one of claims 229-231, wherein the dSaCas9 comprises the sequence set forth in SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

233. The Cas-gRNA combination of any of claims 229-231, wherein the dSaCas9 is set forth in SEQ ID NO:

65.

234. A polynucleotide encoding the epigenetic-modifying DNA-targeting system of any of claims 1-196.

235. A polynucleotide encoding at least one DNA-targeting module of the epigenetic- modifying DNA-targeting system of any one of claims 1-196.

236. A polynucleotide encoding the fusion protein and the at least one gRNA of the epigenetic-modifying DNA-targeting system of any one of claims 1-196.

237. A polynucleotide encoding the gRNA of any one of claims 197-219.

238. A polynucleotide encoding the combination of gRNAs of claim 220 or claim 221. 6037971-sf22474-20029.40 239. A polynucleotide encoding the Cas-gRNA combination of any one of claims 222- 233.

240. A polynucleotide encoding the fusion protein of the epigenetic-modifying DNA- targeting system of any one of claims 1-196, and one or more gRNAs of any one of claims 197- 219.

241. The polynucleotide of claim 236 or claim 240, wherein the polynucleotide encoding the fusion protein is mRNA.

242. A vector comprising the polynucleotide of any one of claims 234-241.

243. The vector of claim 242, wherein the vector is a viral vector.

244. The vector of claim 242 or claim 243, wherein the vector is an adeno-associated virus (AAV) vector.

245. The vector of claim 244, wherein the vector is selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

246. The vector of claim 242, wherein the vector is a non-viral vector.

247. The vector of claim 246, wherein the non-viral vector is selected from: a lipid nanoparticle, a liposome, an exosome, or a cell penetrating peptide.

248. The vector of claim 246 or claim 247, wherein the non-viral vector is a lipid nanoparticle.

249. The vector of any one of claims 242-248, wherein the vector exhibits immune cell tropism, optionally wherein the vector exhibits T-cell tropism.

250. A modified lymphoid cell comprising the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241.

251. A modified lymphoid cell comprising an epigenetic or phenotypic modification resulting from being contacted by the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241.

252. The modified lymphoid cell of claim 250 or claim 251, wherein the modified lymphoid cell is a modified T cell. 6037971-sf22474-20029.40 253. The modified lymphoid cell of claim 250 or claim 251, wherein the modified lymphoid cell is a modified natural killer (NK) cell.

254. The modified lymphoid cell of any one of claims 250-253, wherein the modified lymphoid cell is derived from a primary cell.

255. The modified lymphoid cell of any one of claims 250-254, wherein the modified lymphoid cell is derived from a T or NK cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell.

256. The modified lymphoid cell of any one of claims 250-255, wherein the modified lymphoid cell further comprises a chimeric antigen receptor (CAR).

257. A modified T cell comprising the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241.

258. A modified T cell comprising an epigenetic or phenotypic modification resulting from being contacted by the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241.

259. The modified T cell of claim 257 or claim 258, wherein the modified T cell is derived from a cell from a subject.

260. The modified T cell of any one of claims 257-259, wherein the modified T cell is derived from a primary T cell.

261. The modified T cell of any one of claims 257-260, wherein the modified T cell is derived from a T cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell.

262. The modified T cell of any one of claims 257-260, wherein the T cell is a tumor infiltrating lymphocyte (TIL).

263. The modified T cell of any one of claims 257-261, wherein the modified T cell further comprises an engineered T cell receptor (eTCR) or chimeric antigen receptor (CAR).

264. A method of increasing the transcription of IL-2 in a lymphoid cell, the method comprising introducing into the lymphoid cell the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs 6037971-sf22474-20029.40 of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241, or the vector of any one of claims 242-249.

265. A method of increasing the production of IL-2 in or by a lymphoid cell, the method comprising introducing into the lymphoid cell the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241, or the vector of any one of claims 242-249.

266. The method of claim 264 or 265, wherein the lymphoid cell is a T cell.

267. The method of claim 264 or 265, wherein the lymphoid cell is a natural killer (NK) cell.

268. The method of claim 264 or 265, wherein the lymphoid cell is derived from a primary cell.

269. The method of claim 264 or 265, wherein the lymphoid cell is derived from a T or NK cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell.

270. The method of any one of claims 264-269, wherein the lymphoid cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor (CAR).

271. A method of increasing the transcription of IL-2 in a T cell, the method comprising introducing into a T cell the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241, or the vector of any one of claims 242-249.

272. A method of increasing the production of IL-2 in or by a T cell, the method comprising introducing into a T cell the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241, or the vector of any one of claims 242-249.

273. The method of claim 271 or claim 272, wherein the T cell is a tumor infiltrating lymphocyte (TIL).

274. The method of claim 271 or claim 272, wherein the T cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor or a T cell receptor (eTCR). 6037971-sf22474-20029.40 275. A method of promoting persistence of an immune cell upon repeat stimulations, the method comprising the method comprising introducing into a T cell the epigenetic- modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241, or the vector of any one of claims 242-249., wherein after the introducing the T cell is subjected to a plurality of repeat stimulations that initiate a T cell activating signal.

276. The method of claim 275, wherein the stimulation is with an anti-CD3 and anti- CD28 activation reagent.

277. The method of claim 275 or claim 276, wherein the T cell is a tumor infiltrating lymphocyte (TIL).

278. The method of claim 275 or claim 276, wherein the T cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor or a T cell receptor (eTCR).

279. The method of claim 278, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR) directed against an antigen and the T cell stimulation is an antigen-specific stimulation of the CAR or eTCR, optionally wherein the T cell stimulation is with antigen-expressing target cells.

280. The method of claim 278 or claim 279, wherein the T cell expresses a chimeric antigen receptor (CAR) directed against an antigen and the T cell stimulation is an antigen- specific stimulation of the CAR, optionally wherein the T cell stimulation is with antigen- expressing target cells.

281. The method of any one of claims 275-280, wherein the T cell stimulation is a restimulation after at least one prior T cell stimulation of the T cells.

282. The method of any one of claims 271-281, wherein the T cell is a T cell in a subject and the method is carried out in vivo.

283. The method of any one of claims 271-281, wherein the T cell is a T cell from a subject, or derived from a cell from the subject, and the method is carried out ex vivo.

284. The method of any one of claims 264-281, wherein the method is carried out in vitro.

285. The method of any one of claims 271-284, wherein the T cell is a primary T cell.

286. The method of any one of claims 271-284, wherein the T cell is derived from a T cell progenitor, a pluripotent stem cell, or an induced pluripotent stem cell. 6037971-sf22474-20029.40 287. The method of any one of claims 271-284, wherein the introducing is by transient delivery into the T cell.

288. The method of any one of claims 264-287, wherein the introducing is by electroporation, transfection, or transduction.

289. A modified lymphoid cell produced by the method of any one of claims 264-270.

290. A modified T cell produced by the method of any one of claims 271-288.

291. A pharmaceutical composition comprising a plurality of modified lymphoid cells of any one of claims 250-256 and 289.

292. A pharmaceutical composition comprising a plurality of modified T cells of any one of claims 257-263 and 290.

293. The pharmaceutical composition of claim 291 or claim 292, comprising a pharmaceutically acceptable excipient.

294. A method of treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising the modified lymphoid cell of any one of claims 250-256 and 289 or the pharmaceutical composition of claim 291 or claim 292.

295. A method of treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising the modified T cell of any one of claims 257-263 and 290 or the pharmaceutical composition of claim 291 or claim 292.

296. The method of claim 295, wherein the modified T cells are an adoptive T cell therapy for treating a disease or condition in the subject.

297. The method of claim 295 or claim 296, wherein the modified T cells are tumor infiltrating lymphocytes (TILs).

298. The method of claim 296, wherein the modified T cells express a recombinant receptor specific for a target antigen associated with the disease or condition.

299. A method of treating a disease or condition in a subject, the method comprising administering to a subject: an adoptive T cell therapy for treating a disease or condition in the subject; and the epigenetic-modifying DNA-targeting system of any one of claims 1-196, the gRNA of any one of claims 197-219, the combination of gRNAs of claim 220 or claim 221, the CRISPR Cas-gRNA combination of any one of claims 222-233, or the polynucleotide of any one of claims 234-241, or the vector of any one of claims 242-249. 6037971-sf22474-20029.40 300. The method of claim 299, wherein the T cells are tumor infiltrating lymphocytes (TILs).

301. The method of claim 299, wherein the T cells express a recombinant receptor specific for a target antigen associated with the disease or condition.

302. The method of claim 298 or claim 301, wherein the recombinant receptor is an engineered T cell receptor (eTCR) or chimeric antigen receptor (CAR).

303. The method of any one of claims 298, 301, and 302, wherein the target antigen is a tumor antigen.

304. The method of any one of claims 294-303, wherein the disease or condition is a cancer.

305. The method of claim 304, wherein the cancer is a hematological cancer or is a solid tumor.

306. The method of any one of claims 294-303, wherein the disease or condition is an autoimmune condition and / or an inflammatory condition.

307. The method of any one of claims 295 and 304-306, wherein the administering increases transcription of IL-2 in lymphoid cells.

308. The method of any one of claims 294-306, wherein the administering increases transcription of IL-2 in T cells. 6037971-sf

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