Compositions and methods for modulating IL-2 gene expression

Abstract

An epigenetically modified DNA targeting system, such as a CRISPR-Cas / guide RNA (gRNA) system, is provided that binds to or targets one or more target sites in an IL-2 gene or a regulatory element thereof in a cell, such as a lymphoid cell (e.g., a T cell). In some aspects, the provided epigenetically modified DNA targeting systems modulate lymphoid cell function, such as lymphoid cell phenotype or activity, such as T cell phenotype or activity. Also provided herein, in some aspects, are methods and uses related to the provided compositions, for example, for modulating lymphoid cells, such as T cells, including in combination with methods of adoptive T cell therapy.

Description

Cross-references to related applications

[0001] This application claims the benefits of 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 disclosures of which are incorporated herein by reference in their entirety.

[0002] The sequence list is incorporated by reference. This application is submitted electronically together with the sequence list. The sequence list is provided as a document named 22474_2002940_SeqList.xml, created on July 16, 2024, and is 548,241 bytes in size. The information in the electronic sequence list is incorporated herein by reference in its entirety. Technical Field

[0003] In some aspects, this disclosure relates to epigenetically modified DNA targeting systems, such as CRISPR-Cas / guide RNA (gRNA) systems, which bind to or target the interleukin-2 (IL-2) gene or its regulatory elements in lymphoid cells (e.g., T cells). In some aspects, the epigenetically modified DNA targeting systems provided by this disclosure regulate lymphoid cell function, such as lymphoid cell phenotype or activity, for example, T cell phenotype or activity. In some aspects, the epigenetically modified targeting systems provided by this disclosure enable transcriptional control or regulation of interleukin (IL-2) expression. In some aspects, this disclosure relates to methods and uses associated with the provided compositions, such as for regulating lymphoid cells (e.g., T cells), including in conjunction with methods of lymphoid cell therapy (e.g., adoptive T cell therapy). Background Technology

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

[0005] In some aspects, this document provides an epigenetically modified DNA targeting system comprising multiple 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 a target site of the IL-2 gene; and (b) at least one transcription activator effector domain. In some of the provided embodiments, the DNA-binding domain of each fusion protein comprises: clustered regularly spaced short palindromic repeat-associated (Cas) protein, zinc finger protein (ZFP), transcription activator-like effector (TALE), a wide range of nucleases, homing endonucleases, or I-SceI enzymes, or variants thereof, optionally wherein the DNA-binding domain comprises a non-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 gRNA capable of targeting the Cas protein to a target site.

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

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

[0008] In some of the provided embodiments, the epigenetic marker includes histone H3K27 acetylation. In some of the provided embodiments, at least one transcription activator effector domain catalyzes the acetylation of histone H3 lysine 27 at the target site, or is capable of recruiting an enzyme that catalyzes the acetylation of histone H3 lysine 27 at the target site. In some of the provided embodiments, the enzyme catalyzing acetylation is an acetyltransferase. In some of the provided embodiments, the enzyme catalyzing acetylation is a histone acetyltransferase.

[0009] In some of the provided embodiments, the presumed regulatory region is a promoter or enhancer. In some of the provided embodiments, the target sites are located within a promoter or enhancer.

[0010] In some of the provided embodiments, each target site is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided embodiments, each target site is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided implementations, at least two of the multiple DNA targeting modules target different target sites. In some of the provided embodiments, at least two distinct target sites are located in two distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 the provided embodiments, at least two distinct target sites are located in two distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided implementations, at least three of the multiple DNA targeting modules target different target sites. In some of the provided embodiments, at least three distinct target sites are located in three distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 the provided embodiments, at least three distinct target sites are located in three distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 the provided embodiments, at least four of the multiple DNA targeting modules target different target sites, or at least five of the multiple DNA targeting modules target different target sites. In some of the provided embodiments, at least four or at least five distinct target sites are located in four distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 the provided embodiments, at least four or at least five different target sites are located in four different target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided implementations, each of the multiple DNA targeting modules targets a different target site. In some of the provided embodiments, the target sites are located in different target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided embodiments, the target sites are located in different target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 the provided embodiments, at least two of the plurality of DNA targeting modules target the same target region.

[0015] In some of the provided embodiments, the DNA-binding domain is a zinc finger protein. In some of the provided embodiments, the fusion proteins of the multiple DNA-targeting modules are different. In some of the provided embodiments, each of the DNA-targeting modules shares the same fusion protein and each contains a different guide nucleic acid complementary to a different target site. In some of the provided embodiments, the guide nucleic acid is guide RNA (gRNA).

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

[0017] In some aspects, this document provides an epigenetically modified DNA targeting system comprising: (a) a fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, the DNA-binding domain being a clustered, regularly spaced short palindromic repeat-associated (Cas) protein or a variant thereof; and (b) a plurality of guide RNAs (gRNAs) comprising at least two gRNAs, each gRNA targeting 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 the provided embodiments, the plurality of gRNAs is 2-6 gRNAs. In some of the provided embodiments, the plurality of gRNAs is 2 gRNAs. In some of the provided embodiments, the plurality of gRNAs is 3 gRNAs. In some of the provided embodiments, the plurality of gRNAs is 4 gRNAs or 5 gRNAs.

[0019] In some of the provided embodiments, the target sites are located within the genomic coordinates of the assembled human genome version GRCh38 (hg38) chr4:122,451,261-122,593,946. In some of the provided embodiments, the target sites are located within the putative regulatory region of the IL-2 gene, wherein the putative regulatory region is characterized by having one or more of an epigenetic marker, a regulatory feature, or a transcription factor motif. In some of the provided embodiments, the epigenetic marker includes histone H3K27 acetylation. In some of the provided embodiments, at least one transcription activator effector domain catalyzes the acetylation of histone H3 lysine 27 at the target site, or is capable of recruiting an enzyme that catalyzes the acetylation of histone H3 lysine 27 at the target site. In some of the provided embodiments, the enzyme catalyzing acetylation is an acetyltransferase. In some of the provided embodiments, the enzyme catalyzing acetylation is a histone acetyltransferase.

[0020] In some of the provided embodiments, the presumed regulatory region is a promoter or enhancer. In some of the provided embodiments, the target sites are located within a promoter or enhancer.

[0021] In some of the provided embodiments, each target site is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided embodiments, each target site is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided embodiments, the DNA targeting system targets at least two distinct target sites, optionally two, three, four, or five distinct target sites, wherein each distinct target site is located within a distinct target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 respects, this paper provides an epigenetically modified DNA targeting system comprising: (a) a fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, the DNA-binding domain being a clustered regularly spaced short palindromic repeat-associated (Cas) protein or a variant thereof; and (b) at least one guide RNA (gRNA) targeting a target site of the interleukin-2 (IL-2) gene located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided embodiments, at least one gRNA targets a target site of the interleukin-2 (IL-2) gene located in a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided implementation schemes, at least one gRNA is 1-6 gRNAs.

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

[0026] In some of the provided implementations, the dCas9 protein is a Streptococcus pyogenes protein. Streptococcus pyogenes The dCas9 (dSpCas9) protein. In some of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, referring to the position number of SEQ ID NO: 62. In some of the provided embodiments, dSpCas9 comprises the sequence shown in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some of the provided embodiments, dSpCas9 is shown in SEQ ID NO: 63.

[0027] In some of the provided implementations, the dCas9 protein is from Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus The dCas9 (dSaCas9) protein. In some of the provided embodiments, dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, referring to the position number of SEQ ID NO: 64. In some of the provided embodiments, the dSaCas9 protein comprises the sequence shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some of the provided embodiments, dSaCas9 is shown in SEQ ID NO: 65.

[0028] In some of the provided embodiments, each gRNA comprises a gRNA spacer sequence complementary to the target site of the corresponding gene. In some of the provided embodiments, each gRNA targets a target site in IL-2 comprising the sequence shown 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, any one of which comprises a portion of at least 14 nucleotides (nt), or the complementary sequence of any one of the aforementioned. In some of the provided embodiments, each gRNA targets a target site in IL-2, which is shown 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 the provided embodiments, each gRNA comprises a gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt.

[0030] In some respects, this paper provides an epigenetically modified DNA targeting system comprising: (a) a fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, the DNA-binding domain being a zinc finger protein (ZFP) or a variant thereof; wherein the ZFP targets a target site of the interleukin-2 (IL-2) gene located in a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates of the following groups: (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, (5) chr4: 122,539,300-122,544,050, and (6) chr4: 122,576,890-122,579,315. In some of the provided embodiments, ZFP targets a target site of the interleukin-2 (IL-2) gene located in a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 the provided embodiments, ZFP targets a target site in IL-2 that contains the sequence shown in any of SEQ ID NO: 186-188.

[0031] In some of the provided embodiments, the ZFP targets the target site shown in SEQ ID NO: 186. In some of the provided embodiments, the ZFP includes a zinc finger recognition region comprising six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, selected from the following F1-F6: 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 of the provided embodiments, the ZFP comprises the sequence shown in SEQ ID NO: 189, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some of the provided embodiments, the ZFP comprises the sequence shown in SEQ ID NO: 189. In some of the provided embodiments, the ZFP is encoded by the sequence shown in SEQ ID NO: 192 or a portion thereof, or by an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some of the provided embodiments, the ZFP is encoded by the sequence shown in SEQ ID NO: 192.

[0032] In some of the provided embodiments, the ZFP targets the target site shown in SEQ ID NO: 187. In some of the provided embodiments, the ZFP includes a zinc finger recognition region comprising six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, selected from the following F1-F6: 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 the provided embodiments, the ZFP comprises the sequence shown in SEQ ID NO: 190, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some of the provided embodiments, the ZFP comprises the sequence shown in SEQ ID NO: 190. In some of the provided embodiments, the ZFP is encoded by the sequence shown in SEQ ID NO: 193 or a portion thereof, or by an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some of the provided embodiments, the ZFP is encoded by the sequence shown in SEQ ID NO: 193.

[0033] In some of the provided embodiments, the ZFP targets the target site shown in SEQ ID NO: 188. In some of the provided embodiments, the ZFP includes a zinc finger recognition region comprising six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, selected from the following F1-F6: 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 the provided embodiments, the ZFP comprises the sequence shown in SEQ ID NO: 191, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some of the provided embodiments, the ZFP comprises the sequence shown in SEQ ID NO: 191. In some of the provided embodiments, the ZFP is encoded by the sequence shown in SEQ ID NO: 194 or a portion thereof, or by an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some of the provided embodiments, the ZFP is encoded by the sequence shown in SEQ ID NO: 194.

[0034] In some respects, this paper provides an epigenetically modified DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, the DNA-binding domain being derived from... Streptococcus pyogenes(a) inactivated Cas9 (dSpCas9); 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 shown 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 continuous portion thereof of at least 14 nt.

[0035] In some of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, referring to the position number of SEQ ID NO: 62. In some of the provided embodiments, dSpCas9 comprises the sequence shown in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some of the provided embodiments, dSpCas9 is shown in SEQ ID NO: 63.

[0036] In some of the provided embodiments, each gRNA targets a target site in IL-2, the target site comprising the sequence shown 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, each of the foregoing comprising a portion of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of the provided embodiments, each gRNA targets a target site in IL-2, the target site being shown 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 the provided embodiments, each gRNA comprises a gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt.

[0038] In some respects, this paper provides an epigenetically modified DNA-targeting system comprising: (a) a fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, the DNA-binding domain being derived from... Staphylococcus aureus (a) inactivated Cas9 (dSaCas9); and (b) at least one guide RNA (gRNA) that targets a target site of the interleukin-2 (IL-2) gene, wherein the gRNA contains a gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt.

[0039] In some of the provided embodiments, dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, referring to the position number of SEQ ID NO: 64. In some of the provided embodiments, the dSaCas9 protein comprises the sequence shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some of the provided embodiments, dSaCas9 is shown in SEQ ID NO: 65.

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

[0041] In some of the provided embodiments, each gRNA comprises a gRNA spacer sequence shown 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 continuous portion of at least 14 nt of any of the foregoing.

[0042] In some of the provided embodiments, each gRNA comprises a gRNA spacer sequence shown 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 continuous portion of at least 14 nt of any of the foregoing.

[0043] In some of the provided embodiments, the DNA targeting system comprises at least two gRNAs targeting the same target site. In some of the provided embodiments, the DNA targeting system comprises at least two copies of the same gRNA. In some of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 23. In some of the provided embodiments, the gRNA comprises the gRNA spacer sequence shown in SEQ ID NO: 23.

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

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

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

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

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

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

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

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

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

[0053] In some 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 shown in SEQ ID NO: 11, the second gRNA comprises the spacer sequence shown in SEQ ID NO: 23, the third gRNA comprises the spacer sequence shown in SEQ ID NO: 27, and the fourth gRNA comprises the spacer sequence shown in SEQ ID NO: 37, and optionally further comprises a fifth gRNA, wherein the fifth gRNA comprises the spacer sequence shown in SEQ ID NO: 25.

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

[0055] In some of the provided embodiments, the DNA targeting system further comprises a fusion protein comprising a DNA-binding domain (which is a zinc finger protein (ZFP) or a variant thereof) and at least one transcription activator effector domain, wherein the ZFP targets a target site in IL-2 comprising the sequence shown in any of SEQ ID NO: 186-288.

[0056] In some of the provided embodiments, the effector domains of each transcription activator are NCOA3 domains, FOXO3 domains, NCOA3-FOXO3-NCOA3 domains, VP64 domains, p65 activation domains, p300 domains, Rta domains, CBP domains, VPR domains, VPH domains, HSF1 domains, TET protein domains (optionally where the TET protein is TET1), SunTag domains, or domains, portions, variants, or truncations of any of the foregoing. In some of the provided embodiments, the effector domain of each transcription activator is p300.

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

[0058] In some of the provided embodiments, each transcription activator effector domain includes an NCOA3 domain or a variant or portion thereof exhibiting transcriptional activation activity. In some of the provided embodiments, each transcription activator effector domain includes a FOXO3 domain or a variant or portion thereof exhibiting transcriptional activation activity. In some of the provided embodiments, each transcription activator effector domain includes an NCOA3-FOXO3-NCOA3 domain. In some of the provided embodiments, each transcription activator effector domain is an NCOA3-FOXO3-NCOA3 domain. In some of the provided embodiments, each transcription activator effector domain also includes a VP16 tetramer (VP64) domain.

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

[0060] In some of the provided embodiments, at least one transcription activator effector domain comprises the sequence shown in SEQ ID NO: 181, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the foregoing. In some of the provided embodiments, at least one transcription activator effector domain comprises the sequence shown in SEQ ID NO: 181.

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

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

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

[0064] In some of the provided embodiments, transient delivery of an epigenetically modified DNA targeting system to lymphoid cells promotes increased IL-2 expression, optionally compared to lymphoid cells not delivered with the epigenetically modified DNA targeting system. In some of the provided embodiments, the lymphoid cells are T cells. In some of the provided embodiments, the lymphoid cells are natural killer (NK) cells. In some of the provided embodiments, the lymphoid cells are derived from primary cells. In some of the provided embodiments, the lymphoid cells are derived from T cell progenitor cells or NK cell progenitor cells, pluripotent stem cells, or induced pluripotent stem cells. In some of the provided embodiments, the modified lymphoid cells express engineered antigen receptors, optionally chimeric antigen receptors.

[0065] In some of the provided embodiments, transient delivery of the epigenetically modified DNA targeting system to T cells promotes an increase in IL-2 expression upon T cell stimulation, optionally compared to T cells without delivery of the epigenetically modified DNA targeting system. In some of the provided embodiments, the DNA targeting system increases IL-2 expression in lymphoid cells in contact with the DNA targeting system by a log2 fold change of 1.0 or greater. In some of the provided embodiments, the DNA targeting system increases IL-2 expression in lymphoid cells in contact with the DNA targeting system by a log2 fold change of 2.0 or greater. In some of the provided embodiments, the DNA targeting system increases IL-2 expression in lymphoid cells in contact with the DNA targeting system by a log2 fold change of 2.5 or greater. In some of the provided embodiments, the DNA targeting system increases IL-2 expression in lymphoid cells in contact with the DNA targeting system by a log2 fold change of 2.75 or greater.

[0066] In some of the provided embodiments, T cell stimulation employs anti-CD3 and anti-CD28 activating agents. In some of the provided embodiments, T cells express engineered antigen receptors, optionally chimeric antigen receptors, or T cell receptors (eTCRs). In some of the provided embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR) against an antigen or an engineered T cell receptor (eTCR), and the T cell stimulation is antigen-specific stimulation of the CAR or eTCR, optionally wherein the T cell stimulation uses target cells expressing the antigen. In some of the provided embodiments, T cells express chimeric antigen receptors (CARs) against an antigen, and the T cell stimulation is antigen-specific stimulation of the CAR, optionally wherein the T cell stimulation uses target cells expressing the antigen. In some of the provided embodiments, T cell stimulation is a restimulation of T cells following at least one prior T cell stimulation.

[0067] In some of the provided embodiments, the gRNA further comprises the scaffold sequence shown in SEQ ID NO: 8. In some of the provided embodiments, the gRNA further comprises the scaffold sequence shown in SEQ ID NO: 41.

[0068] In some of the provided implementations, the DNA targeting system does not introduce gene damage or DNA breakage.

[0069] In some respects, this article provides a guide RNA (gRNA) that targets a target site of the interleukin (IL-2) gene, wherein the target site is selected from the sequence represented by 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, the portion of any one of the foregoing containing at least 14 nucleotides (nt), or the target site of the complementary sequence of any one of the foregoing.

[0070] In some of the provided embodiments, the target site is shown in any 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 the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt; optionally, the gRNA comprises 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: 39, or a continuous portion thereof of at least 14 nt; 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 continuous portion thereof of at least 14 nt.

[0071] In some of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 11 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO: 11. In some of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 23 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO: 23. In some of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 25 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO: 25. In some of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 27 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO: 27. In some of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 37 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO: 37.

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

[0073] In some of the provided embodiments, the gRNA also includes the scaffold sequence shown in SEQ ID NO: 8.

[0074] In some aspects, this document provides a guide RNA (gRNA) that targets a target site of the interleukin (IL-2) gene, wherein the target site is selected from the sequence represented by 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 containing at least 14 nucleotides (nt), or a target site of a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site is shown as 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 the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt; optionally, the gRNA comprises the gRNA spacer sequence shown 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 continuous portion thereof of at least 14 nt.

[0075] In some of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 42 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO: 42. In some of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 50 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO: 50.

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

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

[0078] In some of the provided embodiments, the gRNA also includes the scaffold sequence shown in SEQ ID NO: 41.

[0079] In some aspects, this document provides a combination of two or more gRNAs, each gRNA selected from the gRNAs provided herein.

[0080] In some respects, this article provides a Cas-guide RNA (gRNA) ensemble comprising: (a) from purulent Streptococcus(a) a clustered, regularly spaced short palindromic repeat-associated (Cas) protein or a variant thereof; and (b) at least one gRNA provided herein. In some of the provided embodiments, the Cas protein or a variant thereof is an inactive (dSpCas9) protein. In some of the provided embodiments, the dCas protein lacks nuclease activity. In some of the provided embodiments, the dSpCas9 protein contains at least one amino acid mutation selected from D10A and H840A, referring to the position number of SEQ ID NO: 62. In some of the provided embodiments, dSpCas9 contains the sequence shown in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some of the provided embodiments, dSpCas9 is shown in SEQ ID NO: 63.

[0081] In some respects, this article provides a Cas-guide RNA (gRNA) ensemble comprising: (a) from Golden yellow staphylococcus (a) a clustered, regularly spaced short palindromic repeat-associated (Cas) protein or a variant thereof; and (b) at least one gRNA provided herein. In some of the provided embodiments, the Cas protein or a variant thereof is an inactive (dSaCas9) protein. In some of the provided embodiments, the dCas protein lacks nuclease activity. In some of the provided embodiments, the dSaCas9 protein contains at least one amino acid mutation selected from D10A and N580A, referring to the position number of SEQ ID NO: 64. In some of the provided embodiments, dSaCas9 contains the sequence shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some of the provided embodiments, dSaCas9 is shown in SEQ ID NO: 65.

[0082] In some aspects, this article provides a polynucleotide encoding the epigenetically modified DNA targeting system provided herein. In some aspects, this article provides a polynucleotide encoding at least one DNA targeting module of the epigenetically modified DNA targeting system provided herein. In some aspects, this article provides a polynucleotide encoding a fusion protein and at least one gRNA of the epigenetically modified DNA targeting system provided herein.

[0083] In some aspects, this document provides a polynucleotide encoding the gRNA provided herein. In some aspects, this document provides a polynucleotide encoding a combination of the gRNAs provided herein. In some aspects, this document provides a polynucleotide encoding a combination of Cas-gRNAs provided herein. In some aspects, this document provides a polynucleotide encoding a fusion protein of the epigenetic modification 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, this document provides a vector comprising the polynucleotides provided herein. In some of the provided embodiments, the vector is a viral vector. In some of the provided embodiments, the vector is an adeno-associated virus (AAV) vector. In some of the provided embodiments, the vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

[0085] In some of the provided embodiments, the vector is a non-viral vector. In some of the provided embodiments, the non-viral vector is selected from lipid nanoparticles, liposomes, exosomes, or cell-penetrating peptides. In some of the provided embodiments, the non-viral vector is a lipid nanoparticle. In some of the provided embodiments, the vector exhibits immune cell tropism, optionally wherein the vector exhibits T cell tropism.

[0086] In some respects, this article provides a modified lymphoid cell comprising the epigenetic modified DNA targeting system provided herein, the gRNA provided herein, a combination of gRNAs provided herein, a combination of CRISPR Cas-gRNAs provided herein, or a polynucleotide provided herein.

[0087] In some respects, this article provides a modified lymphoid cell containing epigenetic or phenotypic modifications generated through contact with an epigenetic modified DNA targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a combination of CRISPR Cas-gRNAs provided herein, or a polynucleotide provided herein.

[0088] In some of the provided embodiments, the modified lymphoid cells are modified T cells. In some of the provided embodiments, the modified lymphoid cells are modified natural killer (NK) cells. In some of the provided embodiments, the modified lymphoid cells are derived from primary cells. In some of the provided embodiments, the modified lymphoid cells are derived from T cell progenitor cells or NK cell progenitor cells, pluripotent stem cells, or induced pluripotent stem cells. In some of the provided embodiments, the modified lymphoid cells also contain a chimeric antigen receptor (CAR).

[0089] In some aspects, this document provides a modified T cell comprising the epigenetically modified DNA targeting system provided herein, the gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, or a polynucleotide provided herein. In some aspects, this document provides a modified T cell comprising an epigenetic or phenotypic modification generated by contact with the epigenetically modified DNA targeting system provided herein, the gRNA provided herein, a combination of gRNAs provided herein, a CRISPR Cas-gRNA combination provided herein, or a polynucleotide provided herein. In some of the provided embodiments, the modified T cell is derived from a subject's cells. In some of the provided embodiments, the modified T cell is derived from primary T cells. In some of the provided embodiments, the modified T cell is derived from T cell progenitor cells, pluripotent stem cells, or induced pluripotent stem cells. In some of the provided embodiments, the T cell is a tumor-infiltrating lymphocyte (TIL). In some of the provided embodiments, the modified T cells also include engineered T cell receptors (eTCRs) or chimeric antigen receptors (CARs).

[0090] In some respects, this article provides a method for increasing IL-2 transcription in lymphoid cells, comprising introducing the epigenetically modified DNA targeting system provided herein, the gRNA provided herein, a combination of gRNAs provided herein, a combination of CRISPR Cas-gRNAs provided herein, a polynucleotide provided herein, or a vector provided herein into lymphoid cells.

[0091] In some aspects, this document provides a method for increasing the production of IL-2 in or produced by lymphoid cells, the method comprising introducing an epigenetically modified DNA targeting system provided herein, a gRNA provided herein, a combination of gRNAs provided herein, a combination of CRISPR Cas-gRNAs provided herein, a polynucleotide provided herein, or a vector provided herein into lymphoid cells. In some of the provided embodiments, the lymphoid cells are T cells. In some of the provided embodiments, the lymphoid cells are natural killer (NK) cells. In some of the provided embodiments, the lymphoid cells are derived from primary cells. In some of the provided embodiments, the lymphoid cells are derived from T cell progenitor cells or NK cell progenitor cells, pluripotent stem cells, or induced pluripotent stem cells. In some of the provided embodiments, the lymphoid cells express engineered antigen receptors, optionally chimeric antigen receptors (CARs).

[0092] In some aspects, this document provides a method for increasing the transcription of IL-2 in T cells, the method comprising introducing an epigenetically modified 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 into T cells. In some aspects, this document provides a method for increasing the production of IL-2 in or by T cells, the method comprising introducing an epigenetically modified 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 into T cells. 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 engineered antigen receptors, optionally chimeric antigen receptors, or T cell receptors (eTCRs).

[0093] In some aspects, this document provides a method for promoting the persistence of immune cells under repeated stimulation, the method comprising introducing into T cells an epigenetically modified DNA targeting system, a gRNA, a combination of gRNAs, a CRISPR Cas-gRNA combination, a polynucleotide, or a vector provided herein, wherein, upon introduction, the T cells are subjected to repeated stimulation that initiates T cell activation signals. In some of the provided embodiments, the stimulation employs anti-CD3 and anti-CD28 activating agents. In some of the provided embodiments, the T cells are tumor-infiltrating lymphocytes (TILs). In some of the provided embodiments, the T cells express engineered antigen receptors, optionally chimeric antigen receptors, or T cell receptors (eTCRs). In some of the provided embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR) against an antigen, and the T cell stimulation is an antigen-specific stimulation of the CAR or eTCR, optionally wherein the T cell stimulation employs target cells expressing the antigen. In some of the provided embodiments, the T cells express a chimeric antigen receptor (CAR) against the antigen, and the T cell stimulation is antigen-specific stimulation of the CAR, optionally wherein the T cell stimulation employs target cells expressing the antigen. In some of the provided embodiments, the T cell stimulation is a restimulation of the T cells following at least one prior T cell stimulation. In some of the provided embodiments, the T cells are the subject's T cells, and the method is performed in vivo.

[0094] In some of the provided embodiments, the T cells are the subject's T cells or cells derived from the subject, and the method is performed ex vivo. In some of the provided embodiments, the method is performed in vitro.

[0095] In some of the provided embodiments, the T cells are primary T cells. In some of the provided embodiments, the T cells are derived from T cell progenitor cells, pluripotent stem cells, or induced pluripotent stem cells.

[0096] In some of the provided embodiments, introduction is performed via transient delivery to T cells. In some of the provided embodiments, introduction is performed via electroporation, transfection, or transduction.

[0097] In some respects, this paper provides a modified lymphoid cell produced by the methods presented herein.

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

[0099] In some aspects, this document provides a method for treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising modified lymphoid cells provided herein or a pharmaceutical composition provided herein. In some aspects, this document provides a method for treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising modified T cells provided herein or a pharmaceutical composition provided herein.

[0100] In some of the provided embodiments, the T cells or modified T cells are adoptive T-cell therapies for treating a subject's disease or condition. In some of the provided embodiments, the T cells or modified T cells are tumor-infiltrating lymphocytes (TILs). In some of the provided embodiments, the modified T cells express recombinant receptors specific to target antigens associated with the disease or condition.

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

[0102] 2. In some of the provided embodiments, the T cells are tumor-infiltrating lymphocytes (TILs). In some of the provided embodiments, the T cells express recombinant receptors that are specific to target antigens associated with the disease or symptom. In some of the provided embodiments, the recombinant receptors are engineered T cell receptors (eTCRs) or chimeric antigen receptors (CARs).

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

[0104] In some of the provided embodiments, the disease or symptom is cancer. In some of the provided embodiments, the cancer is a hematologic malignancy or a solid tumor.

[0105] In some of the provided implementation schemes, the disease or symptom is an autoimmune symptom and / or an inflammatory symptom.

[0106] In some of the provided implementations, administration increases the transcription of IL-2 in lymphoid cells.

[0107] In some of the provided implementations, administration increases the transcription of IL-2 in T cells. Attached Figure Description

[0108] Figure 1A The IL-2 locus is shown within the genomic coordinates chr4:122,451,261-122,593,946 of the assembled human genome version GRCh38 (hg38), with 7 distinct annotated regions. Figure 1B This plot shows a comparison of the log2 factor change (log2fc) between two different donors. The large black dots highlighted in the box indicate verified hits. Figure 1C The distribution of target sites within each region of the IL-2 locus is shown.

[0109] Figure 2A The percentage of IL-2 expression was shown after the first stimulation of Her2 CAR T cells, which were delivered with mRNA encoding the dSpCas9-2xVP64 effector fusion protein and multiple SpCas9 IL-2 targeting gRNAs. Figure 2B Showing in the pair with Figure 2A The percentage of IL-2 expression in the same Her2 CAR T cells after a second stimulation is shown. Figure 2C Showing in the pair with Figure 2A The figures show the percentage of IL-2 expression in the same Her2 CAR T cells after a third stimulation. The solid line delineates the percentage of IL-2 expression when using the control guide RNA IL-2 gRNA-1. The dashed line delineates the percentage of IL-2 expression in Her2 CAR T cells (“CAR”).

[0110] Figure 3A The study showed the change in the percentage of IL-2 expression between the first and third stimulations for gRNA IL-2_H, gRNA IL-2_gRNA-1, gRNA SpNT, and other gRNAs. Figure 3B This chart shows the fold increase in IL-2+ CAR T cells relative to the non-targeting gRNA SpNT between the first and third stimulations for gRNAs IL-2_H, IL-2_gRNA-1, and others. Data points representing gRNAs IL-2_H and IL-2_gRNA-1 are indicated. Hollow circles represent data using gRNA SpNT.

[0111] Figure 4The mean fluorescence intensity (MFI) is shown for Her2 CAR T cells transiently expressing the dCas9 effector fusion protein to activate IL-2 targeted by guide RNA IL-2_H, compared to other guide sequences. Data points for gRNA IL-2_H and gRNA IL-2_gRNA-1 are indicated. Hollow circle data points represent data using gRNA SpNT.

[0112] Figure 5A The diagram shows an experimental design designed to test the functional enhancement and cytokine secretion of induced pluripotent stem cell (iPSC)-derived natural killer cells after delivery of a DNA-targeting system that targets IL-2. The procedure involves differentiating iPSCs into immune effector cells (induced natural killer cells or iNK cells), followed by delivery of a CAR lentivirus and mRNA encoding the dSpCas9-2xVP64 fusion protein and gRNA targeting IL-2.

[0113] Figure 5B Tumor cell counts over time were shown after co-incubation with induced natural killer (iNK) cells, CAR+ iNK cells (iNK+CAR), or CAR+ iNK cells delivered with mRNA encoding the dSpCas9-2xVP64 fusion protein and gRNA targeting IL-2 (iNK+CAR+IL-2), normalized to the tumor cell count at the start of the experiment (T0).

[0114] Figure 5C It shows the secretion of IL-2 in induced natural killer (iNK) cells or CAR-T cells, as measured in pg / mL. Figure 5D This study demonstrated the secretion of interferon-γ (IFNγ) in induced natural killer (iNK) cells or CAR-T cells, measured in pg / mL. iNK cells under different conditions were tested, including cells electroporated without CAR lentivirus transduction and without an IL-2-targeting DNA-targeting system (-), iNK cells transduced with CAR lentivirus alone (CAR+), and iNK cells electroporated with both CAR lentivirus transduction and an IL-2-targeting DNA-targeting system (+CAR+IL-2). Figure 6A The study showed that, in the exemplary donor, the fold increase in IL-2+ cells relative to IL-2 activation persistence (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 continuous killing). Figure 6B and Figure 6CSimilarly, in two other exemplary donors, the fold increase in IL-2+ cells relative to IL-2 activation persistence is shown. For each exemplary donor, selected data points representing selected combinations of gRNAs are highlighted.

[0115] Figure 7A The percentage of IL-2 expression was shown after the first stimulation of Her2 CAR T cells, which were delivered with mRNA encoding the dSpCas9-2xVP64 effector fusion protein and multiple SpCas9 IL-2 targeting gRNAs or mRNA encoding the dSaCas9-2xVP64 effector fusion protein and multiple SaCas9 IL-2 targeting gRNAs. Figure 7B Showing in the pair with Figure 7A The percentage of IL-2 expression in the same Her2 CAR T cells after a second stimulation is shown.

[0116] Figure 8 The images show normalized tumor target cell counts during two stimulations of T cells derived from two donors (donor 1 and donor 2), which were delivered with chimeric antigen receptor (CAR) mRNA and mRNA encoding the dSpCas9-2xVP64 effector fusion protein, as well as either IL-2-targeting gRNA (bottom left subfigure) or non-targeting (NT) gRNA (top right subfigure). Additionally, as a negative control, T cells received either no mRNA (mimicry; bottom right subfigure) or only CAR mRNA (top left subfigure).

[0117] Figure 9 Two exemplary dSaCas9 fusion proteins for transcriptional activation are depicted: on the left is dSaCas9 (dSaCas9-NFN-VP64) covalently linked to the effector domains NCOA3-FOXO3-NCOA3 (NFN) and VP64, and on the right is dSaCas9 (dSaCas9-2xVP64) covalently linked to two VP64 domains.

[0118] Figure 10 The intracellular expression of IL-2 after the first stimulation is shown in cells delivered with mRNA encoding dSaCas9-2xVP64, dSaCas9-VP64-NFN, or dSpCas9-2xVP64 mRNA along with the corresponding IL-2-targeting gRNA. The values ​​are averages from both donors, as shown in the % of IL-2-positive cells in the left panel and the mean fluorescence level (MFI; corresponding to the mean expression level) in the right panel. Cells without delivery of the exemplary fusion protein (CAR only) served as a negative control.

[0119] Figures 11-13Showing targeting IL-2 gene region 4 ( Figure 11 ), IL-2 region 5 ( Figure 12 ) and IL-2 transcription initiation site (TSS, Figure 13 This is a comparison of subsets of engineered zinc finger protein (ZFP) target sites. Each solid rectangle represents a given target site and the best-performing ZFP (see Table E4), and guide RNAs (gRNAs) are annotated with boxes.

[0120] Figure 14A The figures show IL-2 expression after delivery of the best-performing IL-2-targeting ZFP fusion protein dSpCas9-2xVP64 and gRNA IL2_H (SpCas9) shown in Table E4, or without delivery (CAR only). IL-2 expression is expressed as the % IL-2 positivity after the first stimulation 72 hours post-delivery (top panel), or the cell count of IL-2 positive CAR T cells after the second stimulation (bottom panel).

[0121] Figure 14B The total cell count is shown 72 hours after delivery of the IL-2-targeting ZFP fusion protein, which showed the best performance as shown in Table E4. As a control, cells were delivered with the dSpCas9 fusion protein and the corresponding IL-2 gRNA (SpCas9) or received no fusion protein (CAR only). Detailed Implementation

[0122] This document provides an epigenetically modified DNA targeting system comprising a plurality of DNA targeting modules for increasing transcription of the interleukin (IL-2) gene, each of which comprises a fusion protein comprising: (a) a DNA-binding domain for targeting a target site of the IL-2 gene; and (b) at least one transcription activator effector domain. This document also provides an epigenetically modified DNA targeting system comprising at least one DNA targeting module consisting of a fusion protein comprising: (a) a DNA-binding domain capable of targeting a target site in the interleukin-2 (IL-2) gene or its regulatory DNA elements in lymphoid cells (e.g., T cells or natural killer cells (also known as NK cells)); and (b) at least one transcription activator effector domain capable of activating transcription of the IL-2 gene or its regulatory elements. In some embodiments, the target site is located in the IL-2 gene or its regulatory region, which is found herein to be a positive regulator of lymphoid cell (e.g., T cell or NK cell) function following transient transcriptional regulation of the IL-2 gene. In some such embodiments, at least one effector domain is a transcriptional activation domain, such as VP64. In some embodiments, the target site is located within 1000 base pairs of the transcription start site (TSS) of the IL-2 gene. The target site may be located within a regulatory region, such as the promoter or enhancer of the IL-2 gene. In some embodiments, the target site is located within 50-150 kb upstream of the IL-2 gene.

[0123] In some implementations, the epigenetic modification DNA targeting system is a synthetic transcription factor capable of increasing (or upregulating) the transcription of the IL-2 gene in a targeted manner. This document provides an epigenetic modification DNA targeting system wherein the DNA-binding domain of each fusion protein comprises: clustered regularly spaced short palindromic repeat-associated (Cas) protein, zinc finger protein (ZFP), transcription activator-like effector (TALE), a wide range of nucleases, homing endonucleases, or I-SceI enzymes, or variants thereof, optionally wherein the DNA-binding domain comprises a non-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 gRNA capable of targeting the Cas protein to a target site.

[0124] In some embodiments, the epigenetically modified DNA-binding domain of the DNA targeting system is a clustered, regularly spaced, short palindromic repeat-associated (Cas) protein (e.g., dCas protein) or a variant thereof, inactive with nucleases, complexed with a guide RNA (gRNA). GRNAs are also provided for targeting target sites in the IL-2 gene or its regulatory DNA elements in lymphoid cells (e.g., T cells or NK cells), wherein transient epigenetic regulation of gene transcription has been found to promote lymphoid cell (e.g., T cell or NK cell) function. CRISPR-Cas / gRNA combinations consisting of gRNA and nuclease-inactivated Cas (such as dCas9) are also provided. This document also provides polynucleotides encoding DNA targeting systems or fusion proteins of DNA targeting systems, as well as vectors and cells containing them. This document also provides methods for using epigenetically modified DNA targeting systems to regulate transcription, phenotype, or function of lymphoid cells (e.g., T cells or NK cells), and the resulting modified cells. This document also provides methods for using epigenetically modified DNA targeting systems to increase transcription of the IL-2 gene or its regulatory elements. This document also provides cells modified using any of the compositions and / or methods provided herein, such as lymphoid cells (e.g., T cells or NK cells).

[0125] In some embodiments, the epigenetic modification DNA targeting system contains at least one DNA targeting module, wherein each DNA targeting module of the system is a component of the DNA targeting system capable of independently targeting a target site such as the provided IL-2 gene or its regulatory element. In some embodiments, each DNA targeting module includes: (a) a DNA-binding domain capable of targeting the target site of the IL-2 gene or regulatory element, and (b) an effector domain capable of increasing (e.g., activating) the transcription of the gene. In some embodiments, the epigenetic modification DNA targeting system contains multiple DNA targeting modules. In some embodiments, the multiple DNA targeting modules are 2-6 DNA targeting modules. In a particular embodiment, the multiple DNA targeting modules are 2 DNA targeting modules. In a particular embodiment, the multiple DNA targeting modules are 3 DNA targeting modules. In a particular embodiment, the multiple DNA targeting modules are 4 DNA targeting modules. In a particular embodiment, the multiple DNA targeting modules are 5 DNA targeting modules.

[0126] In some embodiments, the DNA targeting system includes a single DNA targeting module for targeting activation of IL-2 or increasing its expression. In some embodiments, the DNA targeting module includes: (a) a DNA-binding domain capable of targeting a target site of the IL-2 gene or regulatory element, and (b) an effector domain capable of activating transcription of the gene.

[0127] In some embodiments, the DNA targeting system includes multiple DNA targeting modules, each of which is used to target or increase the expression of a different target site of the IL-2 gene or its regulatory elements. In some embodiments, the DNA targeting system is a multiplex DNA targeting system, i.e., targeting multiple target sites of the IL-2 gene or its regulatory elements. Therefore, the term "DNA targeting system" can include multiplex epigenetic modification DNA targeting systems that include more than one DNA targeting module. The multiplex epigenetic modification DNA targeting systems provided herein may include 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 multiplex epigenetic modification DNA targeting system includes between 2 and 6 DNA targeting modules. In some embodiments, the multiplex epigenetic modification target system includes 2 DNA targeting modules. In some embodiments, the multiplex epigenetic modification target system includes 3 DNA targeting modules. In some embodiments, the multiplex epigenetic modification target system includes 4 DNA targeting modules. In some embodiments, the multiplex epigenetic modification target system comprises five DNA targeting modules. In some embodiments, the multiplex epigenetic modification target system comprises six DNA targeting modules. In some embodiments, the multiple DNA targeting modules target multiple (i.e., multiple) target sites of the IL-2 gene or its regulatory elements.

[0128] In some embodiments, any two DNA targeting modules of the DNA targeting system contain independent (i.e., non-overlapping) components. In some embodiments, different DNA targeting modules of the DNA targeting system contain independent (i.e., non-overlapping) components. For example, the DNA targeting system may include a first DNA targeting module and a second DNA targeting module, the first DNA targeting module containing a first fusion protein containing a DNA-binding domain (e.g., a ZFN- or TALE-based DNA-binding domain) targeting a first target site, and the second DNA targeting module containing a second fusion protein containing a second DNA-binding domain (e.g., a ZFN- or TALE-based DNA-binding domain) targeting a second target site.

[0129] In some embodiments, any two DNA targeting modules of the DNA targeting system may contain shared (i.e., overlapping) components. In some embodiments, different DNA targeting modules of the DNA targeting system contain shared (i.e., overlapping) components. For example, in one aspect, the DNA targeting system may include a first DNA targeting module and a second DNA targeting module, the first DNA targeting module comprising (a) a fusion protein containing a Cas protein and a transcriptional effector (e.g., an activator) domain, and (b) a first gRNA complexed with the Cas protein and targeting a first target site; the second DNA targeting module comprising (a) the fusion protein of the first DNA targeting module, and (b) a second gRNA complexed with the Cas protein and targeting a second target site. It should be understood that providing two or more different gRNAs for a given Cas protein allows the same Cas protein to target two or more gRNA target sites. Conversely, different Cas protein variants (e.g., SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs. Therefore, it is possible to engineer a single DNA targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA targeting modules.

[0130] The provided embodiments relate to compositions and methods for promoting lymphoid cell (e.g., T cell or NK cell) function, such as effector functions of one or more lymphoid cell (e.g., T cell or NK cell) cells, achieved through epigenetic modification of a target site in the IL-2 gene or its regulatory elements. In some embodiments, the method may be used in conjunction with lymphoid cell (e.g., T cell) therapy, such as in conjunction with adoptive T cell therapy. In some embodiments, increasing the transcription of the IL-2 gene or its regulatory elements enhances or improves the phenotype or function of one or more lymphoid cell (e.g., T cell or NK cell) cells. In some embodiments, lymphoid cell (e.g., T cell or NK cell) effector functions with increased IL-2 production capacity are enhanced. In some embodiments, lymphoid cell (e.g., T cell or NK cell) effector functions are enhanced, such as the ability to produce cytokines (e.g., IL-2 or IFN-γ (IFNg)), the ability of lymphoid cells (e.g., T cells or NK cells) to proliferate, the ability of lymphoid cells (e.g., T cells or NK cells) to kill target cells, or the ability of lymphoid cells (e.g., T cells or NK cells) to exhibit a sustained immune response. In certain embodiments, activation of the IL-2 gene or its regulatory elements improves lymphoid cell (e.g., T cell or NK cell) effector functions following or at the time of lymphoid cell (e.g., T cell or NK cell) stimulation, including after successive stimulation under conditions of repeated antigen encounters occurring in a mimicry.

[0131] Administering T cells targeting specific antigens, also known as adoptive cell therapy (ACT), is a promising approach for treating diseases such as cancer. However, current ACT treatments face challenges, including suboptimal T cell function, expansion, and persistence. Furthermore, the persistence and functionality of metastatic T cells can vary significantly between different T cell subsets and between T cells from different patients. Recent clinical trials of ACT have shown that the ability to persist long-term in the circulatory system depends on the T cell's differentiation stage, including its ability to retain networks of transcription factors and metabolic regulators (Pilipow K. et al., Journal of Clinical Investigation Insight 2018;3(18):e122299). T cells metastasized to patients are often terminally differentiated and therefore cannot persist long-term, ultimately limiting effective antitumor responses. For example, although the first CAR-T cell therapy was approved by the FDA as a cell and gene therapy in 2017, patients with recurrent cancer or unresponsive to treatment are often hampered by the lack of persistence of CAR T cells (Mueller et al., Blood (2018)). Moreover, durable benefits of CAR T cell therapy have not yet been observed in solid tumors.

[0132] To mitigate these challenges and enhance the persistence, expansion, and antitumor activity of chimeric antigen receptor (CAR) engineered T cells, a variety of strategies have been tested in preclinical and clinical settings. For example, strategies for optimizing in vitro T cell culture conditions have been explored to date, including the addition of cytokines during manufacturing (Besser MJ, Cytotherapy 2009;11(2):206-17); expression of cytokines and / or receptors by CAR T cells (Krenciute G., Cancer Immunol Res. 2017 07;5(7):571-581); the use of pharmacological inhibitors during expansion to inhibit signaling pathways such as the AKT pathway (Urak R. et al., Journal of Immunotherapy Cancer 2017 Mar 21;5:26) or the PI3K pathway (Peterson CT et al., Blood Advances 2018 Feb 13;2(3):210-223); immune clearance and checkpoint blockade (Cherkassky L. et al., Journal of Clinical Investigation 2016 Aug 1;126(8):3130-44). However, existing strategies are not entirely satisfactory. In some cases, concerns about cytokine-induced toxicity or the development of lymphoproliferative disorders resulting from the above strategies have prompted the search for alternative approaches.

[0133] The provided embodiments involve identifying genomic locations in the IL-2 gene or its regulatory elements that are epigenetically modified in lymphoid cells (e.g., T cells or NK cells) to influence or promote lymphoid cell (e.g., T cell or NK cell) effector function, such as after stimulation (e.g., T cell stimulation), including those induced in a TCR and / or CAR-dependent manner, as demonstrated by evaluation of cells that produce IL-2. The provided embodiments involve identifying genomic locations in lymphoid cells (e.g., T cells or NK cells) that are epigenetically modified to influence or promote lymphoid cell (e.g., T cell or NK cell) effector function, including those induced in a TCR and / or CAR-dependent manner after stimulation (e.g., T cell stimulation), as demonstrated by evaluation of cells that produce IL-2, have proliferative capacity, or have the ability to kill target cells. In some embodiments, the stimulating condition or agent includes one or more agents, such as ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or activates the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include antibodies, such as antibodies specific to TCR components and / or co-stimulatory receptors, such as anti-CD3, anti-CD28, for example, binding to a solid support (such as microbeads); and / or one or more cytokines. In some embodiments, one or more agents are PMA and ionomycin. In some embodiments, the stimulation (e.g., T cell stimulation) is antigen-specific stimulation, wherein cells are stimulated with an agent providing an antigen or its epitope that is specific to or recognized by an antigen receptor (e.g., CAR) expressed on lymphoid cells (e.g., T cells or NK cells). For example, the stimulant may include target cells expressing an antigen. In certain embodiments, the phenotype is or includes the production or secretion of cytokines, such as IL-2 or IFN-γ, in response to stimulation (e.g., T cell stimulation). The production and / or secretion of cytokines contribute to the immune response and participate in various processes, including the induction of antiviral proteins and the induction of lymphoid cell (e.g., T cell 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 can be measured, detected, and / or quantified using any suitable technique known in the art.

[0134] In some implementations, lymphoid cells (e.g., T cells or NK cells) function to produce one or more cytokines. In specific implementations, the production of one or more cytokines is measured, detected, and / or quantified by intracellular cytokine staining. Intracellular cytokine staining (ICS) performed by flow cytometry is a technique well-suited for studying cytokine production at the single-cell level. This technique detects the production and accumulation of cytokines within cells (such as in the endoplasmic reticulum) after cell stimulation, allowing the identification of cell populations that are positive or negative for the production of specific cytokines, or the separation of high-producing and low-producing cells based on thresholds. ICS can also be combined with other flow cytometry protocols, used for immunophenotypic analysis with cell surface markers, or combined with MHC multimers to obtain cytokine-producing capacity in specific cell subpopulations, making ICS a flexible and versatile approach. Other single-cell techniques for measuring or detecting cytokine production include, but are not limited to, ELISPOT, extreme dilution, and lymphoid cell (e.g., T cell or NK cell) cloning.

[0135] In some aspects, certain target sites of this disclosure are identified through screening methods involving transient delivery, wherein a DNA-binding domain-effectant fusion protein (also known as an “epigenetic editor”) is transiently delivered to lymphoid cells (e.g., T cells or NK cells) (i.e., by means of a method resulting in transient expression and / or presence of the fusion protein in lymphoid cells (e.g., T cells or NK cells)), followed by primary or successive stimulation of the cells to assess the effect on cytokines of functional lymphoid cells (e.g., T cells or NK cells). This paper finds that transient delivery of epigenetically modified DNA targeting systems allows for the identification of genomic targets whose regulation significantly affects the function of lymphoid cells (e.g., T cells or NK cells), but does not require the permanent presence of the epigenetically modified DNA targeting system and / or stable knockdown or knockout of the IL-2 gene or regulatory elements. This approach is advantageous because it allows for the identification of target sites that offer superior safety profiles because their regulation does not depend on permanent editor integration (such as via lentiviral transduction). Furthermore, the transient screening strategy allows for the identification of target sites where the effects of epigenetically modified DNA targeting systems are persistent and not masked by permanent integration into and expression of the genome. This contrasts with other screening methods that have employed lentiviral delivery of DNA systems (Schmidt et al. 2022 Science, 375, DOI: 10.1126 / science.abj4008; Freimer et al. 2022 Nature Genetics, 54:1133-1144).

[0136] The provided implementation schemes can be used to target the IL-2 gene or its regulatory elements, which, when transcribed through epigenetic modifications, can greatly assist or promote lymphoid cell (e.g., T cell or NK cell) function, including the effector activity required for lymphoid cell (e.g., T cell or NK cell) persistence and function. Such lymphoid cell (e.g., T cell or NK cell) characteristics are expected to produce durable effector functions, such as the ability to produce IL-2 upon TCR or antigen stimulation. Such lymphoid cell (e.g., T cell or NK cell) characteristics are expected to produce durable effector functions with superior fitness / proliferative benefits and the ability to produce proliferative cytokines (e.g., IL-2) and / or cytotoxic cytokines (e.g., IFNg) upon TCR or antigen stimulation. In particular, the provided implementation schemes provide epigenetic DNA-targeting systems (i.e., “epi-editing systems”) and methods that can provide durable effector functions with superior fitness. This approach provides an important clinical solution to circumvent the problems of lymphoid cell (e.g., T cell or NK cell) persistence, suboptimal function, and / or exhaustion. Furthermore, epigenetic modifications to cells do not alter DNA at the sequence level, thus avoiding the safety concerns associated with gene editing methods. The ability to epigenetically control the differentiation fate of lymphoid cells (e.g., T cells or NK cells) provides a favorable method for increasing the percentage or number of lymphoid cells (e.g., T cells or NK cells) in a lymphoid cell (e.g., T cells or NK cells) population.

[0137] All publications (including patent documents, scientific literature, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication were incorporated individually by reference. Where the definitions listed herein contradict or otherwise differ from those listed in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions listed herein shall prevail over those incorporated herein by reference.

[0138] The chapter titles used in this article are for organizational purposes only and are not intended to limit the topics described.

[0139] I. DNA Targeting Systems This document provides an epigenetically modified DNA targeting system comprising multiple 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 a target site of the IL-2 gene; and (b) at least one transcription activator effector domain. In some embodiments, a DNA targeting system is provided capable of specifically targeting a target site of the IL-2 gene or a regulatory element thereof and activating transcription of the IL-2 gene or a regulatory element thereof. In some embodiments, the DNA targeting system targets one or more target sites of the IL-2 gene or a regulatory element thereof and increases (e.g., activates) the transcription of the gene. In some embodiments, the IL-2 gene is located in lymphoid cells (such as T cells). In some embodiments, the IL-2 gene is located in lymphoid cells (such as NK cells). In some embodiments, the target site of the IL-2 gene is a target site in the IL-2 gene or a regulatory DNA element thereof. In some embodiments, transcriptional regulation is an increase in transcription of the IL-2 gene or a regulatory element thereof. In the provided embodiments, for the targeted IL-2 gene or its regulatory element, the DNA targeting system includes a fusion protein comprising a DNA-binding domain that binds to the target site of the gene and an effector domain for increasing transcription of the IL-2 gene or its regulatory element. In some embodiments, the provided DNA targeting system is capable of regulating (e.g., increasing) transcription of the IL-2 gene or its regulatory element in cells. In some embodiments, transcriptional regulation of gene expression via the DNA targeting system provided herein can promote or improve lymphoid cell function. In a particular embodiment, the provided DNA targeting system promotes lymphoid cell (e.g., T cell or NK cell) function, such as one or more lymphoid cell (e.g., T cell or NK cell) effector functions, through epigenetic modification of the target site in the IL-2 gene or its regulatory element.

[0140] In some embodiments, at least one effector domain is a transcription activator effector domain for increasing transcription of the IL-2 gene or its regulatory elements (e.g., activating or increasing transcription of the gene compared to transcription in the absence of a DNA targeting system), such as any effector domain for transcriptional activation. In some embodiments, the effector domain is a transcription activator effector domain.

[0141] In some embodiments, the effector domain directly or indirectly leads to increased transcription of the IL-2 gene or its regulatory elements. In some embodiments, the effector domain induces, catalyzes, or causes transcriptional activation. In some embodiments, the effector domain induces transcriptional activation. In some aspects, the effector domain comprises: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain (optionally wherein the TET protein is TET1), a SunTag domain, or a domain, portion, variant, or truncated form of any of the foregoing. In some embodiments, the effector domain is VP64.

[0142] In some embodiments, the DNA targeting system includes a fusion protein comprising (a) at least one DNA-binding domain capable of targeting a target site; and (b) at least one effector domain capable of increasing transcription of the IL-2 gene or its regulatory elements. In some embodiments, at least one effector domain is a transcription activator effector domain. The fusion protein may be any suitable fusion protein, such as that described in section IF.

[0143] In some embodiments, the DNA-binding domain comprises or is derived from a CRISPR-associated (Cas) protein, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a wide range of nucleases, homing endonucleases, I-SceI enzymes, 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 an inactivated Cas9 (dCas9) protein or a variant thereof, which is catalytically inactive such that it is inactive for nuclease activity and cannot cleave DNA. The DNA-binding domain may be any suitable DNA-binding domain, such as those described in sections IC and ID.

[0144] In some embodiments, the DNA-binding domain comprises or is derived from the Cas protein or a variant thereof, such as Cas or dCas without nuclease activity (e.g., dCas9), and the DNA-targeting system comprises one or more guide RNAs (gRNAs), such as combinations of gRNAs (e.g., two or three gRNAs). In some embodiments, the gRNA comprises a spacer region sequence capable of targeting and / or hybridizing to a target site. In some embodiments, the gRNA is capable of complexing with the Cas protein or a variant thereof. In some aspects, the gRNA guides or recruits the Cas protein or a variant thereof to the target site. The gRNA may be any suitable gRNA, such as that described in section IC2.

[0145] In some embodiments, the DNA targeting system is used to increase the transcription of the IL-2 gene or its regulatory elements, and the fusion protein of its DNA targeting module is a dCas9-VP64 fusion protein, such as the dCas9-2xVP64 fusion protein. In some embodiments, the fusion protein is any fusion protein as described herein (e.g., in section IF).

[0146] The following sections provide exemplary components and characteristics of DNA targeting systems.

[0147] A. DNA targeting modules and multiple DNA targeting systems In some embodiments, the epigenetically modified DNA targeting system contains at least one DNA targeting module, wherein each DNA targeting module of the system is a component of the DNA targeting system capable of independently targeting a target site of the IL-2 gene or its regulatory elements. In some embodiments, each DNA targeting module includes (a) a DNA-binding domain capable of targeting the target site, and (b) an effector domain for increasing transcription of the IL-2 gene or its regulatory elements. In some embodiments, the DNA-binding domain of at least one DNA targeting module comprises: clustered regularly spaced short palindromic repeat-associated (Cas) protein, zinc finger protein (ZFP), transcription activator-like effector (TALE), a wide range of nucleases, homing endonucleases, or I-SceI enzymes, or variants thereof, optionally wherein the DNA-binding domain comprises a non-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 gRNA capable of targeting the Cas protein to the target site.

[0148] In some embodiments, the DNA targeting module is a CRISPR / Cas-based DNA targeting module. In some embodiments, in the CRISPR / Cas-based DNA targeting module, the DNA-binding domain of the fusion protein is a Cas protein or a variant thereof (e.g., a dCas protein, such as dCas9), and the DNA targeting module also includes gRNA for targeting the DNA-binding domain to a target site.

[0149] In some implementations, the DNA targeting module is a zinc finger protein (ZFP)-based DNA targeting module. In some implementations, in the ZFP-based DNA targeting module, the DNA-binding domain of the fusion protein is an engineered zinc finger protein (eZFP).

[0150] In some implementations, the DNA targeting module is a transcription activator-like effector (TALE) based DNA targeting module. In some implementations, in the TALE-based DNA targeting module, the DNA-binding domain of the fusion protein is an engineered TALE.

[0151] In some embodiments, the DNA targeting system includes multiple DNA targeting modules, each targeting a different target site in the IL-2 gene or its regulatory elements. In some embodiments, the DNA targeting system includes multiple DNA targeting modules, each targeting the same target site in the IL-2 gene or its regulatory elements. In some embodiments, the DNA targeting system is a multiplex DNA targeting system. In some embodiments, the multiplex DNA targeting system targets different target sites in the IL-2 gene or its regulatory elements. In some embodiments, the multiplex DNA targeting system targets the same target site in the IL-2 gene or its regulatory elements. Therefore, the term "DNA targeting system" can include a multiplex epigenetic modification DNA targeting system containing more than one DNA targeting module. In some embodiments, the multiplex epigenetic modification DNA targeting system contains 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 modification DNA targeting system contains multiple DNA targeting modules. In some embodiments, the multiple DNA targeting modules are 2-6 DNA targeting modules. In a particular embodiment, the plurality of DNA targeting modules is two DNA targeting modules. In a particular embodiment, the plurality of DNA targeting modules is three DNA targeting modules. In a particular embodiment, the plurality of DNA targeting modules is four DNA targeting modules. In a particular embodiment, the plurality of DNA targeting modules is five DNA targeting modules.

[0152] In some embodiments, any two DNA targeting modules of the DNA targeting system may contain independent (i.e., non-overlapping) components. For example, the DNA targeting system may include a first DNA targeting module and a second DNA targeting module, the first DNA targeting module containing a first fusion protein having a DNA-binding domain (e.g., a ZFN- or TALE-based DNA-binding domain) targeting a first target site, and the second DNA targeting module containing a second fusion protein having a second DNA-binding domain (e.g., a ZFN- or TALE-based DNA-binding domain) targeting a second target site.

[0153] In some embodiments, any two DNA targeting modules of the DNA targeting system may contain shared (i.e., overlapping) components. For example, the DNA targeting system may include: i) a first DNA targeting module comprising (a) a fusion protein containing a Cas protein and an effector domain, and (b) a first gRNA complexed with the Cas protein and targeting 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 complexed with the Cas protein and targeting a second target site. It should be understood that providing two or more different gRNAs for a given Cas protein allows the Cas protein to target two or more gRNA target sites. Conversely, different Cas protein variants (e.g., SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, as described herein. Therefore, it is possible to engineer a single DNA targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA targeting modules.

[0154] In some aspects, this document provides an epigenetically modified DNA targeting system comprising multiple DNA targeting modules for increasing transcription of the IL-2 gene or its regulatory elements. In some embodiments, the multiple DNA targeting modules comprise a first DNA targeting module and a second DNA targeting module, the first DNA targeting module being used to increase transcription of the IL-2 gene or its regulatory elements by targeting a first target site, and the second DNA targeting module being used to increase transcription of the IL-2 gene or its regulatory elements 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 the IL-2 gene or its regulatory elements of the DNA targeting module, and (b) at least one effector domain. In some embodiments, each DNA targeting module comprises a transcription activator effector domain for increasing transcription of the IL-2 gene or its regulatory elements. B. IL-2 gene target sites that promote the activation and function of lymphocytes (e.g., T cells or NK cells). In some respects, this document provides target sites for the IL-2 gene or its regulatory elements, wherein increased transcription of the IL-2 gene or its regulatory elements promotes the activation or function of lymphoid cells (e.g., T cells or NK cells). In some embodiments, any of the provided DNA targeting systems is used to target the target site.

[0155] In some embodiments, the target site is located in the IL-2 gene or its regulatory elements, wherein increased gene expression promotes lymphoid cell activation or function. In some embodiments, the target site is located in the IL-2 gene or its regulatory elements, wherein increased gene expression promotes lymphoid cell activation or function (e.g., T cells or NK cells). In some embodiments, the target site is located in the IL-2 gene or its regulatory elements, wherein increased gene expression promotes natural killer cell activation or function.

[0156] In some embodiments, the target site is targeted by a DNA targeting system, such as by a DNA targeting module of a DNA targeting system, as described herein. In some embodiments, the target site is a target site of the IL-2 gene or a regulatory element thereof. In some embodiments, the target site of the IL-2 gene is located within the IL-2 gene body or its regulatory DNA element. In some embodiments, the target site is a target site within the IL-2 gene. In some embodiments, the gene is the IL-2 gene or a regulatory element thereof. In some embodiments, the gene is the IL-2 gene or a regulatory element thereof in a cell. In some embodiments, the cell is an immune cell, such as a T cell or NK cell. In some embodiments, this document provides a multiple epigenetic modification DNA targeting system that targets a combination of at least two target sites of the IL-2 gene or a regulatory element thereof as described herein.

[0157] In some embodiments, the DNA targeting system targets or binds to a target site in the IL-2 gene or its regulatory elements, such as any target site described herein. In some embodiments, the target site is located in the IL-2 gene or its regulatory elements. In some embodiments, the regulatory DNA element is a sequence that a gene regulatory protein can bind to and influence gene transcription. 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 an 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 a gene. In some embodiments, the target site is located within a promoter, enhancer, exon, intron, untranslated region (UTR), 5' UTR, or 3' UTR of a gene. In some embodiments, the regulatory DNA element is a promoter. In some embodiments, the promoter is a nucleotide sequence that binds to RNA polymerase to initiate gene transcription. In some embodiments, the promoter is a nucleotide sequence located within approximately 100 bp, approximately 500 bp, approximately 1000 bp, or longer from the transcription start site of the gene. In some embodiments, the promoter is located within 500 bp of the transcription start site of the gene. In some embodiments, the target site is located within a sequence suspected of controlling an unknown or known function that controls gene expression. In some embodiments, the target site is located within 50 to 150 kb upstream of the IL-2 gene, including the terminal value.

[0158] 1. Lymphoid cells and regulated effector functions In some embodiments, the provided DNA targeting system and / or DNA targeting provides transcriptional activation to increase the expression of the IL-2 gene or its regulatory elements. In some embodiments, the IL-2 gene is a gene whose expression regulates cellular phenotype. In some embodiments, the IL-2 gene is capable of regulating the phenotype of lymphoid cells (e.g., T cells or NK cells). In some embodiments, increased expression of the IL-2 gene or its regulatory elements regulates the phenotype. In some embodiments, regulated expression of the IL-2 gene promotes enhanced lymphoid cell function. In some embodiments, regulated expression of the IL-2 gene promotes enhanced T cell effector function upon T cell stimulation. In some embodiments, regulated expression of the IL-2 gene promotes enhanced NK cell effector function.

[0159] In some embodiments, the enhanced lymphoid cell effector function is improved compared to lymphoid cells in which IL-2 gene expression is not increased using the provided DNA targeting system. In some embodiments, the enhanced T cell effector function is improved compared to T cells in which IL-2 gene expression is not increased using the provided DNA targeting system. In some embodiments, the enhanced NK cell effector function is improved compared to NK cells in which IL-2 gene expression is not increased using the provided DNA targeting system. Methods for modulating T cell function or other lymphoid cell functions using the provided DNA targeting system are further described below and in Section IV.

[0160] In some embodiments, the IL-2 gene is increased via a DNA-targeting system (such as any DNA-targeting system provided herein). In some embodiments, the DNA-targeting system is transiently delivered to cells. In some embodiments, delivery of the DNA-targeting system (e.g., via transient delivery) promotes enhanced effector function of modified lymphoid cells. In some embodiments, lymphoid cell effector function is enhanced compared to comparable lymphoid cells that have not received the DNA-targeting system. In some embodiments, delivery of the DNA-targeting system (e.g., via transient delivery) promotes enhanced effector function of T cells upon T cell stimulation. In some embodiments, T cell effector function is enhanced compared to comparable T cells that have not received the DNA-targeting system. In some embodiments, delivery of the DNA-targeting system (e.g., via transient delivery) promotes enhanced effector function of natural killer cells. In some embodiments, NK cell effector function is enhanced compared to comparable NK cells that have not received the DNA-targeting system.

[0161] In some respects, transient delivery refers to any delivery method that results in the expression and / or presence of one or more components of a DNA-targeting system in a cell for a limited time. For example, delivering mRNA encoding a fusion protein of a DNA-targeting system (e.g., via electroporation) into a cell can result in transient expression of the fusion protein in the cell, e.g., until the mRNA is degraded. In other instances, the DNA-targeting system may be expressed by one or more nucleic acids encoding the DNA-targeting system, wherein the nucleic acid encoding the DNA-targeting system is not incorporated into the cell's genome and is eventually degraded and / or removed from the cell, so that the expression of the DNA-targeting system is not sustained. In other instances, one or more components of a DNA-targeting system, such as a fusion protein and optionally gRNA, may be synthesized in vitro and delivered directly to cells (e.g., via electroporation) without an expression vector, resulting in the transient presence of the DNA-targeting system, e.g., until the fusion protein and / or gRNA are degraded. In some respects, transient delivery differs from non-transient delivery methods that result in stable expression, such as methods involving the incorporation of an expression vector of a DNA-targeting system or its components into the cell's genome.

[0162] In some embodiments, delivery of a DNA targeting system, such as transient delivery, to cells (e.g., lymphoid cells, such as T cells or NK cells) promotes a phenotype of the cells (e.g., lymphoid cells, such as T cells or NK cells). In some embodiments, the phenotype is activation or enhanced function of the cells (e.g., lymphoid cells, such as T cells or NK cells). In some embodiments, delivery of a DNA targeting system, such as transient delivery, to cells (e.g., modified lymphoid cells, such as T cells or NK cells) promotes activation or enhanced function of the cells (e.g., lymphoid cells, such as T cells or NK cells). In some embodiments, the phenotype is modified lymphoid cell function in response to stimulation by modified lymphoid cells. In some embodiments, the phenotype is enhanced T cell effector function in response to T cell stimulation. In some embodiments, the phenotype is enhanced NK cell effector function in response to NK cell stimulation. In some embodiments, lymphoid cell function is enhanced compared to lymphoid cells without delivery of the epigenetically modified DNA targeting system. In some embodiments, T cell effector function is enhanced compared to T cells that do not deliver an epigenetically modified DNA targeting system. In some embodiments, NK cell effector function is enhanced compared to NK cells that do not deliver an epigenetically modified DNA targeting system. In some embodiments, increased expression (e.g., transcription) of the IL-2 gene or its regulatory elements leads to enhanced T cell effector function upon T cell stimulation. In some embodiments, increased expression (e.g., transcription) of the IL-2 gene or its regulatory elements leads to enhanced NK cell effector function. In some embodiments, T cell effector function is characterized by IL-2 production. In some embodiments, T cell effector function is characterized by activity selected from the group consisting of: IL-2 production, IFN-γ production, TNF-α production, T cell proliferation, or any combination thereof. In some embodiments, NK cell effector function is characterized by IL-2 production. In some embodiments, NK cell effector function is characterized by activity selected from the group consisting of: IL-2 production, IFN-γ production, or any combination thereof.

[0163] In some embodiments, the provided DNA targeting system promotes or enhances improved T cell effector function, as may occur after stimulation in vitro, in vitro, or in vivo. In some embodiments, T cell stimulation is polyclonal T cell stimulation. In some embodiments, T cell stimulation employs anti-CD3 and anti-CD28 activating agents. In some embodiments, T cell stimulation is antigen-specific activity mediated or induced by the specific binding of an antigen to an antigen receptor on the surface of T cells. In some embodiments, T cells express a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR) against the antigen, and T cell stimulation is antigen-specific stimulation of the CAR or eTCR. In some embodiments, T cell stimulation employs target cells expressing the antigen. In some embodiments, T cell stimulation occurs when T cells come into contact with cells expressing the antigen. In some embodiments, T cell stimulation is a restimulation of T cells following at least one prior T cell stimulation. In some embodiments, T cells are stimulated, followed by transient delivery of the provided DNA targeting system, after which T cell effector function or phenotype is assessed.

[0164] In some embodiments, a cell composition containing T cells is stimulated with an anti-CD3 / anti-CD28 activating agent for a period of time, and effector function is measured at one or more time points during or after incubation. In some embodiments, such an activating agent has anti-CD3 / anti-CD28 coated on a support (such as magnetic beads or other matrix). Exemplary activating agents are Dynabeads™ or T-cell TransAct™. In some embodiments, T cells are co-incubated with the activating agent for 3 to 72 hours, such as 12 to 48 hours, for example 12, 18, 24, 36, or 48 hours or any value between the foregoing. In some embodiments, effector function of the cells, such as the ability to produce or proliferate cytokines, can be directly assessed. In some embodiments, the supernatant of the culture can be collected and the amount of soluble factors (e.g., cytokines) can be detected. In some embodiments, T cells can be collected and re-exposed to the activating agent to monitor cell lysis activity. In some embodiments, cells can be restimulated once or multiple times (e.g., by a continuous stimulation method), and effector function can be continuously assessed after each stimulation.

[0165] In some embodiments, antigen-specific activity is measured by co-incubating a cell composition containing lymphoid cells (e.g., T cells or NK cells) expressing an antigen receptor (e.g., CAR) with cells expressing the antigen for a period of time, and effector function is measured at one or more time points during or after incubation. In some embodiments, lymphoid cells (e.g., T cells or NK cells) are co-incubated with an antigen-specific agent (such as cells expressing the antigen) for 3 to 96 hours, such as 12 to 72 hours, for example, 12 hours, 24 hours, 48 ​​hours, 72 hours, or any value between the foregoing. In some embodiments, effector function of the cells, such as the ability to produce or proliferate cytokines, can be directly assessed. In some embodiments, the supernatant of the culture can be collected and the amount of soluble factors (e.g., cytokines) can be detected. In some embodiments, lymphoid cells (e.g., T cells or NK cells) can be collected and re-exposed to target cells expressing the antigen to monitor cytotoxicity (cytolysis activity) of the target cells. In some embodiments, cells may be restimulated once or multiple times (e.g., by a sequential stimulation approach), and effector function may be continuously assessed after each stimulation. In some embodiments, lymphoid cells (e.g., T cells or NK cells) with engineered antigen receptors (e.g., CARs) are co-incubated with a constant number of cells expressing the antigen, such as at an effector cell to target cell (E:T) ratio of 1:4 to 4:1, such as at a ratio of 1:4, 1:3, 1:2, or 1:1.

[0166] In some embodiments, lymphoid cells (e.g., T cells or NK cells) exhibit increased cytokine production. In some embodiments, this increase in 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, cytokine production is increased by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more compared to cells that do not deliver epigenetically modified DNA targeting systems. In some embodiments, cytokine production is the production of IL-2. In some embodiments, cytokine production is the production of IL-2, IFN-γ, TNF-α, or combinations thereof. In some embodiments, T cell effector function is characterized by IL-2 production. In some embodiments, cells (e.g., T cells) exhibit increased IL-2 production. In some embodiments, T cell effector function is characterized by IFN-γ production. In some embodiments, cells (e.g., T cells) exhibit increased IFN-γ production. In some embodiments, T cell effector function is characterized by IL-2 and IFN-γ production. In some embodiments, cells (e.g., T cells) exhibit increased IL-2 and IFN-γ production. In some embodiments, T cell effector function is characterized by the multifunctional production of IL-2, IFN-γ, and TNF-α. In some embodiments, cells (e.g., T cells) exhibit increased production of IL-2, IFN-γ, and TNF-α.

[0167] In some embodiments, NK cell effector function is characterized by IL-2 production. In some embodiments, cells (e.g., NK cells) exhibit increased IL-2 production. In some embodiments, natural killer cell effector function is characterized by IFN-γ production. In some embodiments, cells (e.g., NK cells) exhibit increased IFN-γ production. In some embodiments, NK cell effector function is characterized by both IL-2 and IFN-γ production. In some embodiments, cells (e.g., NK cells) exhibit increased IL-2 and IFN-γ production. In some embodiments, NK cell effector function is characterized by the multifunctional production of IL-2, IFN-γ, and TNF-α. In some embodiments, cells (e.g., NK cells) exhibit increased production of IL-2, IFN-γ, and TNF-α.

[0168] Suitable techniques for measuring the production or secretion of soluble factors (such as cytokines) are known in the art. The production and / or secretion of soluble factors can be measured by determining the concentration or amount of the extracellular quantity of the factor, or by determining the amount of transcriptional activity of the gene encoding the factor. Suitable techniques include, but are not limited to, assays such as: immunoassays, aptamer-based assays, histological or cytological assays, mRNA expression level assays, enzyme-linked immunosorbent assays (ELISA), Western blotting, immunoprecipitation, radioimmunoassays (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence assays, lateral flow immunoassays, inhibition assays or affinity assays, protein microarrays, high-performance liquid chromatography (HPLC), Meso Scale Discovery (MSD) electrochemiluminescence, and microbead-based multiplex immunoassays (MIA). In some embodiments, suitable techniques may employ detectable binding reagents that specifically bind to soluble factors.

[0169] In some implementations, cytokine production is measured as the percentage of cells that are cytokine-positive, for example, by intracellular cytokine staining (ICS) and flow cytometry. Intracellular cytokine staining (ICS) performed by flow cytometry is a technique well-suited for studying cytokine production at the single-cell level. This technique detects the production and accumulation of cytokines in the endoplasmic reticulum after cell stimulation, allowing the identification of cell populations that are positive or negative for specific cytokine production, or the separation of high-producing and low-producing cells based on thresholds. ICS can also be combined with other flow cytometry protocols, used for immunophenotypic analysis with cell surface markers, or combined with MHC multimers to obtain cytokine-producing capacity in specific cell subpopulations, making ICS an extremely flexible and versatile method. Other single-cell techniques for measuring or detecting cytokine production include, but are not limited to, ELISPOT, extreme dilution, and T-cell cloning.

[0170] In some implementations, cytokine production is measured as the amount of cytokines secreted by cells, for example, by ELISA (enzyme-linked immunosorbent assay). ELISA is a plate-based assay designed to detect and quantify substances such as peptides, cytokines, antibodies, and hormones. In ELISA, soluble factors (such as cytokines) must be immobilized on a solid surface and then conjugated with an antibody linked to an enzyme. Detection is performed by assessing the activity of the conjugated enzyme through co-incubation with a substrate to generate a detectable signal.

[0171] In some embodiments, the lymphoid cell (e.g., T cell or NK cell) effector function is characterized by also including the activity of lymphoid cell (e.g., T cell or NK cell) proliferation. In some embodiments, cells (e.g., T cells or NK cells) exhibit increased proliferation. In some embodiments, this increased proliferation occurs upon T cell stimulation. In some embodiments, proliferation increases by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more compared to cells not delivered with the epigenetically modified DNA targeting system. In some embodiments, proliferation is measured as an increase in cell number before and after stimulation. In some embodiments, the increase in proliferation is measured as the number of cells after stimulation of a cell population delivered with the epigenetically modified DNA targeting system and compared to the number of cells after stimulation of a cell population not delivered with the epigenetically modified DNA targeting system. In some embodiments, cells (e.g., T cells or NK cells) do not exhibit increased proliferation.

[0172] In some embodiments, the effector function of lymphoid cells (e.g., T cells or NK cells) is characterized by also including the activity of killing target cells. In some embodiments, the cells (e.g., T cells or NK cells) exhibit increased target cell killing. In some embodiments, this increased target cell killing occurs upon T cell stimulation. In some embodiments, stimulation is performed by contacting the cells (e.g., T cells or NK cells) with the target cells. In some embodiments, lymphoid cells (e.g., T cells or NK cells) are co-incubated with target cells expressing the antigen at ratios such as 4:1 and 1:4 (including end values), such as 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 more compared to cells that have not delivered the epigenetically modified DNA targeting system. In some embodiments, killing is measured as the ability of a cell to kill the target cell when contacted by the target cell.

[0173] In some embodiments, the natural killer cell effector function is characterized by further activity in killing target cells. In some embodiments, cells (e.g., natural killer cells) exhibit increased target cell killing. In some embodiments, this increased target cell killing occurs upon stimulation by natural killer cells. In some embodiments, stimulation is performed by contacting cells (e.g., natural killer cells) with target cells. In some embodiments, natural killer cells are co-incubated with target cells expressing antigens at ratios such as 4:1 and 1:4 (including end values), such as ratios of 1:4, 1:3, 1:2, or 1:1. In some embodiments, target cell killing is increased by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more compared to cells that have not delivered an epigenetically modified DNA targeting system. In some embodiments, killing is measured as the ability of a cell to kill a target cell when contacted by a target cell.

[0174] Target cell killing can be measured by any suitable assay, such as those described in the embodiments herein. In some embodiments, killing is measured in an in vitro assay, wherein cells delivered with an epigenetically modified DNA targeting system are co-cultured with target cells, and the number of target cells is measured over time. In some embodiments, a decrease in the number and / or proliferation of target cells indicates target cell killing. Cell lysis activity can be measured by directly or indirectly measuring the number of target cells over time. For example, target cells may be co-incubated with a detectable marker prior to co-incubation with cells expressing an antigen receptor (e.g., CAR), such a marker may then be detected upon lysis of the target cells, or a detectable marker detectable in live target cells. These readouts provide, directly or indirectly, the number of target cells and / or target cell death, and can be measured at different time points during the assay. A decrease in the number of target cells and / or an increase in target cell death indicates the cell lysis activity. Suitable methods for performing cell lysis assays are known in the art and include, but are not limited to, chromium-51 release assays, non-radioactive chromium assays, and flow cytometry assays using fluorescent dyes such as carboxyfluorescein succinimide (CFSE), PKH-2, and PKH-26.

[0175] In some embodiments, the lymphoid cell (e.g., T cell or NK cell) effector function is characterized by also including persistent activity of lymphoid cells (e.g., T cell or NK cell). In some embodiments, the cells (e.g., T cell or NK cell) exhibit persistent (e.g., persistent T cell or NK cell) enhancement. In some embodiments, persistence involves 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 the cells to a subject. Persistence can be measured by any suitable method, such as that described in Section IV.

[0176] In some embodiments, the ability of lymphoid cells (e.g., T cells or NK cells) to persist can be measured as a pharmacokinetic property of the cellular composition after its administration to a subject. In some embodiments, pharmacokinetic parameters may include exposure, quantity, concentration, persistence, and proliferation. In some cases, pharmacokinetics can be assessed by measuring parameters such as peak plasma concentration (C) after administration. max Peak time (i.e., when the highest plasma concentration (C) occurs) max ) time; T max ), lowest plasma concentration (i.e., the lowest plasma concentration between administrations of the therapeutic agent (e.g., CAR+ T cells); C min Eliminate half-life (T) 1 / 2 The area under the curve (AUC) is calculated by plotting the plasma concentration of the therapeutic agent CAR+ T cells over time. Parameters of the administered engineered lymphoid cells (e.g., NK or T cells) can be measured in a blood sample from the subject. For example, nucleic acid-based methods such as quantitative PCR (qPCR) or flow cytometry-based methods, or other assays such as immunoassays, ELISA, or chromatography / mass spectrometry-based assays can be used.

[0177] In some aspects, nucleic acid-based methods, such as quantitative PCR (qPCR), are used to assess the amount of cells expressing antigen receptors (e.g., CAR-expressing cells administered in response to T-cell-based therapies) in a subject's blood or serum or organ or tissue sample (e.g., a disease site, such as a tumor sample). In some aspects, persistence is quantified as the copy number of DNA or plasmid encoding the receptor (e.g., CAR) per microgram of DNA, or as the number of cells expressing the antigen receptor (e.g., CAR) per microliter of sample (e.g., blood or serum), or per microliter of sample based on the total number of peripheral blood mononuclear cells (PBMCs) or leukocytes or lymphoid cells (e.g., T cells or NK cells). In some embodiments, primers or probes used for qPCR or other nucleic acid-based methods are specific for binding, recognizing, and / or amplifying nucleic acids encoding antigen receptors and / or other components or elements of plasmids and / or vectors (including regulatory elements, such as promoters, transcriptional and / or post-transcriptional regulatory elements, or response elements) or markers (e.g., surrogate markers). In some implementations, primers may be specific to regulatory elements such as marmot hepatitis virus posttranscriptional regulatory elements (WPREs).

[0178] In some embodiments, any of the phenotypes described herein, such as increased IL-2 production, is 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-γ production, increased IL-2 and IFN-γ production, increased production of IL-2, IFN-γ and TNF-α, increased proliferation or no proliferation, increased target cell killing, and / or sustained enhancement, are observed after stimulation (e.g., T cell stimulation).

[0179] In some embodiments, a phenotype, such as any of the phenotypes described herein, including enhanced lymphoid cell (e.g., T cell or NK cell) effector function, appears 48 hours or longer after transient delivery of the epigenetically modified DNA targeting system to lymphoid cells (e.g., T cells or NK cells). In some embodiments, a phenotype, such as enhanced lymphoid cell (e.g., T cell or NK cell) effector function, appears at most 6 days, 9 days, 12 days, 15 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 71 days or longer after transient delivery of the epigenetically modified DNA targeting system to lymphoid cells (e.g., T cells or NK cells).

[0180] In some aspects, the phenotype is a phenotype characterized by cell surface phenotypes. In some embodiments, the phenotype includes the expression of IL-2+. In some embodiments, the phenotype includes the expression of one or more cell surface markers selected from IL-2+, TNFα+, IFNg+, or any combination thereof. In some embodiments, the phenotype is a lymphoid cell phenotype (e.g., T cells or NK cells), such as CD3+ T cells, which may be CD4+ T cells or CD8+ T cells. Therefore, in some embodiments, the phenotype includes the expression of one or more cell surface markers selected from CD3+, CD4+, CD8+, IL-2+, TNFα+, IFNg+, or any combination thereof. In some aspects, the phenotype includes the expression of IL-2+. In some embodiments, the phenotype includes the expression of both IL-2+ and IFNg+.

[0181] It should be understood that implementations of the provided epigenetically modified DNA targeting system are not limited to increasing (e.g., activating) the expression of the IL-2 gene or its regulatory elements and promoting the T cell phenotype. In addition to T cells, lymphoid cells may include NK cells, NKT cells, any cells that have differentiated from stem cells into such lymphoid cells and / or have differentiated from progenitor cells, such as common lymphoid progenitor cells (CLPs). In some implementations, lymphoid cells are differentiated from stem cells (such as hematopoietic stem cells or progenitor cells) or progenitor cells. In some implementations, lymphoid cells are transdifferentiated from non-hematopoietic lineage non-pluripotent cells.

[0182] In some embodiments, the lymphoid cells used for regulation are isolated or enriched populations of lymphoid immune cells, such as isolated or enriched populations of T cells, NK cells, and / or NKT cells. In some embodiments, the cells used for regulation are isolated or enriched T cells. In some embodiments, the cells used for regulation are isolated or enriched NK cells. In some embodiments, the cells used for regulation are isolated or enriched NK T cells. In some embodiments, isolated or enriched populations or subsets of immune cells containing T cells, NK cells, and / or NKT cells for regulation can be obtained from a unit of blood using a variety of techniques known to those skilled in the art, such as Ficoll™ isolation. In one embodiment, T cells, NK cells, or NKT cells from an individual's circulating blood are obtained via apheresis and separated from other nucleated leukocytes, erythrocytes, and platelets, such as by Ficoll™ isolation or affinity-based selection. In some embodiments, the cells are primary cells. In some embodiments, primary cells are isolated or enriched from a peripheral blood sample of a subject (such as a human subject).

[0183] In some embodiments, the lymphoid cells used for regulation are differentiated from stem cells or progenitor cells in vitro. In some embodiments, the lymphoid cells (such as T cells, NK cells, or NKT cells, or their lineages) may be differentiated from stem cells, hematopoietic stem cells, or progenitor cells (HSCs) or progenitor cells. Progenitor cells may be CD34+ hematopoietic endothelial cells, pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, or NKT cell progenitor cells. In some embodiments, the progenitor cells are lymphoid progenitor cells, such as common lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T progenitor cells, NK progenitor cells, or NKT progenitor cells. Stem cells may be pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). iPSCs are reprogrammed pluripotent cells that are not naturally occurring. Once a subject's cells have been reprogrammed into a pluripotent state, the cells can be programmed or differentiated into the desired cell type or subtype, such as T cells, NK cells, or NKT cells.

[0184] In some implementations, iPSCs differentiate into T cells, NK cells, or NKT cells via a multi-stage differentiation platform, wherein cells from various developmental stages can be induced to present a hematopoietic phenotype, ranging from mesodermal stem cells to fully differentiated T cells, NK cells, or NKT cells (see, for example, U.S. Patent No. 10,626,372).

[0185] In some embodiments, a population or subset of lymphoid cells is transdifferentiated in vitro from non-hematopoietic non-pluripotent cells to hematopoietic lineage cells, or from non-pluripotent cells of a first hematopoietic cell type to different hematopoietic cell types, which may be T progenitor cells, NK progenitor cells, or NKT progenitor cells, or fully differentiated specific immune cell types, such as T cells, NK cells, or NKT cells (see, for example, U.S. Patent No. 9,376,664 and U.S. Application Serial No. 15 / 072,769, the disclosures of which are incorporated herein by reference in their entirety). In some embodiments, the non-hematopoietic non-pluripotent cells are somatic cells, such as skin fibroblasts, adipose tissue-derived cells, and human umbilical vein endothelial cells (HUVECs). The somatic cells available for transdifferentiation may be immortalized somatic cells.

[0186] Various strategies are being explored to induce pluripotency or enhance 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 to improve the efficiency of reprogramming (Shi et al., Cell Stem Cell 2, 525-528 (2008a); Shi et al., Cell Stem Cell 2, 525-528 (2008a)). 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 The publications of these references are incorporated herein by reference in their entirety by way of citation.

[0187] It should be understood that a cell that is positive (+) for a specific cell surface marker is a cell that expresses that marker at a detectable level on its surface. Similarly, it should be understood that a cell that is negative (-) for a specific cell surface marker is a cell that expresses that marker at an undetectable level on its surface. Antibodies and other binding entities can be used to detect the expression level of a marker protein to identify or detect a given cell surface marker. Suitable antibodies may include polyclonal antibodies, monoclonal antibodies, fragments (such as Fab fragments), single-chain antibodies, and other forms of specific binding molecules. Antibody reagents for the above cell surface markers are readily known to those skilled in the art. A variety of well-known methods can be used to assess the expression level of a surface marker or protein, such as by affinity-based methods, for example, immunoaffinity-based methods, such as flow cytometry in the case of surface markers. In some embodiments, the label is a fluorophore, and the method used to detect or identify cell surface markers on cells (e.g., T cells or NK cells) is flow cytometry. In some embodiments, multicolor flow cytometry uses different labels for each of the different markers. In some implementations, 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.

[0188] In some embodiments, a cell (e.g., a T cell or NK cell) is positive (pos or +) for a specific marker if a detectable marker (which may be an intracellular or surface marker) is present on or within the cell. In some embodiments, surface expression is positive if the level of staining detectable by flow cytometry is significantly higher than that detected under the same procedure with an isotype-matched control under otherwise identical conditions, and / or the level is substantially similar to or, in some cases, higher than, cells known to be positive for the marker, and / or the level is higher than that of cells known to be negative for the marker.

[0189] In some embodiments, a cell (e.g., T cells or NK cells) is negative (neg or -) for a specific marker if the marker is not detectably present on or within the cell. This specific marker can be an intracellular or surface marker. In some embodiments, surface expression is negative if the level of staining that is undetectable by flow cytometry is significantly higher than staining detected under the same procedure with isotype-matched controls under otherwise identical conditions, and / or significantly lower than that of cells known to be positive for the marker, and / or substantially similar to that of cells known to be negative for the marker.

[0190] In some aspects, a phenotype can be characterized by one or more functions of a cell. In some aspects, a phenotype is characterized by the multifunctional activity of lymphoid cells (e.g., T cells or NK cells) producing more than one type of lymphoid cell-stimulating cytokine, as determined in a multifunctional cytokine secretion assay after stimulation of lymphoid cells with a stimulator. In some embodiments, lymphoid cells (e.g., T cells or NK cells) are multifunctional for producing two or more cytokines. In some embodiments, lymphoid cells (e.g., T cells or NK cells) are multifunctional for producing two or more cytokines selected from interferon-γ (IFN-γ), interleukin-2 (IL-2), and TNF-α. In some embodiments, multifunctional lymphoid cells (e.g., T cells or NK cells) produce IFN-γ, IL-2, and TNF-α. In some embodiments, the stimulator is a nonspecific or non-antigen-dependent lymphoid cell stimulator. In some embodiments, the nonspecific or non-antigen-dependent lymphoid cell stimulator is a polyclonal stimulator. In some embodiments, the nonspecific or non-antigen-dependent stimulant includes PMA / ionomycin, anti-CD3 / anti-CD28, phytohemagglutinin (PHA), or concanavalin A (ConA). In some embodiments, the nonspecific or non-antigen-dependent lymphoid cell stimulant contains PMA / ionomycin.

[0191] In certain implementations, the production of one or more cytokines is measured, detected, and / or quantified by intracellular cytokine staining. Intracellular cytokine staining (ICS) performed by flow cytometry is a technique well-suited for studying cytokine production at the single-cell level. This technique detects the production and accumulation of cytokines in the endoplasmic reticulum after cell stimulation, allowing the identification of cell populations that are positive or negative for specific cytokine production, or the separation of high-producing and low-producing cells based on thresholds. In some implementations, as described above, stimulation can be performed using non-specific stimulation, such as non-antigen-specific stimulation. For example, PMA / ionomycin can be used for non-specific cell stimulation. ICS can also be combined with other flow cytometry protocols, used for immunophenotypic analysis using cell surface markers, or combined with MHC multimers to obtain cytokine-producing capacity in specific cell subpopulations, making ICS an extremely flexible and versatile method. Other single-cell techniques for measuring or detecting cytokine production include, but are not limited to, ELISPOT, extreme dilution, and T-cell cloning. In some implementations, the assay for measuring the secretion of multiple cytokines may include multiplex assays or other assays for assessing versatility (see, for example, Xue et al., (2017) Journal for ImmunoTherapy of Cancer 5:85).

[0192] 2. Increase the target sites for IL-2 transcription In some embodiments, delivery of a DNA-targeting system increases transcription of the IL-2 gene or its regulatory elements. In some embodiments, this document provides target sites for IL-2 that promote cellular phenotypes for their increased transcription. In some embodiments, the target site may be located on the IL-2 gene body or on the regulatory DNA element of IL-2 as described herein. In some embodiments, this document provides target sites for the IL-2 gene or its regulatory DNA element as described herein that promote enhanced lymphoid cell (e.g., T cell or NK cell) effector function for their increased transcription. In some embodiments, the increased transcription promotes enhanced lymphoid cell (e.g., T cell or NK cell) effector function in response to cellular stimulation (e.g., T cell stimulation).

[0193] In some embodiments, each target site of the epigenetic modified DNA targeting system is located within the genomic coordinates of the assembled human genome version GRCh38 (hg38) chr4:122,451,261-122,593,946. In some embodiments, each target site is located within a putative regulatory region of the IL-2 gene, wherein the putative regulatory region is characterized by one or more of an epigenetic marker, regulatory feature, or transcription factor motif. In some embodiments, the putative regulatory region is a promoter or enhancer. In some embodiments, the target site is located within a promoter or enhancer.

[0194] In some embodiments, the DNA targeting system comprises multiple DNA targeting modules. In some embodiments, each DNA targeting module targets a target site. In some embodiments, the multiple DNA targeting modules target at least a first target site of the IL-2 gene or its regulatory elements and a second target site of the IL-2 gene or its regulatory elements. 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.

[0195] In some embodiments, multiple 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 its regulatory elements. In some embodiments, the first target site, the second target site, and the third target site are all different from each other.

[0196] In some embodiments, the DNA targeting system targets a combination of target sites of the IL-2 gene or regulatory elements described herein to achieve transcriptional activation. In some embodiments, the target site is located within the genomic coordinates of the assembled human genome version GRCh38 (hg38) chr4:122,451,261-122,593,946. In some embodiments, the target site is located within a 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 at an IL-2 gene locus. In some embodiments, the target site is a regulatory element located at an IL-2 gene locus. In some embodiments, the regulatory element is a sequence that a gene regulatory protein can bind to and influence gene transcription. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of the IL-2 gene.

[0197] In some embodiments, seven identified target regions exist within the IL-2 locus. In some embodiments, the target regions exhibit characteristics indicating regulatory regions. In a particular embodiment, this characteristic may be an epigenetic marker. In a particular embodiment, this characteristic may be a regulatory feature. In a particular embodiment, this characteristic may be an associated transcription factor (TF) motif.

[0198] In some embodiments, the target site is located on the IL-2 gene body. In some embodiments, the target site is located within 100 kb of the IL-2 transcription start site (TSS). In some embodiments, the target site is located 50 to 150 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 located 50 to 150 kb upstream of the IL-2 gene and the IL-2 TSS.

[0199] In some implementations, each target site of the epigenetic modified DNA targeting system is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 implementations, each target site of the epigenetic modified DNA targeting system is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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.

[0200] In some embodiments, the target site is located on the IL-2 genomic body. In some embodiments, the target site is located 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 the target region identified as Region 1. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 12, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 12, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 12. In some embodiments, the target site is shown in SEQ ID NO: 12. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 14, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 14, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 14. In some embodiments, the target site is shown in SEQ ID NO: 14. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 16, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 16, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 16. In some embodiments, the target site is shown in SEQ ID NO: 16.In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 18, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 18, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 18. In some embodiments, the target site is shown in SEQ ID NO: 18. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 20, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 20, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 20. In some embodiments, the target site is shown in SEQ ID NO: 20. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 43, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 43, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 43. In some embodiments, the target site is shown in SEQ ID NO: 43. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 45, a continuous portion of at least 14 nucleotides thereof, a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 45, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 45. In some embodiments, the target site is shown in SEQ ID NO: 45. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 47, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 47, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 47. In some implementations, the target site is shown in SEQ ID NO: 47.

[0201] In some embodiments, the target site is located within the genomic coordinates of the human genome assembly version GRCh38 (hg38) chr4: 122,465,000-122,472,000. In some embodiments, the target site is located within the genomic coordinates of the human genome assembly version GRCh38 (hg38) chr4: 122,479,410-122,482,750. In some embodiments, the target site is located within the genomic coordinates of the human genome assembly version GRCh38 (hg38) chr4: 122,488,840-122,491,890.

[0202] In some embodiments, the target site is located 50 to 150 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 located 50 to 150 kb upstream of the IL-2 gene and the IL-2 TSS. In some embodiments, the regulatory DNA element is a distal regulatory element of the 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 of the assembled human genome version GRCh38 (hg38) chr4: 122,507,000-122,508,985. In some embodiments, the target site is located in the target region identified as Region 4. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 22, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 22, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 22. In some embodiments, the target site is shown in SEQ ID NO: 22. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 24, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 24, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 24. In some embodiments, the target site is shown in SEQ ID NO: 24. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 26, a continuous portion of at least 14 nucleotides thereof, a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 26, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 26. In some embodiments, the target site is shown in SEQ ID NO: 26. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 49, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 49, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 49. In some embodiments, the target site is shown in SEQ ID NO: 49. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 51, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 51, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 51. In some embodiments, the target site is shown in SEQ ID NO: 51. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 53, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 53 having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 53. In some embodiments, the target site is shown in SEQ ID NO: 53.In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 55, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 55, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 55. In some embodiments, the target site is shown in SEQ ID NO: 55. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 186, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 186 having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 186. In some embodiments, the target site is shown in SEQ ID NO: 186. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 187, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 187 having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 187. In some embodiments, the target site is shown in SEQ ID NO: 187. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 188, a continuous portion of at least 14 nucleotides thereof, a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing.In some embodiments, the target site is a continuous portion of SEQ ID NO: 188, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 188. In some embodiments, the target site is shown in SEQ ID NO: 188.

[0203] In some embodiments, the target site is located within the genomic coordinates of the assembled human genome version GRCh38 (hg38)chr4: 122,539,300-122,544,050. In some embodiments, the target site is located within the target region identified as Region 5. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 28, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 28, with a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 28. In some embodiments, the target site is shown in SEQ ID NO: 28. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 30, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 30, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 30. In some embodiments, the target site is shown in SEQ ID NO: 30. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 32, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 32, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 32. In some embodiments, the target site is shown in SEQ ID NO: 32.In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 57, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 57, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 57. In some embodiments, the target site is shown in SEQ ID NO: 57. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 59, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 59 having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 59. In some embodiments, the target site is shown in SEQ ID NO: 59.

[0204] In some embodiments, the target site is located within the genomic coordinates of the assembled human genome version GRCh38 (hg38)chr4: 122,576,890-122,579,315. In some embodiments, the target site is located within the target region identified as Region 6. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 34, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 34, with a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 34. In some embodiments, the target site is shown in SEQ ID NO: 34. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 36, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 36, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 36. In some embodiments, the target site is shown in SEQ ID NO: 36. In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 38, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 38 having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 38. In some embodiments, the target site is shown in SEQ ID NO: 38.In some embodiments, the target site comprises the sequence shown in SEQ ID NO: 40, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of SEQ ID NO: 40, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is located within a sequence comprising the sequence shown in SEQ ID NO: 40. In some embodiments, the target site is shown in SEQ ID NO: 40.

[0205] Exemplary target sites for IL-2 are shown in Table 1.

[0206] Table 1. Target Region and Target Sites

[0207] In some implementations, at least two of the multiple DNA targeting modules of the epigenetic modified DNA targeting system target the same target site. In some implementations, at least two identical target sites of the epigenetic modified DNA targeting system are located in the same target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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, at least two identical target sites of the epigenetic modified DNA targeting system are located in the same target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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.

[0208] In some embodiments, the DNA targeting system targets a first target site and a second target site of IL-2, wherein the first and second target sites are identical. In a particular embodiment, each of the identical target sites of IL-2 comprises the sequence shown in SEQ ID NO: 12. In a particular embodiment, each of the identical target sites of IL-2 comprises the sequence shown in SEQ ID NO: 24. In a particular embodiment, each of the identical targets of IL-2 comprises the sequence shown in SEQ ID NO: 26.

[0209] In some implementations, at least two of the multiple DNA targeting modules of the epigenetic modified DNA targeting system target different target sites. In some implementations, at least two distinct target sites of the epigenetic modified DNA targeting system are located in two distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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, at least two distinct target sites of the epigenetic modified DNA targeting system are located in two distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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.

[0210] In some embodiments, the DNA targeting system targets a first target site and a second target site, wherein the first target site and the second target site are different. In a particular embodiment, the first target site of IL-2 comprises the sequence shown in SEQ ID NO: 12, and the second target site of IL-2 comprises the sequence shown in SEQ ID NO: 24. In a particular embodiment, the first target site of IL-2 comprises the sequence shown in SEQ ID NO: 12, and the second target site of IL-2 comprises the sequence shown in SEQ ID NO: 26. In a particular embodiment, the first target site of IL-2 comprises the sequence shown in SEQ ID NO: 12, and the second target site of IL-2 comprises the sequence shown in SEQ ID NO: 28. In a particular embodiment, the first target site of IL-2 comprises the sequence shown in SEQ ID NO: 12, and the second target site of IL-2 comprises the sequence shown in SEQ ID NO: 38. In a particular embodiment, the first target site of IL-2 comprises the sequence shown in SEQ ID NO: 24, and the second target site of IL-2 comprises the sequence shown in SEQ ID NO: 26. In a particular embodiment, the first target site of IL-2 comprises the sequence shown in SEQ ID NO: 12, and the second target site of IL-2 comprises the sequence shown in SEQ ID NO: 28. In a particular embodiment, the first target site of IL-2 comprises the sequence shown in SEQ ID NO: 12, and the second target site of IL-2 comprises the sequence shown in SEQ ID NO: 38.

[0211] Exemplary pairings of target sites are shown in Table 2.

[0212] Table 2. Exemplary pairings of target sites

[0213] In some implementations, 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 implementations, at least three of the multiple DNA targeting modules of the epigenetic modified DNA targeting system target different target sites. In some implementations, at least three distinct target sites of the epigenetic modified DNA targeting system are located in three distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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, at least three distinct target sites of the epigenetic modified DNA targeting system are located in three distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 implementations, 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 implementations, at least four of the multiple DNA targeting modules of the epigenetic modified DNA targeting system target different target sites. In some implementations, at least four distinct target sites of the epigenetic modified DNA targeting system are located in four distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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, at least four distinct target sites of the epigenetic modified DNA targeting system are located in four distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 a particular embodiment, the first target site of IL-2 contains the sequence shown in SEQ ID NO: 12 (GACTTAGTGCAATGCAAGAC), the second target site of IL-2 contains the sequence shown in SEQ ID NO: 24 (CTCTCTCTGCAGACAGGGCA), the third target site of IL-2 contains the sequence shown in SEQ ID NO: 28 (GGCAGGGTAGAGAAGTAGAG), and the fourth target site of IL-2 contains the sequence shown in SEQ ID NO: 38 (GGAAATGACATGCTTGAAGT).

[0216] In some implementations, 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 implementations, at least five of the multiple DNA targeting modules of the epigenetic modified DNA targeting system target different target sites. In some implementations, at least five distinct target sites of the epigenetic modified DNA targeting system are located in five distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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, at least five distinct target sites of the epigenetic modified DNA targeting system are located in five distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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 a particular embodiment, the first target site of IL-2 contains the sequence shown in SEQ ID NO: 12 (GACTTAGTGCAATGCAAGAC), the second target site of IL-2 contains the sequence shown in SEQ ID NO: 24 (CTCTCTCTGCAGACAGGGCA), the third target site of IL-2 contains the sequence shown in SEQ ID NO: 26 (GGCAGGGTAGAGAAGTAGAG), the fourth target site of IL-2 contains the sequence shown in SEQ ID NO: 28 (GGCAGGGTAGAGAAGTAGAG), and the fifth target site of IL-2 contains the sequence shown in SEQ ID NO: 38 (GGAAATGACATGCTTGAAGT).

[0218] In some embodiments, delivery of the DNA-targeting system increases the expression (e.g., transcription) of the IL-2 gene or its regulatory elements. In some embodiments, the increase in gene expression in cells (e.g., T cells or NK cells) is a fold change of more than 1.0 log2. In some embodiments, the increase in IL-2 expression in cells (e.g., T cells or NK cells) is a fold change of more than 1.0 log2. For example, compared to the expression level of the IL-2 gene in control cells, the log2 fold change is greater than or equal to about 1.5, about 2.0, about 2.5, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, or any value between the foregoing.

[0219] C. CRISPR / Cas-based DNA targeting systems and DNA-binding domains This document provides a CRISPR / Cas-based multiplex epigenetic targeting DNA targeting system, specifically a CRISPR / Cas-based DNA targeting system capable of binding to target sites of the IL-2 gene or its regulatory elements, or combinations of target sites, such as combinations of target sites within the IL-2 gene or its regulatory elements. Exemplary target sites include any or any combination of those described in Section IB above. In some embodiments, the CRISPR / Cas DNA-binding domain is nuclease-free, such as including dCas (e.g., dCas9), such that the system binds to target sites of the IL-2 gene or its regulatory elements without mediating nucleic acid cleavage at the target site. The CRISPR / Cas-based DNA targeting system can be used to activate or increase the expression of the IL-2 gene or its regulatory elements in cells such as lymphoid cells (e.g., T cells or NK cells). In some embodiments, the CRISPR / Cas-based DNA targeting system may contain any known Cas enzyme, and is typically nuclease-free or dCas. In some embodiments, the CRISPR / Cas-based DNA targeting system comprises a fusion protein of a nuclease-free Cas protein or a variant thereof and an effector domain, and at least one gRNA. In some implementations, the effector domain increases transcription of the IL-2 gene or its regulatory elements (e.g., the effector domain is a transcription activator, such as any transcription activator described in section IE1).

[0220] The CRISPR system (also known as the CRISPR / Cas system or CRISPR-Cas system) refers to a conserved microbial nuclease system found in the genomes of bacteria and archaea, providing a form of acquired immunity against invading bacteriophages and plasmids. Clustered regularly spaced short palindromic repeats (CRISPR) are loci containing multiple repetitive DNA elements separated by non-repetitive DNA sequences called spacers. Spacers are short sequences of foreign DNA incorporated into the genome between CRISPR repeat sequences, serving as a “memory” of past exposures. The spacers encode the DNA-targeting portion of RNA molecules, thus conferring the specificity of nucleic acid cleavage via the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes, which act as RNA-guided nucleases mediating cleavage; and non-protein-coding DNA elements that encode RNA molecules capable of being programmed for the specificity of CRISPR-mediated nucleic acid cleavage.

[0221] In a type II CRISPR / Cas system containing the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to guide Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer region sequence complementary to the target nucleic acid sequence (target site) and encodes the sequence specificity of the complex. Trans-activated crRNA (tracrRNA) pairs with a subset of the bases of the crRNA, forming a structure that complexes with the Cas9 protein, thus creating the Cas / RNA RNP complex.

[0222] Naturally occurring CRISPR / Cas systems (such as those with Cas9) have been engineered to allow Cas / RNA RNPs to be efficiently programmed to target desired sequences in target cells, enabling both gene editing and regulation of gene expression. tracrRNA and crRNA have been engineered to form single chimeric guide RNA molecules, commonly referred to as guide RNAs (gRNAs), as described, for example, 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 selected by the user to target the Cas / gRNA RNP complex to desired loci, such as desired target sites within a target gene.

[0223] Cas proteins have also been engineered to be catalytically inactive or nuclease-free to allow targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or eliminate their nuclease activity, rendering them catalytically inactive. Cas proteins with reduced or eliminated nuclease activity are referred to as inactive Cas (dCas) or nuclease-free Cas (iCas) proteins; these terms are used interchangeably in this article. (Source: [Original Source Name]) Streptococcus pyogenes Exemplary inactivated Cas9 (dCas9) contains silencing mutations (D10A and H840A) in the RuvC and HNH nuclease domains, as described, for example, 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) can be readily identified in Cas orthogonal homologs and can be mutated to produce inactive variants, as described, for example, in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0224] dCas fusion proteins with transcriptional and / or epigenetic regulatory factors have been used as a universal platform for ectopic regulation of gene expression in target cells. These include fusions of Cas with effector domains such as transcriptional activators. For example, fusing dCas9 with a transcriptional activator such as VP64 (a polypeptide consisting of four tandem copies of VP16, which is the 16-amino acid transactivation domain of herpes simplex virus) results in potent induction of gene expression. Various dCas fusion proteins with effector domains can be engineered for gene expression regulation, such as 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, ML et al. Nat. Methods 10, ...) As described in 977–979 (2013), Gilbert, LA et al. Cell 154(2):442–451 (2013), and Nuñez, JK et al. Cell 184(9):2503–2519 (2021).

[0225] In some aspects, a DNA targeting system comprising a fusion protein is provided, the fusion protein comprising a DNA-binding domain and an effector domain, the DNA-binding domain comprising a nuclease-free Cas protein or a variant thereof, the effector domain being configured to increase transcription or induce transcriptional activation (i.e., act as a transcription activator) upon targeting an IL-2 gene or regulatory element in a target cell (e.g., lymphoid cells (e.g., T cells or NK cells)). In some embodiments, the dCas protein is any suitable dCas protein, such as any one described in section IC1. In some embodiments, the dCas protein is a dCas9 protein, such as dSpCas9 or dSaCas9. In some embodiments, at least one effector domain is any suitable transcription activator effector domain, such as any transcription activator effector domain described in section IE1, such as VP64. In some embodiments, the epigenetic marker includes histone H3K27 acetylation. In some embodiments, the effector domain catalyzes the acetylation of histone H3 lysine 27 at the target site, or is capable of recruiting an enzyme that catalyzes the acetylation of histone H3 lysine 27 at the target site. In some embodiments, the enzyme catalyzing acetylation is an acetyltransferase.

[0226] In some embodiments, at least one effector domain is VP64. In some embodiments, the fusion protein is a dCas9-VP64 fusion protein, such as that described in section IF. In such embodiments, the DNA targeting system further comprises one or more gRNAs (such as those described in section IC2) provided in combination with or as a complex with a dCas protein or a variant thereof for targeting the DNA targeting system to a target site of the IL-2 gene or its regulatory element. In some embodiments, the fusion protein is guided to a specific target site sequence of the IL-2 gene or its regulatory element via a guide RNA, wherein the effector domain mediates targeted epigenetic modifications to increase or activate transcription of the IL-2 gene or its regulatory element. 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 modifications to increase or activate transcription of the combination of target sites present on the IL-2 gene or its regulatory element. Any of the plurality of effector domains that increase or activate transcription may be used as further described below.

[0227] 1. CRISPR / Cas-based DNA binding domains In some respects, the DNA-binding domain contains a CRISPR-associated (Cas) protein or a variant thereof, or is derived from a Cas protein or a variant thereof. In a particular embodiment herein, the Cas protein is non-nuclease-active (i.e., a dCas protein).

[0228] In some embodiments, the Cas protein is derived from a class 1 CRISPR system (i.e., a multi-Cas protein system), such as type I, type III, or type IV CRISPR systems. In some embodiments, the Cas protein is derived from a class 2 CRISPR system (i.e., a single-Cas protein system), such as type II, type V, or type VI CRISPR systems. In some embodiments, the Cas protein is derived from a type V CRISPR system. In some embodiments, the Cas protein is derived from the Cas12 protein (i.e., Cpf1) or a variant thereof, such as 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 implementations, the Cas protein is derived from the Cas9 protein or a variant thereof, 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 related Cas proteins used for gene editing and regulation have been described, for example, in Moon, SB et al. Exp. Mol. Med. 51, 1–11 (2019), Zhang, FQ Rev. Biophys. 52, E6 (2019), and Makarova KS et al. Methods Mol. Biol. 1311:47-75 (2015).

[0229] In some embodiments, the dCas9 protein may comprise a sequence derived from a naturally occurring Cas9 molecule or a variant thereof. In some embodiments, the dCas9 protein may comprise a sequence derived from a naturally occurring Cas9 molecule or a variant thereof from the following species: Streptococcus pyogenes Streptococcus thermophilus ( Staphylococcus thermophilus), Staphylococcus aureus Campylobacter jejuni (C. jejuni), Neisseria meningitidis (N. meningitidis The new culprit, Francis bacteria. (F. novicida), Streptococcus canis (S. canis), Staphylococcus aureus (S. auricularis) In some implementations, the dCas9 protein contains components derived from... gold Staphylococcus aureus The sequence of the naturally occurring Cas9 molecule. In some embodiments, the dCas9 protein contains components derived from... change Streptococcus pyogenes The sequence of the naturally occurring Cas9 molecule.

[0230] Non-limiting examples of Cas9 orthologs from other bacterial strains include, but are not limited to, Cas proteins identified in: marine unicellular cyanobacteria (Acaryochloris marina) MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; and Alicyclobacillus acidocaldarius subsp.* *Acidocaldarius* DSM 446; *Allochromatium vinosum* DSM 180; *Ammonifex degensii* KC4; *Anabaena variabilis* ATCC 29413; *Arthrospira maxima* CS-328; *Arthrospira platensis* strain Paraca; *Arthrospira* species PCC 8005; *Bacillus pseudomycoides* DSM 12442; *Bacillus sselenitireducens* MLS10; *Burkholderiales bacterium* 1_1_47; *Caldicelulosiruptor becscii* DSM 6725; Provisional species *Candidatus desulforudis audaxviator* MP104C; *Caldicellulosiruptorhydrothermalis* 108; *Clostridium* bacteriophage c-st; *Clostridium botulinum* A3 strain Loch Maree; *Clostridium botulinum* Ba4 strain 657; *Clostridium difficile* QCD-63q42; *Crocosphaera watsonii* WH 8501; *Cyanothece* species ATCC 51142; *Cyanothece* species CCY0110; *Cyanothece* species PCC 7424; *Cyanothece* species PCC7822; *Exiguobacterium sibiricum* 255-15; *Finegoldia* magna) ATCC 29328; Ktedonobacter racemifer DSM44963; Lactobacillus delbrueckii subsp.bulgaricus) PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya species PCC 8106; Marinobacter species ELB17; Methanohalobium evestigatum Z-7303; Microcystis bacteriophage Ma-LMM01; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; *Nocardiopsis dassonvillei* subsp. *dassonvillei* DSM 43111; *Nodularia spumigena* CCY9414; *Nostoc* species PCC 7120; *Oscillatoria* species PCC 6506; *Pelotomaculum thermopropionicum* SI; *Petrotoga mobilis* SJ95; *Polaromonas naphthalenivorans* CJ2; *Polaromonas* species JS666; *Pseudoalteromonas haloplanktis* TAC125; *Streptomyces pristinaespiralis* ATCC 25486; *Streptomyces pristinaespiralis* ATCC 25486; *Streptococcus thermophilus*; *Streptomyces viridochromogenes* DSM 40736; *Streptosporangium roseum* DSM 43021; ​​*Synechococcus* species PCC7335; and *Thermosipho africanus* TCF52B (Chylinski et al., RNABiol)., 2013; 10(5): 726-737).

[0231] In some respects, the Cas protein is a variant lacking nuclease activity (i.e., a dCas protein). In some embodiments, the Cas protein is mutated to reduce or eliminate nuclease activity. Such Cas proteins are referred to as inactive Cas or dead Cas (dCas) or Cas proteins without nuclease activity (iCas), and the two terms are used interchangeably herein. In some embodiments, the variant Cas protein is a variant Cas9 protein lacking nuclease activity or an inactive Cas9 (dCas9 or iCas9) protein.

[0232] In some implementations, the Cas9 protein or its variants are derived from... Staphylococcus aureus Cas9 (SaCas9) protein or its variants. In some implementations, the variant Cas9 is... Staphylococcus aureus The dCas9 protein (dSaCas9) contains at least one amino acid mutation selected from D10A and N580A, as indicated by the position number in SEQ ID NO: 64. In some embodiments, the variant Cas9 protein contains the sequence shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0233] In some implementations, the Cas9 protein or its variants are derived from... Streptococcus pyogenes Cas9 (SpCas9) protein or its variants. In some embodiments, the variant Cas9 is... Streptococcus pyogenes The dCas9 (dSpCas9) protein contains at least one amino acid mutation selected from D10A and H840A, as indicated by the position number in SEQ ID NO: 62. In some embodiments, the variant Cas9 protein comprises the sequence shown in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0234] 2. Guide RNA (gRNA) In some embodiments, the gRNA is capable of complexing with a Cas protein or a variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (also called a spacer sequence or guide sequence) capable of hybridizing with or being complementary to a target site, such as any target site described herein, or any target site in the genome. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to a Cas protein. In some embodiments, gRNAs specific to a target locus of interest (e.g., regulatory DNA elements of the IL-2 locus) are used to recruit RNA-guided proteins (e.g., Cas proteins) or variants thereof, or fusion proteins containing such RNA-guided proteins (e.g., Cas peptides), to the target site.

[0235] In some embodiments, the Cas protein (e.g., dCas9) is combined with one or more guide RNAs (gRNAs) or provided as a complex with gRNA. In some aspects, the gRNA is a nucleic acid that promotes the specific targeting or homing of the gRNA / Cas RNP complex to target sites of the IL-2 gene or its regulatory elements (such as any target sites described above in Section IB). In some embodiments, the target site of the gRNA may be referred to as the protospacer region.

[0236] This document provides gRNAs, such as gRNAs that target or bind to target sites of the IL-2 gene or its regulatory elements (such as any target sites described herein, for example, in Section IB). This document provides gRNAs, such as gRNAs that target or bind to regulatory DNA elements of the IL-2 gene locus. In some embodiments, the gRNA binds to a target site located in the IL-2 gene and / or a regulatory DNA element of the IL-2 gene. In some embodiments, the gRNA binds to a target site located in the IL-2 gene. In some embodiments, the gRNA binds to a target site 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 a variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e., a spacer sequence or guide sequence) capable of hybridizing with or being complementary to a 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 respects, a "gRNA molecule" is a nucleic acid that promotes the specific targeting or homing of the gRNA / Cas9 molecule complex to a target nucleic acid (such as a locus on the genomic DNA of a cell). Typically, the spacer region sequence of a guide RNA is any polynucleotide sequence that contains at least one target polynucleotide sequence (such as a locus on the genomic DNA of a cell). IL-2The guide RNA (guide RNA) contains a sequence portion at the target locus that is sufficiently complementary to the target sequence, thereby hybridizing at the target site and guiding the CRISPR complex to bind specifically to the target sequence. In some implementations, in the case of CRISPR complex formation, the "target sequence" is a spacer sequence designed to be complementary to it, wherein hybridization between the target sequence and the spacer sequence of the guide RNA promotes CRISPR complex formation. Perfect complementarity is not required, as long as there is sufficient complementarity to induce hybridization and promote CRISPR complex formation. Typically, the spacer sequence is chosen to minimize the degree of secondary structure within the spacer sequence. Secondary structure can be determined using any suitable multinucleotide folding algorithm.

[0239] In some implementations, guide RNAs (gRNAs) specific to the target loci of interest (e.g., in humans) are used. IL-2 The gRNA (at the locus) is used in conjunction with an RNA-guided nuclease or a variant thereof (e.g., a Cas variant without nuclease activity) to target the provided DNA targeting system to a target site or location. Methods for designing gRNAs and exemplary spacer region sequences are known. Exemplary gRNA structures that can be associated with specific RNA-guided nucleases or variants thereof (e.g., Cas variants without nuclease activity), and with specific domains and scaffold regions, are also known. In some aspects, the gRNA molecule contains a scaffold sequence, such as a sequence that can complex with a Cas protein. In some aspects, the scaffold sequence is specific to the Cas protein.

[0240] In some embodiments, the gRNAs provided herein are chimeric gRNAs. Generally, gRNAs can be monomolecular (i.e., composed of a single RNA molecule) or modular (containing more than one, and typically two, separate RNA molecules). Modular gRNAs can be engineered to be monomolecular, wherein sequences from separate modular RNA molecules are contained within a single gRNA molecule; these are sometimes referred to as chimeric gRNAs, synthetic gRNAs, or monogRNAs. In some embodiments, a chimeric gRNA is a fusion of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence, as described, for example, in WO 2013 / 176772 or Jinek, M. et al., Science 337(6096):816-21 (2012). In some embodiments, the chimeric gRNA mimics a naturally occurring crRNA:tracrRNA duplex involved in type II effector systems, wherein the naturally occurring crRNA:tracrRNA duplex acts as a guide sequence for the Cas9 protein. Exemplary types of CRISPR / Cas systems and associated gRNA structures include those described, for example, in the following literature: Moon et al. Exp. Mol. Med. 51, 1–11 (2019), Zhang, FQ 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] Guide RNAs may contain at least a spacer sequence that hybridizes to the target nucleic acid sequence of interest and a CRISPR repeat sequence. In type II systems, gRNAs also contain a second RNA sequence called tracrRNA. In type II guide RNAs (gRNAs), the CRISPR repeat sequence and the tracrRNA sequence hybridize to form a double strand. In type V guide RNAs (gRNAs), the crRNA forms a double strand. In both systems, the double strand can bind to a site-directed peptide, causing the guide RNA and the site-directed peptide to form a complex. The gRNA provides target specificity to the complex by binding to the site-directed peptide. Therefore, the gRNA can guide the activity of the site-directed peptide.

[0242] In some embodiments, the spacer region sequence of the gRNA is a polynucleotide sequence containing at least a portion sufficiently complementary to the target site to hybridize with the target site in the target gene and guide the Cas / gRNA (or CRISPR) complex to bind sequence-specifically to the target site sequence. Perfect complementarity is not required, as long as sufficient complementarity is sufficient to induce hybridization. In some embodiments, the gRNA contains a spacer region sequence complementary to the target site, for example, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% complementary (e.g., perfect complementarity). The target nucleic acid strand containing the target site sequence may be referred to as the “complementary strand” of the target nucleic acid. In some embodiments, the spacer region sequence is a user-defined sequence. Guidelines for the selection of the spacer region sequence can be found, for example, in Fuji et al., Nat Biotechnol 2014 32:279–284 and Sternberg et al., Nature 2014 507:62-67.

[0243] In some respects, gRNA targets a target site in double-stranded DNA. Therefore, in some respects, the sequence of the target site may be defined by the sequence hybridized to the gRNA spacer region, or by a sequence complementary to the sequence hybridized to the gRNA spacer region. In some respects, the sequence of the target site may be defined by the sequence substituted by the gRNA spacer region for hybridization with DNA. In some embodiments, the sequence of the target site is the sequence hybridized to the gRNA.

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

[0245] In some implementations, the gRNA is a tandem of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence. The gRNA can target a desired DNA sequence by exchanging sequences encoding a 20 bp protospacer region, which imparts targeting specificity through complementary base pairing with the desired DNA target. The gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in type II CRISPR / Cas systems (e.g., Cas9). This duplex may include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acting as a guide sequence for Cas9 protein cleavage of target nucleic acids. As used interchangeably herein, “target region,” “target sequence,” or “protospacer region” refers to a region of the IL-2 gene or regulatory element targeted by a CRISPR / Cas9-based system. A CRISPR / Cas9-based system may include two or more gRNAs targeting 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 or different genes. The target sequence or protospacer region is followed by a PAM sequence located at the 3' end of the protospacer region. Different Type II systems have different PAM requirements. For example, Streptococcus pyogenes The type II system uses the “NGG” sequence, where “N” can be any nucleotide.

[0246] The target site of gRNA may be referred to as the protospacer region. In some embodiments, the spacer region is designed to target a protospacer region with a specific protospacer adjacent motif (PAM), which is a sequence adjacent to the protospacer region 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 implementations, Streptococcus pyogenes Cas9 uses PAM 5'-NGG-3' (SEQ ID NO: 68), where N is any nucleotide. In some embodiments, the PAM for the gRNA complexed with a type V CRISPR / Cas system (such as Cas12a (also known as Cpf1) or a variant thereof) uses TTTV (SEQ ID NO: 67), where V is A, C, or G. In some embodiments, Golden grapes Staphylococcus aureus Cas9 uses PAM 5'-NNGRRT-3' (SEQ ID NO: 69), where N is any nucleotide and R is G or A. In some embodiments, Neisseria meningitidis Cas9 uses PAM 5'-NNNNGATT-3' (SEQ ID NO: 70), where N is any nucleotide. In some embodiments, Campylobacter jejuniCas9 uses 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, Streptococcus thermophilus Using PAM5'-NNAGAAW-3' (SEQ ID NO: 72), where N is any nucleotide and W is A or T. In some embodiments, The new culprit, Francis bacteria Cas9 uses PAM 5'-NGG-3' (SEQ ID NO: 68), where N is any nucleotide. In some embodiments, *Treponema pallidum* (…) T. denticola Cas9 uses PAM 5'-NAAAAC-3' (SEQ ID NO: 73), where N is any nucleotide. In some embodiments, Cas12a (also known as Cpf1) from multiple species uses PAM 5'-TTTV-3' (SEQ ID NO: 74). In some embodiments, the Cas protein can be used or engineered to use a different PAM than those listed above. For example, the mutant SpCas9 protein can use PAM 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, for use with... suppuration Streptococcus The protospacer adjacent motif (PAM) of the gRNA complexed with Cas9 or its variants is NGG, as shown in SEQ ID NO: 68. In some embodiments, it is used to... Staphylococcus aureus The PAM for gRNA complexed with Cas9 or its variants is NNGRRT, as shown in SEQ ID NO: 69. Methods for designing or identifying gRNA spacer sequences and / or protospacer sequences in specific regions are known. The gRNA spacer sequence and / or protospacer sequence can be determined based on the type of Cas protein used and the associated PAM sequence.

[0247] The spacer sequence can be selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure can be determined using any suitable polynucleotide folding algorithm.

[0248] In some embodiments, the gRNA (including the guide sequence) will contain the base uracil (U), while the DNA encoding the gRNA molecule will contain the base thymine (T). Although not wishing to be bound by theory, in some embodiments, it is believed that complementarity between the guide sequence and the target sequence contributes to the specificity of the interaction between the gRNA / Cas molecule complex and the target nucleic acid. It should be understood that in the guide sequence and target sequence pair, the uracil base in the guide sequence will pair with the adenine base in the target sequence. The gRNA spacer sequence described herein can be defined by the DNA sequence encoding the gRNA spacer and / or the RNA sequence encoding the spacer.

[0249] In some embodiments, the gRNA contains modified nucleotides, for example, to improve stability. In some embodiments, one, more, or all of the nucleotides of the gRNA may be modified, for example, to make the gRNA less susceptible to degradation and / or to improve biocompatibility. By way of non-limiting example, the backbone of the gRNA may be modified with phosphate thioesters or other modifications. In some cases, the nucleotides of the gRNA may contain 2' modifications, such as 2-acetylation, 2' methylation, or other modifications.

[0250] Methods for designing gRNAs and exemplary targeting domains may include, for example, International PCT publications 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, and WO Those described in 2017 / 193107 and WO 2017 / 093969.

[0251] In some respects, the gRNA contains a scaffold sequence. In some respects, the scaffold sequence (in some cases including crRNA and / or tracrRNA sequences) will vary depending on the Cas protein. In some respects, different CRISPR / Cas systems have different gRNA scaffold sequences for binding to the Cas protein. In some embodiments, Staphylococcus aureusAn exemplary scaffold sequence of Cas9 comprises the sequence shown in SEQ ID NO: 41, or a sequence having sequence identity with SEQ ID NO: 41 of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, Staphylococcus aureus An exemplary scaffold sequence of Cas9 includes the sequence shown in SEQ ID NO: 41. In some embodiments, Streptococcus pyogenes An exemplary scaffold sequence of Cas9 comprises the sequence shown in SEQ ID NO: 8, or a sequence having sequence identity with SEQ ID NO: 8 of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, Streptococcus pyogenes An exemplary scaffold sequence of Cas9 includes the sequence shown in SEQ ID NO: 8.

[0252] In some implementations, amino acid cocci species ( Acidaminococcus sp.The exemplary stent sequence of Cas12a comprises the sequence shown in SEQ ID NO: 123, or a sequence having sequence identity of SEQ ID NO: 123 with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, the exemplary stent sequence of CasPhi-2 comprises the sequence shown in SEQ ID NO: 124, or a sequence having sequence identity of SEQ ID NO: 124 with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, the exemplary stent sequence of Un1Cas12f1 comprises the sequence shown in SEQ ID NO: 125, the sequence “GGAATGAAC” (SEQ ID NO: 126), or the sequence “TTTTATTTT” (SEQ ID NO: 127), or a sequence having sequence identity of SEQ ID NO: 125, 126, or 127 with a sequence identity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, the exemplary stent sequence of Un1Cas12f1 comprises the sequence shown in SEQ ID NO: 213, or a sequence having sequence identity of SEQ ID NO: 213 with a sequence identity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, the exemplary stent sequence of Un1Cas12f1 comprises the sequence shown in SEQ ID NO: 126, or a sequence having sequence identity of SEQ ID NO: 126 with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, the exemplary stent sequence of Un1Cas12f1 comprises the sequence shown in SEQ ID NO: 127, or a sequence having sequence identity of SEQ ID NO: 127 with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, Campylobacter jejuniAn exemplary stent sequence of Cas9 comprises the sequence shown in SEQ ID NO: 128, or a sequence having sequence identity of SEQ ID NO: 128 with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, an exemplary stent sequence of Cas12k comprises the sequence shown in SEQ ID NO: 129, or a sequence having sequence identity of SEQ ID NO: 129 with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some implementations, the exemplary scaffold sequence of CasMini includes the sequence shown in SEQ ID NO: 130, or a sequence having sequence identity with SEQ ID NO: 130 of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.

[0253] In some respects, gRNAs can target DNA targeting systems to direct the activity of related peptides (e.g., fusion proteins, DNA targeting systems, effector domains, etc.) to specific target sites within target nucleic acids (e.g., regulatory DNA elements at the IL-2 locus).

[0254] a. gRNAs used for transcriptional activation In some embodiments, the gRNAs provided herein target a site on the IL-2 gene or a regulatory element thereof to activate transcription. In some embodiments, the target site is located on the IL-2 gene. In some embodiments, the target site is located within a regulatory DNA element of the gene. In some embodiments, the regulatory DNA element is a sequence that a gene regulatory protein can bind to and affect gene transcription. In some embodiments, the regulatory DNA element is a sequence that a gene regulatory protein can bind to and affect IL-2 gene transcription. Exemplary target sites and combinations of target sites for gRNAs with multiple DNA binding systems include any of those described in Section IB.

[0255]

[0256] In some embodiments, the gRNA targets a target site comprising a sequence selected from any of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, as shown in Table 1, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the aforementioned sequences, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the aforementioned sequences. In some embodiments, the target site is a continuous portion of any one of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site is shown as any one of SEQ ID NO: 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 comprising the sequence shown in SEQ ID NO: 12, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 12, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown in SEQ ID NO: 12.

[0258] In some embodiments, the gRNA targets a target site comprising the sequence shown in SEQ ID NO: 24, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 24, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown in SEQ ID NO: 24.

[0259] In some embodiments, the gRNA targets a target site comprising the sequence shown in SEQ ID NO: 26, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 26, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown in SEQ ID NO: 26.

[0260] In some embodiments, the gRNA targets a target site comprising the sequence shown in SEQ ID NO: 28, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 28, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown in SEQ ID NO: 28.

[0261] In some embodiments, the gRNA targets a target site comprising the sequence shown in SEQ ID NO: 38, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 38, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown 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 shown in SEQ ID NO: 8 (GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC), or a sequence having all or part of it with sequence identity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, the scaffold sequence is shown in SEQ ID NO: 8.

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

[0264] In some implementations, this article provides a multiple epigenetic modification DNA targeting system comprising a combination of gRNAs.

[0265] In some embodiments, the gRNA comprises a spacer sequence selected from any of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, as shown in Table 3, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a continuous portion of any of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, with a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in any one of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39.

[0266] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 11, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 11, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 11.

[0267] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 23, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 23, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 23.

[0268] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 25, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 25, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 25.

[0269] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 27, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 27, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 27.

[0270] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 37, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 37, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 37.

[0271] Table 3. Exemplary guide RNAs (gRNAs) for use in combination with Cas (e.g., SpCas9) to transcribe and activate IL-2.

[0272] In some embodiments, this document provides combinations of gRNAs, each targeting a site of the IL-2 gene or its regulatory elements for transcriptional activation. In some embodiments, this document provides a multiple epigenetic modification DNA targeting system comprising combinations of gRNAs.

[0273] In some embodiments, the combination of gRNAs comprises at least two gRNAs that target at least two different target sites to transcribe and activate IL-2. In some embodiments, the combination of gRNAs comprises at least two gRNAs that target two identical target sites to perform transcriptional activation. In some embodiments, the combination of target sites targeted by the gRNAs is selected from the combinations of target sites listed in Table 1. In some embodiments, the gRNAs target the combinations of target sites listed in Table 2.

[0274] In some implementations, each gRNA in the combination is selected from any of the gRNAs described herein for targeting transcriptional activation. Exemplary combinations of gRNA pairings are listed in Table 4.

[0275] Table 4. Exemplary pairing combinations of SpCas9 gRNA

[0276] In some embodiments, the combination of gRNAs comprises at least two gRNAs that target the same target site. In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first target site and a second gRNA targeting the first target site. In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first target site and a second gRNA targeting a second target site, wherein these target sites are identical.

[0277] In some embodiments, the combination of gRNAs comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 11, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 11. In some embodiments, the combination of gRNAs comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 23, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 23. In some embodiments, the combination of gRNAs comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 25, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 25.

[0278] In some embodiments, the combination of gRNAs comprises at least two gRNAs that target at least two target sites. In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first target site and a second gRNA targeting a second target site, wherein these target sites are different.

[0279] In some embodiments, the gRNA combination comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 11, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 23. In some embodiments, the gRNA combination comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 11, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 25. In some embodiments, the gRNA combination comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 11, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 27. In some embodiments, the gRNA combination comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 11, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 37. In some embodiments, the combination of gRNAs comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 23, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 25. In some embodiments, the combination of gRNAs comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 23, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 27. In some embodiments, the combination of gRNAs comprises a first gRNA and a second gRNA, wherein the first gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 23, and the second gRNA targets a target site of IL-2 and contains the sequence shown in SEQ ID NO: 37.

[0280] In some embodiments, the combination of gRNAs comprises at least three gRNAs that target at least three target sites. In a particular embodiment, the at least three target sites are different target sites. In a particular embodiment, the combination of gRNAs comprises a first gRNA targeting a first target site, a second gRNA targeting a second target site, and a third gRNA targeting a third target site. In some embodiments, at least two of the three target sites may be the same. In some embodiments, the combination of gRNAs comprises at least three gRNAs that target at least two target sites.

[0281] In some embodiments, the combination of gRNAs comprises at least four gRNAs that target at least four target sites. In a particular embodiment, the at least four target sites are different target sites. In a particular embodiment, the combination of gRNAs comprises a first gRNA targeting a first target site, a second gRNA targeting a second target site, a third gRNA targeting a third target site, and a fourth gRNA targeting a fourth target site. In some embodiments, at least two of the four target sites may be the same. In some embodiments, the combination of gRNAs comprises at least four gRNAs that target at least three target sites.

[0282] In a particular embodiment, the first gRNA contains the spacer sequence shown in SEQ ID NO: 11 (GACUUAGUGCAAUGCAAGAC), the second gRNA contains the spacer sequence shown in SEQ ID NO: 23 (CUCUCUCUGCAGACAGGGCA), the third gRNA contains the spacer sequence shown in SEQ ID NO: 27 (AGAGGGAAGUGUCACAUAAU), and the fourth gRNA contains the spacer sequence shown in SEQ ID NO: 37 (GGAAAUGACAUGCUUGAAGU).

[0283] In some embodiments, the combination of gRNAs comprises at least five gRNAs that target at least five target sites. In a particular embodiment, the at least five target sites are different target sites. In a particular embodiment, the combination of gRNAs comprises a first gRNA targeting a first target site, a second gRNA targeting a second target site, a third gRNA targeting a third target site, a fourth gRNA targeting a fourth target site, and a fifth target gRNA targeting a fifth target site. In some embodiments, at least two of the five target sites may be the same. In some embodiments, the combination of gRNAs comprises at least five gRNAs that target at least four target sites.

[0284] In a particular embodiment, the first gRNA contains the spacer sequence shown in SEQ ID NO: 11 (GACUUAGUGCAAUGCAAGAC), the second gRNA contains the spacer sequence shown in SEQ ID NO: 23 (CUCUCUCUGCAGACAGGGCA), the third gRNA contains the spacer sequence shown in SEQ ID NO: 25 (GGCAGGGUAGAGAAGUAGAG), the fourth gRNA contains the spacer sequence shown in SEQ ID NO: 27 (AGAGGGAAGUGUCACAUAAU), and the fifth gRNA contains the spacer sequence shown in SEQ ID NO: 37 (GGAAAUGACAUGCUUGAAGU).

[0285] In some implementations, combinations of gRNAs targeting specific sites are used to activate transcription, as shown in Table 2, for example.

[0286] Exemplary combinations of paired SpCas9 guide RNA (gRNA) and target sites are shown in Table 5.

[0287] Table 5. Exemplary combinations of paired SpCas9 guide RNA (gRNA) and target sites for transcriptional activation

[0288] In some embodiments, any of the provided gRNA sequences is provided complexed with or in combination with a fusion protein containing Cas9. In some embodiments, Cas9 is dCas9. In some embodiments, dCas9 is dSaCas9, such as dSaCas9 shown in SEQ ID NO: 65.

[0289] In some embodiments, the gRNA targets a target site comprising a sequence selected from any of SEQ ID NO: 43, 45, 47, 49, 51, 53, 55, 57, and 59, as shown in Table 1, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the aforementioned sequences, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the aforementioned sequences. In some embodiments, the target site is a continuous portion of any of SEQ ID NO: 43, 45, 47, 49, 51, 53, 55, 57, and 59, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some implementations, the target site is shown in any one of SEQ ID NO: 43, 45, 47, 49, 51, 53, 55, 57 and 59.

[0290] In some embodiments, the gRNA targets a target site comprising the sequence shown in SEQ ID NO: 43, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 43, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown in SEQ ID NO: 43.

[0291] In some embodiments, the gRNA targets a target site comprising the sequence shown in SEQ ID NO: 51, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 51, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown in SEQ ID NO: 51.

[0292] In some embodiments, the gRNA targets a target site comprising the sequence shown in SEQ ID NO: 57, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 57, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown in SEQ ID NO: 57.

[0293] In some embodiments, the gRNA targets a target site comprising the sequence shown in SEQ ID NO: 59, a continuous portion of at least 14 nucleotides thereof, a complementary sequence to any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the target site is a continuous portion of the sequence shown in SEQ ID NO: 59, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the target site sequence is shown in SEQ ID NO: 59.

[0294] In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence shown in SEQ ID NO: 41 (GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU), or a sequence having all or part of it with sequence identity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. In some embodiments, the scaffold sequence is shown in SEQ ID NO: 41.

[0295] In some embodiments, the gRNA comprises a spacer sequence selected from any of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, and 58, as shown in Table 6, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a continuous portion of any of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, and 58, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some implementations, the spacer sequence of the gRNA is shown in any one of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56 and 58.

[0296] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 42, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 42, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 42.

[0297] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 50, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 50, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 50.

[0298] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 56, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 56, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 56.

[0299] In some embodiments, the gRNA comprises the spacer region sequence shown in SEQ ID NO: 58, or a continuous portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any of the foregoing. In some embodiments, the spacer region sequence of the gRNA is a continuous portion of SEQ ID NO: 58, having a length of 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the spacer region sequence of the gRNA is shown in SEQ ID NO: 58.

[0300] Table 6. Exemplary SaCas9 guide RNAs (gRNAs) for transcriptional activation

[0301] In some embodiments, this document provides combinations of gRNAs, each targeting a site of the IL-2 gene or its regulatory elements for transcriptional activation. In some embodiments, this document provides a multiple epigenetic modification DNA targeting system comprising combinations of gRNAs.

[0302] In some embodiments, the combination of gRNAs comprises at least two gRNAs that target at least two different target sites for transcriptional activation. In some embodiments, the combination of gRNAs comprises at least two gRNAs that target two identical target sites for transcriptional activation. In some embodiments, the gRNA targeting is selected from combinations of target sites listed in Table 1. In some embodiments, the gRNA targeting is selected from combinations of target sites from the group consisting of SEQ ID NOs: 43, 45, 47, 49, 51, 53, 55, 57, and 59.

[0303] In some embodiments, each gRNA in the combination of gRNAs is selected from any of the gRNAs described herein for targeting transcriptional activation. In some embodiments, the combination of gRNAs is selected from the gRNAs listed in Table 6. In some embodiments, the gRNAs have a spacer region sequence selected from the group consisting of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, and 58.

[0304] In some embodiments, the combination of gRNAs comprises at least two gRNAs that target the same target site. In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first target site and a second gRNA targeting the first target site. In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first target site and a second gRNA targeting a second target site, wherein these target sites are identical.

[0305] In some embodiments, the combination of gRNAs comprises at least two gRNAs that target at least two target sites. In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first target site and a second gRNA targeting a second target site, wherein these target sites are different.

[0306] In some embodiments, the combination of gRNAs comprises at least three gRNAs that target at least three target sites. In a particular embodiment, the at least three target sites are different target sites. In a particular embodiment, the combination of gRNAs comprises a first gRNA targeting a first target site, a second gRNA targeting a second target site, and a third gRNA targeting a third target site. In some embodiments, at least two of the three target sites may be the same. In some embodiments, the combination of gRNAs comprises at least three gRNAs that target at least two target sites.

[0307] In some embodiments, the combination of gRNAs comprises at least four gRNAs that target at least four target sites. In a particular embodiment, the at least four target sites are different target sites. In a particular embodiment, the combination of gRNAs comprises a first gRNA targeting a first target site, a second gRNA targeting a second target site, a third gRNA targeting a third target site, and a fourth gRNA targeting a fourth target site. In some embodiments, at least two of the four target sites may be the same. In some embodiments, the combination of gRNAs comprises at least four gRNAs that target at least three target sites.

[0308] In some embodiments, the combination of gRNAs comprises at least five gRNAs that target at least five target sites. In a particular embodiment, the at least five target sites are different target sites. In a particular embodiment, the combination of gRNAs comprises a first gRNA targeting a first target site, a second gRNA targeting a second target site, a third gRNA targeting a third target site, a fourth gRNA targeting a fourth target site, and a fifth target gRNA targeting a fifth target site. In some embodiments, at least two of the five target sites may be the same. In some embodiments, the combination of gRNAs comprises at least five gRNAs that target at least four target sites.

[0309] D. Other DNA-binding domains and DNA targeting systems In some of the provided embodiments, the DNA-binding domain includes zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), a wide range of nucleases, homing endonucleases, or I-SceI enzymes, or variants thereof. In some embodiments, the DNA-binding domain comprises a non-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 the DNA-binding domains described herein, such as the DNA-binding domain as an engineered zinc finger protein (eZFP) or TALE.

[0310] In some implementations, a ZFP, zinc finger DNA-binding protein, or zinc finger DNA-binding domain is a domain within a protein or larger protein that binds DNA in a sequence-specific manner via one or more zinc fingers. A zinc finger is an amino acid sequence region within the binding domain whose structure is stabilized by coordination with zinc ions. The term "zinc finger DNA-binding protein" is often abbreviated as zinc finger protein or ZFP. ZFPs include artificial or engineered ZFPs (eZFPs) that contain a ZFP domain typically 9-18 nucleotides in length, assembled from a single finger that targets a specific DNA sequence. ZFPs include those where the single-finger domain is approximately 30 amino acids in length and contains an α-helix and a β-turn, and has two, three, four, five, or six fingers. The two constant histidine residues in the α-helix are coordinated with two cysteine ​​residues at a single β-turn via zinc. Generally, the sequence specificity of a ZFP can be altered by amino acid substitutions at the four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Therefore, for example, ZFP or molecules containing ZFP are not naturally occurring, such as eZFP engineered to bind to selected target sites.

[0311] In some implementations, the zinc finger is custom-designed (i.e., designed by the user) or obtained from commercial sources. Various methods for designing zinc finger proteins are available. For example, methods for designing zinc finger proteins to bind to target DNA sequences of interest are described in, for example, Liu, Q. et al., PNAS, 94(11):5525-30 (1997); Wright, DA et al., Nat. Protoc., 1(3):1637-52 (2006); Gersbach, CA et al., Acc. Chem. Res., 47(8):2309-18 (2014); Bhakta MS et al., Methods Mol. Biol., 649:3-30 (2010); and Gaj et al., Trends Biotechnol, 31(7):397-405 (2013). Furthermore, various network-based tools for designing zinc finger proteins to bind to target DNA sequences of interest are publicly available. See, for example, Scripps' ZincFinger Tools design website: scrips.edu / barbas / zfdesign / zfdesignhome.php. Various commercial services for designing zinc finger proteins to bind to DNA target sequences of interest are also available. See, for example, commercially available services or kits from Creative Biolabs (creative-biolabs.com / Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium Modular Assembly kit from Addgene (addgene.org / kits / zfc-modular-assembly / ), or Sigma Aldrich's CompoZr Custom ZFN Service (sigmaaldrich.com / life-science / zinc-finger-nuclease-technology / custom-zfn.html).

[0312] In some implementations, the fusion protein of the DNA targeting system includes an eZFP DNA-binding domain and an effector domain.

[0313] Transcription activator-like effectors (TALEs) are found in Xanthomonas spp. XanthomonasA protein naturally occurring in bacteria. TALE comprises multiple repeating amino acid sequences, each repeating sequence having binding specificity to one base in the target sequence. Each repeating sequence contains a pair of variable residues (repeated variable double residues; RVD) at positions 12 and 13, which determines the nucleotide specificity of the repeating sequence. In some embodiments, the RVDs associated with recognizing 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, 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 to other amino acid residues to modulate their specificity for nucleotides A, T, C, and G, and particularly to enhance this specificity. Binding domains with similar modular, base-by-base nucleic acid binding properties can also originate from different bacterial species. These alternative modular proteins can exhibit higher sequence variability than TALE repetitive sequences.

[0314] In some embodiments, a “TALE DNA-binding domain” or “TALE” is a polypeptide containing one or more TALE repeat domains / units. Each repeat domain, containing a repeating variable double residue (RVD), is involved in the binding of TALE to its homologous target DNA sequence. A single “repeat unit” (also called a “repeat sequence”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within naturally occurring TALE proteins. TALE proteins can be programmed to bind to target sites using classical or non-classical RVDs within the repeat unit. See, for example, U.S. Patents 8,586,526 and 9,458,205.

[0315] In some implementations, the fusion protein of the DNA targeting system includes a TALE DNA-binding domain and an effector domain.

[0316] Zinc finger and TALE DNA-binding domains can be engineered to bind predetermined nucleotide sequences, for example, by engineering the recognition helical region of naturally occurring zinc finger proteins (altering one or more amino acids), by engineering amino acids (repetitive variable double residues or RVD regions) in TALE repeat sequences involved in DNA binding, or by systematically sequencing modular DNA-binding domains (such as TALE repeat sequences or ZFP domains). Therefore, engineered zinc finger or TALE proteins are non-naturally occurring proteins. Non-limiting examples of methods for engineering zinc finger and TALE proteins are design and selection. The engineered protein is a protein that does not exist in nature, and its design / composition is primarily derived from rational criteria. Rational design criteria include applying substitution rules and using computerized algorithms to process information in databases storing existing ZFP and / or TALE designs (classical and non-classical RVD) and binding data. See, for example, U.S. Patents 9,458,205, 8,586,526, 6,140,081, 6,453,242, and 6,534,261; also see WO 98 / 53058, WO 98 / 53059, WO 98 / 53060, WO 02 / 016536, and WO 03 / 016496.

[0317] 1. Exemplary ZFP In some respects, the DNA-binding domain contains zinc finger proteins (ZFPs). In some respects, this document provides exemplary ZFPs that are capable of binding to or can bind to target sites, such as any target sites provided herein in Section IB. In some respects, exemplary ZFPs can facilitate specific targeting of effector domains to transcriptionally activate target sites provided in Section I, such as target sites in the IL-2 gene, to achieve gene-specific transcriptional activation of IL-2. Therefore, in some respects, exemplary ZFPs contribute to promoting lymphoid activation and function.

[0318] In some embodiments, the target site of the ZFP provided herein is located within the IL-2 gene. In some embodiments, the target site within the IL-2 gene is located around or including the transcription start site (TSS) in region 4, region 5, or the region surrounding the TSS. In some embodiments, the region surrounding or including the TSS is located 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 of the assembled human genome version GRCh38 (hg38) chr4: 122,507,000-122,508,985. In some embodiments, the target site within the IL-2 gene is located within region 4 or region 5.

[0319] In some embodiments, the target site of the ZFP provided herein comprises the nucleotide sequence shown in any of SEQ ID NO: 186-188, a continuous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the target site of the ZFP provided herein comprises the nucleotide sequence shown in any of SEQ ID NO: 186-188. In some embodiments, the target site is contained 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, the ZFP binds to the target site without binding to other sites containing different sequences. For example, in some embodiments, a single ZFP disclosed herein binds to the target site shown in SEQ ID NO: 186 and does not bind to different target sites (such as the target site shown in SEQ ID NO: 188). In some embodiments, the target site of the ZFP provided herein comprises the sequences shown in Table 7.

[0320] Table 7. ZFP target sequences

[0321] In some embodiments, the target site of the ZFP provided herein comprises the nucleotide sequence shown in SEQ ID NO: 186, a continuous portion thereof of at least 12 nt, or a complementary sequence to any of the foregoing. In some embodiments, the target site of the ZFP provided herein comprises the sequence shown in SEQ ID NO: 186.

[0322] In some embodiments, the target site of the ZFP provided herein comprises the nucleotide sequence shown in SEQ ID NO: 187, a continuous portion thereof of at least 12 nt, or a complementary sequence to any of the foregoing. In some embodiments, the target site of the ZFP provided herein comprises the sequence shown in SEQ ID NO: 187.

[0323] In some embodiments, the target site of the ZFP provided herein comprises the nucleotide sequence shown in SEQ ID NO: 188, a continuous portion thereof of at least 12 nt, or a complementary sequence to any of the foregoing. In some embodiments, the target site of the ZFP provided herein comprises the sequence shown in SEQ ID NO: 188.

[0324] In some embodiments, the characteristics of the ZFPs targeting specific target sites provided herein are shown in Table 8. In some embodiments, the ZFP comprises six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, each zinc finger containing a corresponding recognition region F1-F6, as shown in Table 8. In some embodiments, recognition regions F1-F6 facilitate specific binding to the target site sequences indicated in Table 8. In some embodiments, the ZFP comprises an amino acid sequence containing the recognition region, as shown in Table 8. In some embodiments, the ZFP may be encoded by the DNA sequence shown in Table 8.

[0325] Table 8. Exemplary ZFP engineered zinc finger proteins

[0326] In some embodiments, this document provides a ZFP, such as IL2_R4_A as described herein. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence shown in SEQ ID NO: 186, a continuous portion thereof of at least 12 nt, or a complementary sequence to any of the foregoing. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence shown in SEQ ID NO: 186. In some embodiments, the target site is double-stranded DNA. In some embodiments, the ZFP comprises six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, each zinc finger containing a corresponding zinc finger recognition region F1 to F6, and the amino acid sequences of each zinc finger recognition region are 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 shown in SEQ ID NO: 189 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the ZFP comprises the amino acid sequence shown in SEQ ID NO: 189. In some embodiments, the ZFP is encoded by the nucleotide sequence shown in SEQ ID NO: 192 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the ZFP is encoded by the nucleotide sequence shown in SEQ ID NO: 192.

[0327] In some embodiments, this document provides a ZFP, such as IL2_R4_B as described herein. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence shown in SEQ ID NO: 187, a continuous portion thereof of at least 12 nt, or a complementary sequence to any of the foregoing. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence shown in SEQ ID NO: 187. In some embodiments, the target site is double-stranded DNA. In some embodiments, the ZFP comprises six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, each zinc finger containing a corresponding zinc finger recognition region F1 to F6, and the amino acid sequences of each zinc finger recognition region are 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 shown in SEQ ID NO: 190 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the ZFP comprises the amino acid sequence shown in SEQ ID NO: 190. In some embodiments, the ZFP is encoded by the nucleotide sequence shown in SEQ ID NO: 191 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the ZFP is encoded by the nucleotide sequence shown in SEQ ID NO: 191.

[0328] In some embodiments, this document provides a ZFP, such as IL2_R5_A as described herein. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence shown in SEQ ID NO: 188, a continuous portion thereof of at least 12 nt, or a complementary sequence to any of the foregoing. In some embodiments, the ZFP targets a target site comprising the nucleotide sequence shown in SEQ ID NO: 188. In some embodiments, the target site is double-stranded DNA. In some embodiments, the ZFP comprises six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, each zinc finger containing a corresponding zinc finger recognition region F1 to F6, and the amino acid sequences of each zinc finger recognition region are 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 shown in SEQ ID NO: 191 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the ZFP comprises the amino acid sequence shown in SEQ ID NO: 191. In some embodiments, the ZFP is encoded by the nucleotide sequence shown in SEQ ID NO: 194 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the ZFP is encoded by the nucleotide sequence shown in SEQ ID NO: 194.

[0329] E. Effect domain In some aspects, the DNA targeting systems provided herein also include one or more effector domains. In some embodiments, the one or more effector domains are transcription activator effector domains. In some embodiments, in a DNA targeting system having multiple effector domains, each effector domain is a transcription activator. In some embodiments, this document provides a DNA targeting system comprising a fusion protein, the fusion protein comprising: (a) a DNA-binding domain capable of targeting a target site in the IL-2 gene or its regulatory DNA elements, such as any of those described in Section IC or Chapter ID above; and (b) at least one effector domain.

[0330] 1. Effector domains for transcriptional activation In some aspects, the DNA targeting systems provided herein also include one or more effector domains, such as transcription activator effector domains. In some embodiments, this document provides a DNA targeting system comprising a fusion protein that includes: (a) a DNA-binding domain capable of targeting a target site in the IL-2 gene or its regulatory elements or regulatory DNA elements thereof, such as any DNA-binding domain described in Section ID or Chapter IE of the preceding article; and (b) at least one effector domain. In some aspects, the effector domain is capable of increasing IL-2 transcription. In some aspects, the effector domain includes a transcription activation domain.

[0331] In some embodiments, when ectopically recruited to a gene or its DNA regulatory elements, the effector domain activates, induces, catalyzes, or causes an increase in gene transcription. In some embodiments, the effector domain activates, induces, catalyzes, or causes the following: transcriptional activation, transcriptional co-activation, transcriptional elongation, transcriptional derepression, 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 causes transcriptional activation, transcriptional co-activation, or transcriptional elongation. In some embodiments, the effector domain induces transcriptional activation. In some embodiments, the effector domain itself has one of the aforementioned activities (i.e., direct action). In some embodiments, the effector domain recruits a polypeptide domain having one of the aforementioned activities and / or interacts with that polypeptide domain (i.e., indirect action).

[0332] Gene expression of endogenous mammalian genes (such as human genes) can be achieved by targeting mammalian genes or their regulatory DNA elements (e.g., promoters or enhancers) via one or more gRNAs with a fusion protein containing a DNA-binding domain (such as dCas9) and an effector domain (such as a transcription activation domain). Any of a variety of effector domains for transcriptional activation (e.g., transcription activation domains) is known and can be used according to the provided implementation scheme. The activation of target genes by transcriptional activation domains and through Cas fusion proteins (containing multiple Cas molecules) and transcriptional activation domains is described in, for example, the following publications: 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. Methods10, 973–976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833–838. (2013), and Maeder, ML et al. Nat. Methods 10, 977–979 (2013).

[0333] In some embodiments, the transcriptional activation domain comprises a domain of a protein selected from VP64, p65, Rta, p300, CBP, VPR, VPH, HSF1, TET protein (e.g., TET1), a partial or complete functional fragment or domain thereof, or a combination of any of the foregoing. In some embodiments, the transcriptional activation domain further comprises at least one domain of a protein selected from FOXO3 and NCOA3, which exhibits transcriptional activation, is capable of inducing or activating gene transcription, is a functional transcriptional activation domain, and / or has transcriptional activation function. In some embodiments, the transcriptional activation domain further comprises at least one domain selected from FOXO3 and NCOA3.

[0334] In some embodiments, the epigenetic marker includes histone H3K27 acetylation. In some embodiments, the effector domain catalyzes the acetylation of histone H3 lysine 27 at the target site, or is capable of recruiting an enzyme that catalyzes the acetylation of histone H3 lysine 27 at the target site. In some embodiments, the enzyme catalyzing acetylation is an acetyltransferase. In some embodiments, the transcriptional activation domain includes the VP64 domain. For example, dCas9-VP64 can activate a gene by targeting a target site via one or more gRNAs. VP64 is a polypeptide consisting of four tandem copies of VP16, which is a 16-amino acid transactivation domain of herpes simplex virus. The VP64 domain included in dCas fusion proteins has 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 shown 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 66 or a portion thereof. In some embodiments, the transcriptional activation domain is shown in SEQ ID NO: 66.

[0335] In some embodiments, the transcriptional activation domain comprises the p65 activation domain (p65AD). p65AD is the major trans-activation domain of the 65 kDa polypeptide in the nuclear form of the NF-KB transcription factor. Exemplary sequences of the human transcription factor p65 are available in the Uniprot database with accession number Q04206. The p65 domain included in dCas fusion proteins has been described, for example, in WO2017 / 180915 and Chavez, A. et al., Nat. Methods 12, 326–328 (2015). An exemplary p65 activation domain is shown in SEQ ID NO: 79. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 79 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 79 or a portion thereof. In some embodiments, the transcriptional activation domain is shown in SEQ ID NO: 79.

[0336] 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 cleavage gene expression and triggers viral reactivation. Rta domains included 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 Rta domain is shown 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 80 or a portion thereof. In some embodiments, the transcriptional activation domain is shown in SEQ ID NO: 80.

[0337] 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 associated with CBP. Both CBP and p300 interact with a variety of transcriptional activators to influence gene transcription (Gerritsen, ME et al. PNAS 94(7):2927-2932 (1997)). In some embodiments, the transcriptional activation domain comprises a p300 domain. The p300 domain (such as the catalytic core of p300) included in dCas fusion proteins for gene activation has been described, for example, in WO 2016 / 130600, WO 2017 / 180915 and Hilton, IB et al., Nat. Biotechnol. 33(5):510-517 (2015). An exemplary human CBP sequence is shown in SEQ ID NO: 81. An exemplary human p300 sequence is shown in SEQ ID NO: 82. An exemplary p300 domain is shown in SEQ ID NO: 83. In some embodiments, the transcriptional activation domain comprises any one or a portion of SEQ ID NO: 81-83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one or a portion of SEQ ID NO: 81-83. In some embodiments, the transcription activation domain comprises SEQ ID NO: 83 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 83 or a portion thereof. In some embodiments, the transcription activation domain is shown in SEQ ID NO: 83.

[0338] In some embodiments, the transcriptional activation domain comprises an HSF1 domain. In some aspects, HSF1 refers to the heat shock factor protein 1 protein encoded by the human HSF1 gene. HSF1 included 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 shown in SEQ ID NO: 84. An exemplary HSF1 domain sequence is shown in SEQ ID NO: 84. In some embodiments, the transcriptional activation domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 84 or SEQ ID NO: 1775 or a portion thereof. In some embodiments, the transcription activation domain comprises SEQ ID NO: 84 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 84 or a portion thereof. In some embodiments, the transcription activation domain is shown in SEQ ID NO: 84.

[0339] In some embodiments, the transcriptional activation domain comprises a triplet activator VP64-p65-Rta (also known as a VPR). The VPR comprises three transcriptional activation domains (VP64, p65, and Rta) fused together via a short amino acid linker and can efficiently upregulate target gene expression. VPRs included in dCas fusion proteins for gene activation have been described, for example, in WO2021 / 226555 and Chavez, A. et al., Nat. Methods 12, 326–328 (2015). An exemplary VPR polypeptide is shown 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 85 or a portion thereof. In some embodiments, the transcriptional activation domain is shown in SEQ ID NO: 85.

[0340] In some embodiments, the transcription activation domain comprises a VPH. The VPH is a triple activator polypeptide comprising VP64, mouse p65, and HSF1. VPHs included in dCas fusion proteins for gene activation have been described, for example, in WO 2021 / 226555. An exemplary VPH polypeptide is shown in SEQ ID NO: 86. In some embodiments, the transcription activation domain comprises SEQ ID NO: 86 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 86 or a portion thereof. In some embodiments, the transcription activation domain is shown in SEQ ID NO: 86.

[0341] In some embodiments, the transcriptional activation effector domain has demethylase activity. The effector domain may include an enzyme that removes methyl (CH3-) groups from nucleic acids, proteins (particularly histones), and other molecules. The effector domain may convert methyl groups to hydroxymethylcytosine in mechanisms that demethylate DNA. Alternatively, the transcriptional activation domain may convert methyl groups to hydroxymethylcytosine in mechanisms that demethylate DNA. The effector domain may catalyze this reaction. For example, the transcriptional activation domain catalyzing this reaction may contain a domain from a TET protein, such as TET1 (decano-undecapodimethylcytosine 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) to 5-hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation. TET1, included in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555. An exemplary human TET1 sequence is shown in SEQ ID NO: 87. An exemplary TET1 catalytic domain is shown in SEQ ID NO: 88. In some embodiments, the transcriptional activation domain comprises an amino acid sequence of SEQ ID NO: 87 or SEQ ID NO: 88 or a portion thereof, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 87 or a portion thereof. In some embodiments, the transcriptional activation domain comprises an amino acid sequence of SEQ ID NO: 88 or a portion thereof, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 88 or a portion thereof. In some embodiments, the transcriptional activation domain is shown in SEQ ID NO: 88.

[0342] In some embodiments, the effector domain may include a SunTag domain. A SunTag is an array of repeating peptides that recruits multiple copies of an antibody-fusion protein to which the repeating peptides bind. The antibody-fusion protein may include additional effector domains, such as transcriptional activation domains (e.g., VP64), to induce increased transcription of a target gene. SunTags included in dCas fusion proteins for gene activation have been described, for example, in WO 2016 / 011070 and Tanenbaum, M. et al. Cell. 159(3):635–646 (2014). An exemplary SunTag effector domain comprises a repeating GCN4 peptide having the amino acid sequence LLPKNYHLENEVARLKKLVGER (SEQ ID NO: 89) separated by a linker having the amino acid sequence GGSGG (SEQ ID NO: 90). In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 89, its domain, a portion thereof, or a variant thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the foregoing. In some embodiments, the SunTag effector domain recruits an antibody-fusion protein containing a transcription activator effector domain (e.g., VP64) and binding to a GCN4 peptide, thereby activating transcription at the target site and acting as a transcription activator effector domain.

[0343] In some embodiments, the transcriptional activation domain includes a FOXO3 domain, i.e., a domain derived 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 shown in SEQ ID NO: 219. Exemplary FOXO3 domain sequences are shown in SEQ ID NO: 220 and SEQ ID NO: 221. In some embodiments, the transcriptional activation domain comprises the sequence shown in any of SEQ ID NO: 219-221 or a domain or portion thereof (such as a continuous portion 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 with SEQ ID NO: The sequence or its domains or portions (such as a continuous portion 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)) shown in any of SEQ ID NO: 219 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 219. In some embodiments, the transcription activation domain is or contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 219. In some embodiments, the transcription activation domain contains a continuous portion of at least 80 amino acids in length of SEQ ID NO: 219. In some embodiments, the transcription activation domain comprises SEQ ID NO: 220. In some embodiments, the transcription activation domain is shown in SEQ ID NO: 220. An exemplary nucleotide sequence encoding the transcription activation domain shown in SEQ ID NO: 220 is shown in SEQ ID NO: 222. In some embodiments, the transcription activation domain is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 221. In some embodiments, the transcription activation domain comprises a continuous portion of SEQ ID NO: 219 with a length of at least 42 amino acids. In some embodiments, the transcription activation domain comprises SEQ ID NO: 221. In some embodiments, the transcription activation domain is shown in SEQ ID NO: 221.

[0344] In some embodiments, the transcriptional activation domain includes the NCOA3 domain, i.e., a domain derived 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 of both steroid receptors and nuclear receptors. An exemplary human NCOA3 sequence is shown in SEQ ID NO: 223. Exemplary NCOA3 domain sequences are shown in SEQ ID NO: 224 and SEQ ID NO: 184. In some embodiments, the transcriptional activation domain comprises the sequence shown in any of SEQ ID NO: 184, 223, and 224, or a domain or portion thereof (such as a continuous portion 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 with SEQ ID NO: The sequence or its domains or portions (such as a continuous portion 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)) shown in any of SEQ ID NO: 224 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 224. In some embodiments, the transcription activation domain is or contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 224. In some embodiments, the transcription activation domain contains a continuous portion of SEQ ID NO: 223 with a length of at least 80 amino acids. In some embodiments, the transcription activation domain comprises SEQ ID NO: 224. In some embodiments, the transcription activation domain is shown in SEQ ID NO: 224. An exemplary nucleotide sequence encoding the transcription activation domain shown in SEQ ID NO: 224 is shown in SEQ ID NO: 185. In some embodiments, the transcription activation domain is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 184. In some embodiments, the transcription activation domain comprises a continuous portion of SEQ ID NO: 223 with a length of at least 49 amino acids. In some embodiments, the transcription activation domain comprises SEQ ID NO: 184.In some implementations, the transcriptional activation domain is shown in SEQ ID NO: 184.

[0345] In some embodiments, the transcription activation domain comprises a fusion of NCOA3 and FOXO3 domains as described herein, such as the NCOA3 domain shown in SEQ ID NO: 184 and the FOXO3 domain shown in SEQ ID NO: 221. In some embodiments, the transcription activation domain comprises a fusion of two NCOA3 domains and one FOXO3. The fusion protein comprises these domains, which may be arranged in any order. In some embodiments, the transcription activation domain is arranged from the N-terminus to the C-terminus as follows: a first NCOA3 domain, a FOXO3 domain, and a second NCOA3 domain, also referred to as the NCOA3-FOXO3-NCOA3 domain (NFN). In some embodiments, the NFN domain is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with 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 implementations, the domains are directly connected to each other, or they are connected by linkers (such as peptide linkers).

[0346] In some embodiments, the fusion protein includes a transcriptional activation domain, which is an NFN domain. In some embodiments, the fusion protein also includes an additional transcriptional activation domain, which is a VP64 domain. In some embodiments, the NFN domain and the VP64 domain are directly connected to each other, or they are connected by a linker, or they are separated by a DNA-binding domain. In some embodiments, the NFN domain and the VP64 domain are separated by a DNA-binding domain.

[0347] F. Fusion protein In some embodiments, the fusion protein comprises: (a) a DNA-binding domain capable of targeting multiple target sites of the IL-2 gene or its regulatory elements, and (b) at least one transcription activator effector domain for increasing transcription of the IL-2 gene or its regulatory elements.

[0348] In some embodiments, the fusion protein comprises at least one of any DNA-binding domains described in Section IC or Section ID herein, and at least one of any effector domains described in Section IE herein. In some embodiments, the fusion protein comprises a CRISPR / Cas-based DNA-binding domain, such as that described in Section IC; and at least one effector domain for transcriptional activation, as described in Section IE1. In some embodiments, the fusion protein comprises a ZFP DNA-binding domain, such as that described in Section ID or ID1; and at least one effector domain for transcriptional activation, as described in Section IE. In some aspects, the fusion protein targets a target site in the IL-2 gene or its regulatory elements, resulting in increased transcription or activation of the gene. In some aspects, the fusion protein targets a target site in a combination of target sites on the IL-2 gene or its regulatory elements, resulting in increased transcription or activation of the IL-2 gene.

[0349] In some implementations, the DNA-binding domain and effector domain of the fusion protein are heterologous, i.e., these domains originate from different species, or at least one domain 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.

[0350] In some embodiments, at least one effector domain is fused to the N-terminus, C-terminus, or both of the N-terminus and C-terminus of a DNA-binding domain or a component thereof. At least one effector domain may be fused directly to the DNA-binding domain or fused via any intervening amino acid sequence, such as a linker sequence or nuclear localization sequence (NLS).

[0351] In some embodiments, the fusion protein of the provided DNA-binding system or its DNA-targeting module comprises, from the N-terminus to the C-terminus, a transcription activator effector domain and a DNA-binding domain.

[0352] In some embodiments, at least one effector domain of the fusion protein includes more than one effector domain. In some embodiments, the fusion protein comprises 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 is different. In some embodiments, at least one effector domain includes two effector domains, wherein these two effector domains are different. In some embodiments, the effector domains and the DNA-binding domain may be arranged in any order.

[0353] In some respects, each of the effector domains is a transcriptional activator effector domain.

[0354] In some embodiments, at least one effector domain of the fusion protein comprises two distinct effector domains. The two distinct effector domains and the DNA-binding domain may be arranged in any order. In some embodiments, each of the effector domains is located at the N-terminus of the DNA-binding domain, wherein the 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 the provided DNA-binding system or its DNA-targeting module comprises, from N-terminus to C-terminus, a first effector domain, a second effector domain, and a DNA-binding domain. In some embodiments, each of the effector domains is located at the C-terminus of the DNA-binding domain, wherein the 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 the provided DNA-binding system or its DNA-targeting module comprises, from N-terminus to C-terminus, a DNA-binding domain, a first effector domain, and a second effector domain. In some embodiments, the DNA-binding domain is located between effector domains, with one effector domain fused to the N-terminus of the DNA-binding domain and the other effector domain fused to the C-terminus of the DNA-binding domain. In some embodiments, the fusion protein of the provided DNA-binding system or its DNA-targeting module comprises, from N-terminus to C-terminus, a first effector domain, a DNA-binding domain, and a second effector domain. In some embodiments, one or more of these components may be fused directly to each other or fused via any intercalary amino acid sequence, such as via a linker sequence or nuclear localization sequence (NLS).

[0355] In some embodiments, the fusion protein comprises one or more linkers. In some embodiments, the linker is a peptide linker. In some embodiments, one or more linkers connect a DNA-binding domain or a component thereof to at least one effector domain. The linker may be included at any position in the polypeptide sequence of the fusion protein, for example, between the effector domain and a DNA-binding domain or a component thereof. The linker may have any length and is designed to facilitate or restrict the mobility of components in the fusion protein. The linker may contain 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. The linker may contain an amino acid sequence of at least about 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids. The linker may contain an amino acid sequence of less than about 100, 90, 80, 70, 60, 50, or 40 amino acids. Those skilled in the art can readily select an appropriate linker for the connection of the two domains. In some embodiments, the linker is a flexible linker. Flexible linkers are typically composed of small nonpolar or polar residues, such as glycine, serine, or threonine. Linkers may comprise sequential or tandem repeats of amino acid sequences ranging from 2 to 20 amino acids in length. Linkers may be enriched with the amino acids glycine (G), serine (S), and / or alanine (A). Linkers may include, for example, GS linkers. An exemplary GS linker is represented by the sequence GGGGS (SEQ ID NO: 91). Linkers may contain repeats of the sequence, such as those represented by the formula (GGGGS)n, where n is an integer representing the number of repetitions of the GGGGS sequence (e.g., between 1 and 10). The number of repetitions of the linker sequence can be adjusted to optimize the linker length and achieve appropriate separation of functional domains. For example, in some embodiments, the linker is (GGGGS). n Connector, where n is an integer from 1 to 10. Other instances of connectors 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).

[0356] In some embodiments, artificially linked sequences may be used. In some embodiments, the linker is EASGSGRASPGIPGSTR (SEQ ID NO: 96). In some embodiments, the 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).

[0357] In some implementations, the inclusion of a linker in the fusion protein leads to enhanced activation of the IL-2 gene or its regulatory elements.

[0358] In some embodiments, the linker is an XTEN linker. In some aspects, the XTEN linker is a recombinant polypeptide lacking hydrophobic amino acid residues (e.g., an unstructured recombinant peptide). Exemplary XTEN linkers are described, for example, in Schellenberger et al., Nature Biotechnology 27, 1186-1190 (2009) or WO 2021 / 247570. In some embodiments, the linker comprises the sequence shown in SEQ ID NO: 100 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 100. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 77, or a continuous portion of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids of SEQ ID NO: 100. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 100 or a continuous portion of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids of SEQ ID NO: 100. In some embodiments, the linker comprises the sequence shown in SEQ ID NO: 100. In some embodiments, the linker comprises the sequence shown in SEQ ID NO: 101 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the foregoing. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 101, or a continuous portion of at least 5, 10, or 15 amino acids of SEQ ID NO: 101. In some aspects, the linker consists of the sequence shown in SEQ ID NO: 101 or a continuous portion of at least 5, 10, or 15 amino acids of SEQ ID NO: 101. In some embodiments, the linker comprises the sequence shown in SEQ ID NO: 101. In some embodiments, the linker consists of the sequence shown in SEQ ID NO: 101. Suitable linkers can be selected or designed based on reasonable criteria known in the art, such as those described in Chen et al. Adv. Drug Deliv. Rev. 65(10):1357-1369 (2013). In some embodiments, the linker comprises the linker described in WO 2021 / 247570.

[0359] In some embodiments, the fusion protein of the DNA targeting system or its DNA targeting module comprises one or more nuclear localization signals (NLS). In some embodiments, the fusion protein described herein comprises one or more nuclear localization sequences (NLS), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. When more than one NLS is present, each can be selected independently of the others, such that a single NLS can exist in more than one copy and / or be combined with one or more other NLS to exist in one or more copies. Non-limiting examples of NLS include NLS sequences derived from: NLS of the SV40 viral large T antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 102); NLS of nucleoplasmic proteins (e.g., nucleoplasmic protein bitype NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 103)); c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 104) or RQRRNELKRSP (SEQ ID NO: 105); hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 106); sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 107) from the IBB domain of the input protein-α; and sequence VSRKRPRP (SEQ ID NO: 107) from the fibroid T protein. Sequences of human p53 (SEQ ID NO: 108) and PPKKARED (SEQ ID NO: 109); sequence of human p53 (SEQ ID NO: 110); sequence of mouse c-abl IV (SEQ ID NO: 111); sequence of influenza virus NS1 (SEQ ID NO: 112) and PKQKKRK (SEQ ID NO: 113); sequence of hepatitis D virus antigen (SEQ ID NO: 114); sequence of mouse Mx1 protein (SEQ ID NO: 115); sequence of human poly(ADP-ribose) polymerase (SEQ ID NO: 116); and sequence of steroid hormone receptor (human) glucocorticoid (SEQ ID NO: 117). NLS may contain portions of any of the aforementioned. Generally, one or more NLS have sufficient strength to drive the fusion protein to accumulate in a detectable amount in the nucleus of a eukaryotic cell.Generally, the intensity of nuclear localization activity can be derived from the number of NLSs in the fusion protein, the specific NLS used, or a combination of these factors. Accumulation in the nucleus can be detected using any suitable technique. For example, a detectable marker can be fused to the fusion protein to visualize its intracellular location, such as in conjunction with means for detecting nuclear location (e.g., nucleus-specific staining agents such as DAPI). The nucleus can also be isolated from the cell and its contents analyzed using any suitable protein detection method (e.g., immunohistochemistry, Western blotting, or enzyme activity assay). Accumulation in the nucleus can also be determined indirectly, such as by measuring the effect of the fusion protein compared to control conditions (e.g., untransformed cells) (e.g., measuring changes in gene expression activity in cells transformed with a DNA targeting system containing the fusion protein).

[0360] In some embodiments, the NLS is linked to the N-terminus or C-terminus of the DNA-binding domain via a linker. In some embodiments, the NLS is linked to the N-terminus or C-terminus of the effector domain via a linker. The linker can be any linker as described above. In some embodiments, the linker is GIHGVPAA (SEQ ID NO: 97). In some embodiments, the NLS and the linker have the sequence PKKKRKVGIHGVPAA (SEQ ID NO: 118).

[0361] In some configurations, the N-terminus or C-terminus of the fusion protein may be linked to a moiety for detection and / or purification. In some aspects, this moiety is or includes a Flag tag DYKDDDDK (SEQ ID NO: 119), a 3xFlag tag MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 120), an HA tag YPYDVPDYA (SEQ ID NO: 121), or a His tag such as HHHHHH (SEQ ID NO: 122).

[0362] In some embodiments, the fusion protein is a dCas-VP64 fusion protein, such as dSpCas9-2xVP64, which is a fusion of dSpCas9 with two copies of VP64. In some embodiments, the fusion protein is dSpCas9-2xVP64. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 5. In some embodiments, the fusion protein is encoded by the nucleotide sequence shown in SEQ ID NO: 4.

[0363] In some embodiments, the fusion protein is a dCas-VP64 fusion protein, such as dSaCas9-2xVP64, which is a fusion of two copies of dSaCas9 and VP64. In some embodiments, the fusion protein is dSaCas9-2xVP64. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 61, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 61. In some embodiments, the fusion protein is encoded by the nucleotide sequence shown in SEQ ID NO: 60.

[0364] In some embodiments, the fusion protein is a dCas-NFN-VP64 fusion protein, such as dSaCas9-NFN-VP64, which is a fusion of one copy of dSaCas9 with one copy of VP64 and one copy of NCOA3-FOXO3-NCOA3 (NFN). In some embodiments, the fusion protein is dSaCas9-NFN-VP64. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 182, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 182. In some embodiments, the fusion protein is encoded by the nucleotide sequence shown in SEQ ID NO: 183.

[0365] In some embodiments, the fusion p...

Claims

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

2. The epigenetic modified DNA targeting system of claim 1, wherein the DNA-binding domain of each fusion protein comprises: clustered regularly spaced short palindromic repeat-associated (Cas) proteins, zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), a wide range of nucleases, homing endonucleases, or I-SceI enzymes, or variants thereof, optionally wherein the DNA-binding domain comprises a non-catalytically inactive variant of any of the foregoing. When the DNA-binding domain of each fusion protein contains a Cas protein, the DNA targeting system further includes at least two gRNAs, each of which can target the Cas protein to a target site.

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

4. The epigenetic modified DNA targeting system according to any one of claims 1-3, wherein the plurality of DNA targeting modules is two DNA targeting modules.

5. The epigenetic modified DNA targeting system according to any one of claims 1-3, wherein the plurality of DNA targeting modules are three DNA targeting modules.

6. The epigenetic modified DNA targeting system according to any one of claims 1-3, wherein the plurality of DNA targeting modules is 4 DNA targeting modules or 5 DNA targeting modules.

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

8. The epigenetic modified DNA targeting system according to any one of claims 1-7, wherein each target site is located in the putative regulatory region of the IL-2 gene, wherein the putative regulatory region is characterized by having one or more of an epigenetic marker, a regulatory feature, or a transcription factor motif.

9. The epigenetic modified DNA targeting system of claim 8, wherein the epigenetic marker comprises histone H3K27 acetylation.

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

11. The epigenetic DNA targeting system of claim 10, wherein the enzyme catalyzing the acetylation is an acetyltransferase.

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

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

14. The epigenetic modified DNA targeting system according to any one of claims 1-13, wherein each target site is located in a promoter or enhancer.

15. The epigenetic DNA targeting system according to any one of claims 1-14, wherein each target site is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 modified DNA targeting system of any one of claims 1-15, wherein each target site is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 modified DNA targeting system according to any one of claims 1-16, wherein at least two of the plurality of DNA targeting modules target different target sites.

18. The epigenetic DNA targeting system according to any one of claims 1-15 and 17, wherein at least two distinct target sites are located in two distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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.

19. The epigenetic modified DNA targeting system according to any one of claims 1-16, 17 and 18, wherein the at least two distinct target sites are located in two distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 modified DNA targeting system according to any one of claims 1-15, wherein at least three of the plurality of DNA targeting modules target different target sites.

21. The epigenetic DNA targeting system according to any one of claims 1-15 and 20, wherein at least three distinct target sites are located in three distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 modified DNA targeting system according to any one of claims 1-16, 20 and 21, wherein the at least three distinct target sites are located in three distinct target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 modified DNA targeting system according to any one of claims 1-15, wherein at least four of the plurality of DNA targeting modules target different target sites, or at least five of the plurality of DNA targeting modules target different target sites.

24. The epigenetic DNA targeting system according to any one of claims 1-15 and 23, wherein at least four or at least five different target sites are located in four different target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 modified DNA targeting system according to any one of claims 1-16, 23 and 24, wherein the at least four different target sites or the at least five different target sites are located in four different target regions of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 modified DNA targeting system of any one of claims 1-15, wherein each of the plurality of DNA targeting modules targets a different target site.

27. The epigenetic DNA targeting system according to any one of claims 1-15 and 26, wherein each target site is located in a different target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 modified DNA targeting system according to any one of claims 1-16, 26 and 27, wherein each target site is located in a different target region of the human genome assembly version 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 modified DNA targeting system according to any one of claims 1-25, wherein at least two of the plurality of DNA targeting modules target the same target region.

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

31. The epigenetic modified DNA targeting system according to any one of claims 1-30, wherein the fusion proteins of the plurality of DNA targeting modules are different.

32. The epigenetic modified DNA targeting system according to any one of claims 1-29, wherein each of the DNA targeting modules shares the same fusion protein and each comprises different guide nucleic acids complementary to different target sites.

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

34. The epigenetic modified DNA targeting system of any one of claims 31-33, wherein the DNA-binding domain of the fusion protein is a clustered regularly spaced short palindromic repeat (Cas) protein or a variant thereof.

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

39. An epigenetic DNA-targeting system comprising: (a) A fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, wherein the DNA-binding domain is a clustered, regularly spaced short palindromic repeat-associated (Cas) protein or a variant thereof; and (b) Multiple guide RNAs (gRNAs) comprising at least two gRNAs, each gRNA targeting a target site of the interleukin-2 (IL-2) gene.

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

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

42. The epigenetic DNA targeting system according to any one of claims 39-41, wherein the plurality of gRNAs are two gRNAs.

43. The epigenetic modification DNA targeting system according to any one of claims 39-41, wherein the plurality of gRNAs are 3 gRNAs.

44. The epigenetic modified DNA targeting system according to any one of claims 39-41, wherein the plurality of gRNAs is 4 gRNAs or 5 gRNAs.

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

46. ​​The epigenetic modified DNA targeting system according to any one of claims 39-45, wherein each target site is located in the putative regulatory region of the IL-2 gene, wherein the putative regulatory region is characterized by having one or more of an epigenetic marker, a regulatory feature, or a transcription factor motif.

47. The epigenetic modified DNA targeting system of claim 46, wherein the epigenetic marker comprises histone H3K27 acetylation.

48. The epigenetic modified DNA targeting system of any one of claims 39-47, wherein the at least one transcription activator effector domain catalyzes the acetylation of histone H3 lysine 27 at the target site, or is capable of recruiting an enzyme that catalyzes the acetylation of histone H3 lysine 27 at the target site.

49. The epigenetic DNA targeting system of claim 48, wherein the enzyme catalyzing the acetylation is an acetyltransferase.

50. The epigenetic modified DNA targeting system of claim 48 or claim 49, wherein the enzyme catalyzing the acetylation is a histone acetyltransferase.

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

52. The epigenetic modified DNA targeting system according to any one of claims 39-51, wherein each target site is located in a promoter or enhancer.

53. The epigenetic DNA targeting system according to any one of claims 39-52, wherein each target site is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates selected from the following groups: (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 modified DNA targeting system of any one of claims 39-53, wherein each target site is independently located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 in a different target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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.

56. An epigenetic DNA-targeting system, comprising: (a) A fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, wherein the DNA-binding domain is a clustered, regularly spaced short palindromic repeat-associated (Cas) protein or a variant thereof; and (b) At least one guide RNA (gRNA) targeting a target site of the interleukin-2 (IL-2) gene, said target site being located within a target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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, (5) chr4: 122,539,300–122,544,050, and (6) chr4: 122,576,890-122,579,315.

57. The epigenetic modified DNA targeting system of claim 56, wherein the at least one gRNA targets a target site of the interleukin-2 (IL-2) gene, the target site being located in a target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 DNA targeting system of claim 56 or claim 57, wherein the at least one gRNA is 1-6 gRNAs.

59. The epigenetic modified DNA targeting system of any one of claims 34-58, wherein the Cas protein or a variant thereof is a variant Cas protein as an inactivating (dCas) protein.

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

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

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

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

64. The epigenetic modified DNA targeting system of claim 63, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, referring to the position number of SEQ ID NO:

62.

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

66. The epigenetic modified DNA targeting system according to any one of claims 63-65, wherein the dSpCas9 is shown in SEQ ID NO:

63.

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

68. The epigenetic modified DNA targeting system of claim 67, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, referring to the position number of SEQ ID NO:

64.

69. The epigenetic modified DNA targeting system of claim 67 or claim 68, wherein the dSaCas9 protein comprises the sequence shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

70. The epigenetic modified DNA targeting system of any one of claims 67-69, wherein the dSaCas9 is shown in SEQ ID NO:

65.

71. The epigenetic modification DNA targeting system according to any one of claims 33-70, wherein each gRNA comprises a gRNA spacer sequence complementary to the target site of the corresponding gene.

72. The epigenetic modified DNA targeting system according to any one of claims 33-55 and 59-71, wherein each gRNA targets a target site in IL-2, said target site comprising the sequence shown 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, wherein any one of the foregoing contains a portion of at least 14 nucleotides (nt), or a complementary sequence of any one of the foregoing.

73. The epigenetic modified DNA targeting system according to any one of claims 33-55 and 59-73, wherein each gRNA targets a target site in IL-2, said target site being shown 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.

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, said gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt.

75. An epigenetic DNA-targeting system, comprising: (a) A fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, wherein the DNA-binding domain is a zinc finger protein (ZFP) or a variant thereof; The ZFP targets the interleukin-2 (IL-2) gene at a target site located in the target region of the human genome assembly version GRCh38 (hg38) corresponding to the genomic coordinates selected from the following groups: (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, (5) chr4: 122,539,300-122,544,050, and (6) chr4: 122,576,890-122,579,315.

76. The epigenetic modified DNA targeting system of claim 75, wherein the ZFP targets a target site of the interleukin-2 (IL-2) gene, the target site being located in a target region of the human genome assembly version GRCh38 (hg38) corresponding to genomic coordinates 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 modified DNA targeting system of claim 75 or claim 76, wherein the ZFP targets a target site in IL-2, the target site comprising the sequence shown in any one of SEQ ID NO: 186-188.

78. The epigenetic modified DNA targeting system according to any one of claims 75-77, wherein the ZFP targets the target site shown in SEQ ID NO:

186.

79. The epigenetic modified DNA targeting system of claim 78, wherein the ZFP includes a zinc finger recognition region comprising six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, selected from the following F1-F6: 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 modified DNA targeting system of claim 78 or 79, wherein the ZFP comprises the sequence shown in SEQ ID NO: 189, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

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

189.

82. The epigenetic modified DNA targeting system according to any one of claims 78-81, wherein the ZFP is encoded by the sequence shown in SEQ ID NO: 192 or a portion thereof, or by an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

83. The epigenetic modified DNA targeting system according to any one of claims 78-82, wherein the ZFP is encoded by the sequence shown in SEQ ID NO:

192.

84. The epigenetic modified DNA targeting system according to any one of claims 75-77, wherein the ZFP targets the target site shown in SEQ ID NO:

187.

85. The epigenetic modified DNA targeting system of claim 84, wherein the ZFP includes a zinc finger recognition region, the zinc finger recognition region comprising six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, selected from the following F1-F6: 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 modified DNA targeting system of claim 84 or 85, wherein the ZFP comprises the sequence shown in SEQ ID NO: 190, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

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

190.

88. The epigenetic modified DNA targeting system according to any one of claims 84-87, wherein the ZFP is encoded by the sequence shown in SEQ ID NO: 193 or a portion thereof, or by an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

89. The epigenetic modified DNA targeting system according to any one of claims 84-88, wherein the ZFP is encoded by the sequence shown in SEQ ID NO:

193.

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

188.

91. The epigenetic modified DNA targeting system of claim 90, wherein the ZFP includes a zinc finger recognition region, the zinc finger recognition region comprising six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus, selected from the following F1-F6: 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 modified DNA targeting system of claim 90 or 91, wherein the ZFP comprises the sequence shown in SEQ ID NO: 191, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

93. The epigenetic modified DNA targeting system according to any one of claims 90-92, wherein the ZFP comprises the sequence shown in SEQ ID NO:

191.

94. The epigenetic modified DNA targeting system according to any one of claims 90-93, wherein the ZFP is encoded by the sequence shown in SEQ ID NO: 194 or a portion thereof, or by an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

95. The epigenetic modified DNA targeting system according to any one of claims 90-94, wherein the ZFP is encoded by the sequence shown in SEQ ID NO:

194.

96. An epigenetic DNA-targeting system, comprising: (a) A fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, wherein the DNA-binding domain is derived from... Streptococcus pyogenes Inactivated Cas9 (dSpCas9); 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 shown 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 continuous portion thereof of at least 14 nt.

97. The epigenetic modified DNA targeting system of claim 96, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, referring to the position number of SEQ ID NO:

62.

98. The epigenetic modified DNA targeting system of claim 96 or claim 97, wherein the dSpCas9 comprises the sequence shown in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

99. The epigenetic modified DNA targeting system according to any one of claims 96-98, wherein the dSpCas9 is shown in SEQ ID NO:

63.

100. The epigenetic modified DNA targeting system of any one of claims 33-55 and 59-71, wherein each gRNA targets a target site in IL-2, said target site comprising the sequence shown 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, wherein any one of the foregoing contains a portion of at least 14 nucleotides (nt), or a complementary sequence of any one of the foregoing.

101. The epigenetic modified DNA targeting system of any one of claims 33-55, 59-71, and 100, wherein each gRNA targets a target site in IL-2, said target site being shown 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 according to any one of claims 33-55, 59-61, 67-71, 100, and 101, wherein each gRNA comprises a gRNA spacer sequence, said gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt.

103. An epigenetic DNA-targeting system, comprising: (a) A fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, wherein the DNA-binding domain is derived from... Staphylococcus aureus Inactivated Cas9 (dSaCas9); 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 shown 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 continuous portion thereof of at least 14 nt.

104. The epigenetic modified DNA targeting system of claim 103, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, referring to the position number of SEQ ID NO:

64.

105. The epigenetic modified DNA targeting system of claim 103 or claim 104, wherein the dSaCas9 protein comprises the sequence shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

106. The epigenetic modified DNA targeting system according to any one of claims 103-105, wherein the dSaCas9 is shown in SEQ ID NO:

65.

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

108. The epigenetic modified DNA targeting system according to any one of claims 33-74 and 96-107, wherein each gRNA independently comprises a spacer region sequence of length between 16 nt and 22 nt.

109. The epigenetic modified DNA targeting system according to any one of claims 33-74 and 96-108, wherein each gRNA independently comprises a spacer region sequence of length 18 nt, 19 nt, 20 nt, 21 nt or 22 nt.

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 shown 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 continuous portion 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 shown 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 continuous portion of at least 14 nt of any of the foregoing.

112. The epigenetic DNA targeting system according to any one of claims 33-74 and 96-111, wherein the DNA targeting system comprises at least two gRNAs targeting 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, the gRNA spacer sequence comprising the sequence shown in SEQ ID NO:

23.

115. The epigenetic DNA targeting system of claim 113 or claim 114, wherein the gRNA comprises the gRNA spacer sequence shown in SEQ ID NO:

23.

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

117. The epigenetic DNA targeting system according to 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, 59-74, and 96-117, wherein at least one gRNA targets a target site corresponding to the genomic coordinates of the human genome assembly version 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, the gRNA spacer sequence comprising the sequences shown 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 continuous 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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 11 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown in SEQ ID NO:

11.

121. The DNA targeting system of claim 118, wherein the at least one gRNA comprises a gRNA spacer sequence, the gRNA spacer sequence comprising the sequences shown in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46 or a continuous 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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 42 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown in SEQ ID NO:

42.

123. The DNA targeting system according to 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 transcription 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 the genomic coordinates of the human genome assembly version GRCh38 (hg38) chr4: 122,488,840-122,491,890.

125. The DNA targeting system of claim 124, wherein the at least one gRNA comprises a gRNA spacer sequence, the gRNA spacer sequence comprising the sequences shown in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or a continuous 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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 23 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown 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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 25 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown in SEQ ID NO:

25.

128. The DNA targeting system of claim 124, wherein the at least one gRNA comprises a gRNA spacer sequence, the gRNA spacer sequence comprising the sequences shown in SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or a continuous 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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 50 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown 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 the genomic coordinates of the human genome assembly version 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, the gRNA spacer sequence comprising the sequences shown in SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or a continuous 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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 27 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown in SEQ ID NO:

27.

133. The DNA targeting system of claim 130, wherein the at least one gRNA comprises a gRNA spacer sequence, the gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 56, SEQ ID NO: 58 or a continuous portion thereof of at least 14 nt.

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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 56 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown 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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 58 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown 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 the genomic coordinates of the human genome assembly version 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, the gRNA spacer sequence comprising the sequences shown in SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39 or a continuous 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, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 37 or a continuous portion thereof of at least 14 nt, optionally wherein said gRNA spacer sequence is shown 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 region sequence shown in SEQ ID NO: 11 and the second gRNA comprises the spacer region sequence shown 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 region sequence shown in SEQ ID NO: 11 and the second gRNA comprises the spacer region sequence shown 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 region sequence shown in SEQ ID NO: 11 and the second gRNA comprises the spacer region sequence shown 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 region sequence shown in SEQ ID NO: 11 and the second gRNA comprises the spacer region sequence shown 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 region sequence of SEQ ID NO: 23 and the second gRNA comprises the spacer region sequence of 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 region sequence shown in SEQ ID NO: 23 and the second gRNA comprises the spacer region sequence shown 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 region sequence of SEQ ID NO: 23 and the second gRNA comprises the spacer region sequence of SEQ ID NO:

27.

146. The DNA targeting system according to 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, represented by three gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence shown in SEQ ID NO: 11, a gRNA comprising the spacer sequence shown in SEQ ID NO: 23, a gRNA comprising the spacer sequence shown in SEQ ID NO: 27, and a gRNA comprising the spacer sequence shown 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 region sequence shown in SEQ ID NO: 11, the second gRNA comprises the spacer region sequence shown in SEQ ID NO: 23, the third gRNA comprises the spacer region sequence shown in SEQ ID NO: 27, and the fourth gRNA comprises the spacer region sequence shown in SEQ ID NO: 37, optionally further comprising a fifth gRNA, wherein the fifth gRNA comprises the spacer region sequence shown 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, represented by two gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence shown in SEQ ID NO: 42, a gRNA comprising the spacer sequence shown in SEQ ID NO: 50, a gRNA comprising the spacer sequence shown in SEQ ID NO: 56, and a gRNA comprising the spacer sequence shown in SEQ ID NO: 58, optionally wherein the two gRNAs are: (i) gRNAs containing the spacer region sequence shown in SEQ ID NO: 42 and gRNAs containing the spacer region sequence shown in SEQ ID NO: 50, (ii) gRNA containing the spacer region sequence shown in SEQ ID NO: 42 and gRNA containing the spacer region sequence shown in SEQ ID NO: 56; (iii) gRNA containing the spacer region sequence shown in SEQ ID NO: 42 and gRNA containing the spacer region sequence shown in SEQ ID NO: 58; (iv) gRNA containing the spacer region sequence shown in SEQ ID NO: 50 and gRNA containing the spacer region sequence shown in SEQ ID NO: 56; or (v) gRNA containing the spacer sequence shown in SEQ ID NO: 50 and gRNA containing the spacer sequence shown 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, 128-130, 133, and 134, wherein the DNA targeting system comprises a first gRNA, a second gRNA, and a third gRNA, shown by three gRNAs selected from the group consisting of: a gRNA comprising the spacer sequence shown in SEQ ID NO: 42, a gRNA comprising the spacer sequence shown in SEQ ID NO: 50, a gRNA comprising the spacer sequence shown in SEQ ID NO: 56, and a gRNA comprising the spacer sequence shown in SEQ ID NO: 58, optionally wherein the three gRNAs are: (i) gRNA containing the spacer sequence shown in SEQ ID NO: 42, gRNA containing the spacer sequence shown in SEQ ID NO: 50, and gRNA containing the spacer sequence shown in SEQ ID NO: 56; or (ii) gRNA containing the spacer sequence shown in SEQ ID NO: 42, gRNA containing the spacer sequence shown in SEQ ID NO: 50, and gRNA containing the spacer sequence shown in SEQ ID NO:

58.

150. The epigenetic modified DNA targeting system according to any one of claims 96-149, wherein the DNA targeting system further comprises a fusion protein, the fusion protein comprising a DNA-binding domain and at least one transcription activator effector domain, the DNA-binding domain being a zinc finger protein (ZFP) or a variant thereof. The ZFP targets a target site in IL-2, the target site comprising the sequence shown in any of SEQ ID NO: 186-188.

151. The epigenetic modified DNA targeting system according to any one of claims 1-150, wherein each transcription activator effector domain is an NCOA3 domain, a FOXO3 domain, an 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, or a TET protein domain, optionally wherein the TET protein is a TET1 domain, a SunTag domain, or a domain, portion, variant, or truncated form of any of the foregoing.

152. The epigenetic modified DNA targeting system according to any one of claims 1-151, wherein each transcription activator effector domain is p300.

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

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

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

156. The epigenetic modification DNA targeting system according to any one of claims 1-151, wherein each transcription activator effector domain comprises an NCOA3 domain or a variant or portion thereof exhibiting transcriptional activation activity.

157. The epigenetic modified DNA targeting system of any one of claims 1-151, wherein each transcription activator effector domain comprises a FOXO3 domain or a variant or portion thereof exhibiting transcriptional activation activity.

158. The epigenetic modified DNA targeting system according to any one of claims 1-151, 156 and 157, wherein each transcription activator effector domain comprises an NCOA3-FOXO3-NCOA3 domain.

159. The epigenetic modified DNA targeting system according to any one of claims 1-151 and 156-158, wherein each transcription activator effector domain is an NCOA3-FOXO3-NCOA3 domain.

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

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

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

66.

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

164. The epigenetic modified 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 shown in SEQ ID NO:

181.

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

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

168. The epigenetic modified DNA targeting system according to 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 shown in SEQ ID NO: 100, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

169. The epigenetically modified DNA targeting system according to 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 shown in SEQ ID NO:

100.

170. The epigenetic modified DNA targeting system according to any one of claims 1-158 and 160-167, wherein the fusion protein comprises any one of the sequences shown in SEQ ID NO: 5, 61, 182 and 213-215, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

171. The epigenetic modified DNA targeting system of any one of claims 1-158, 160-167 and 170, wherein the fusion protein comprises any one of the sequences shown in SEQ ID NO: 5, 61, 182 and 213 to 215.

172. The epigenetically modified DNA targeting system according to 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 shown in SEQ ID NO:

5.

173. The epigenetically modified DNA targeting system according to 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 shown in SEQ ID NO:

61.

174. The epigenetically modified DNA targeting system according to 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, 153-158, and 160-167, wherein the fusion protein comprises the sequence shown in SEQ ID NO:

182.

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

213.

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

214.

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

215.

178. The epigenetically modified DNA targeting system of any one of claims 1-177, wherein transient delivery of the epigenetically modified DNA targeting system to lymphoid cells promotes an increase in IL-2 expression, optionally compared to lymphoid cells not delivered the epigenetically modified DNA targeting system.

179. The epigenetic modified DNA targeting system of claim 178, wherein the lymphoid cells are T cells.

180. The epigenetic modified DNA targeting system of claim 178, wherein the lymphoid cells are natural killer (NK) cells.

181. The epigenetic modified DNA targeting system of any one of claims 178-180, wherein the lymphoid cells are derived from primary cells.

182. The epigenetic modified DNA targeting system according to any one of claims 178-181, wherein the lymphoid cells are derived from T cell progenitor cells or NK cell progenitor cells, pluripotent stem cells or induced pluripotent stem cells.

183. The epigenetically modified DNA targeting system of any one of claims 178-182, wherein the lymphoid cells express engineered antigen receptors, optionally chimeric antigen receptors.

184. The epigenetically modified DNA targeting system of claim 179, wherein transient delivery of the epigenetically modified DNA targeting system to the T cells promotes an increase in IL-2 expression upon T cell stimulation, optionally compared to T cells not delivered the epigenetically modified DNA targeting system.

185. The epigenetically modified DNA targeting system of any one of claims 178-184, wherein the DNA targeting system increases the expression of IL-2 in the lymphoid cells in contact with the DNA targeting system by a log2 fold change of 1.0 or more.

186. The epigenetically modified DNA targeting system of any one of claims 178-185, wherein the DNA targeting system increases the expression of IL-2 in the lymphoid cells in contact with the DNA targeting system by a log2 fold change of 2.0 or more.

187. The epigenetically modified DNA targeting system of any one of claims 178-186, wherein the DNA targeting system increases the expression of IL-2 in the lymphoid cells in contact with the DNA targeting system by a log2 fold change of 2.5 or more.

188. The epigenetically modified DNA targeting system of any one of claims 178-187, wherein the DNA targeting system increases the expression of IL-2 in the lymphoid cells in contact with the DNA targeting system by a log2 fold change of 2.75 or more.

189. The epigenetic modified DNA targeting system according to any one of claims 184-188, wherein the T cell stimulation employs anti-CD3 and anti-CD28 activating agents.

190. The epigenetically modified DNA targeting system of any one of claims 184-189, wherein the T cells express engineered antigen receptors, optionally chimeric antigen receptors, or T cell receptors (eTCRs).

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

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

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

194. The epigenetically modified DNA targeting system according to any one 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 the scaffold sequence shown in SEQ ID NO:

8.

195. The epigenetically modified DNA targeting system according to any one 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 the scaffold sequence shown in SEQ ID NO:

41.

196. The epigenetic DNA targeting system according to any one of claims 1-195, wherein the DNA targeting system does not introduce gene damage or DNA breakage.

197. A guide RNA (gRNA) that targets a target site of the interleukin (IL-2) gene, wherein the target site is selected from sequences comprising 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 one of the foregoing comprising at least 14 nucleotides (nt), or a target site of a complementary sequence of any one of the foregoing.

198. The gRNA of claim 197, wherein the target site is shown 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 one of the foregoing.

199. The gRNA of claim 197 or claim 198, wherein the gRNA comprises a gRNA spacer sequence, the gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt, optionally wherein the gRNA comprises 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: 39, or a continuous portion thereof of at least 14 nt, ... The gRNA spacer sequence shown in 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 continuous portion thereof of at least 14 nt.

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

11.

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

23.

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

25.

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

27.

204. The gRNA of any one of claims 197-200, wherein the gRNA comprises a gRNA spacer sequence, the gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 37 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO:

37.

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

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

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

208. The gRNA of any one of claims 197-207, wherein the gRNA further comprises the scaffold sequence shown 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 the sequence represented by 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, the portion of any one of the foregoing containing at least 14 nucleotides (nt), or the target site of a complementary sequence of any one of the foregoing.

210. The gRNA of claim 209, wherein the target site is shown 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 one of the foregoing.

211. The gRNA of claim 209 or claim 210, wherein the gRNA comprises a gRNA spacer sequence, the gRNA spacer sequence comprising the sequence shown 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 continuous portion thereof of at least 14 nt; optionally wherein the gRNA comprises the gRNA spacer sequence shown 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 continuous portion thereof of at least 14 nt.

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

42.

213. The gRNA of any one of claims 209-211, wherein the gRNA comprises a gRNA spacer sequence, the gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 50 or a continuous portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence is shown in SEQ ID NO:

50.

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

56.

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

58.

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

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

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

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

41.

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

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

222. A Cas-guide RNA (gRNA) assembly comprising: (a) From Streptococcus pyogenes Clustered, regularly spaced short palindromic repeats associated with (Cas) proteins or their variants; and (b) At least one gRNA as claimed in any one of claims 197-208.

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

224. The Cas-gRNA combination of claim 223, wherein the dCas protein lacks nuclease activity.

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, referring to the position number of SEQ ID NO:

62.

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

227. The Cas-gRNA combination according to any one of claims 223-225, wherein the dSpCas9 is shown in SEQ ID NO:

63.

228. A Cas-guide RNA (gRNA) assembly comprising: (a) From Staphylococcus aureus Clustered, regularly spaced short palindromic repeats associated with (Cas) proteins or their variants; and (b) At least one gRNA as described in any one of claims 209-219.

229. The Cas-gRNA combination of claim 228, wherein the Cas protein or a variant thereof is an inactivated (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, referring to the position number of SEQ ID NO:

64.

232. The Cas-gRNA combination according to any one of claims 229-231, wherein the dSaCas9 comprises the sequence shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

233. The Cas-gRNA combination according to any one of claims 229-231, wherein the dSaCas9 is shown in SEQ ID NO:

65.

234. A polynucleotide encoding an epigenetic DNA targeting system as described in any one of claims 1-196.

235. A polynucleotide encoding at least one DNA targeting module of an epigenetic modified DNA targeting system as described in any one of claims 1-196.

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

237. A polynucleotide encoding a gRNA as described in any one of claims 197-219.

238. A polynucleotide encoding a combination of gRNAs as described in claim 220 or claim 221.

239. A polynucleotide encoding a combination of Cas-gRNAs as described in any one of claims 222-233.

240. A polynucleotide encoding the fusion protein of the epigenetic modified DNA targeting system as described in any one of claims 1-196 and one or more gRNAs as described in 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 as described in 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 carrier of claim 244, wherein the carrier is selected from 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 carrier of claim 246, wherein the non-viral carrier is selected from: lipid nanoparticles, liposomes, exosomes, or cell-penetrating peptides.

248. The carrier of claim 246 or claim 247, wherein the non-viral carrier 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 an epigenetically modified DNA targeting system as described in any one of claims 1-196, a gRNA as described in any one of claims 197-219, a combination of gRNAs as described in claim 220 or claim 221, a CRISPR Cas-gRNA combination as described in any one of claims 222-233, or a polynucleotide as described in any one of claims 234-241.

251. A modified lymphoid cell comprising epigenetic or phenotypic modifications resulting from contact with an epigenetic DNA-targeting system as described in any one of claims 1-196, a gRNA as described in any one of claims 197-219, a combination of gRNAs as described in claim 220 or claim 221, a CRISPR Cas-gRNA combination as described in any one of claims 222-233, or a polynucleotide as described in any one of claims 234-241.

252. The modified lymphoid cells of claim 250 or claim 251, wherein the modified lymphoid cells are modified T cells.

253. The modified lymphoid cells of claim 250 or claim 251, wherein the modified lymphoid cells are modified natural killer (NK) cells.

254. The modified lymphoid cells as described in any one of claims 250-253, wherein the modified lymphoid cells are derived from primary cells.

255. The modified lymphoid cells according to any one of claims 250-254, wherein the modified lymphoid cells are derived from T cell progenitor cells or NK cell progenitor cells, pluripotent stem cells or induced pluripotent stem cells.

256. The modified lymphoid cells according to any one of claims 250-255, wherein the modified lymphoid cells further comprise a chimeric antigen receptor (CAR).

257. A modified T cell comprising an epigenetically modified DNA targeting system as described in any one of claims 1-196, a gRNA as described in any one of claims 197-219, a combination of gRNAs as described in claim 220 or claim 221, a CRISPR Cas-gRNA combination as described in any one of claims 222-233, or a polynucleotide as described in any one of claims 234-241.

258. A modified T cell comprising epigenetic or phenotypic modifications resulting from contact with an epigenetic DNA-targeting system as described in any one of claims 1-196, a gRNA as described in any one of claims 197-219, a combination of gRNAs as described in claim 220 or claim 221, a CRISPR Cas-gRNA combination as described in any one of claims 222-233, or a polynucleotide as described in 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 the subject's cells.

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

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

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

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

264. A method for increasing IL-2 transcription in lymphoid cells, the method comprising introducing an epigenetically modified DNA targeting system as described in any one of claims 1-196, a gRNA as described in any one of claims 197-219, a combination of gRNAs as described in claims 220 or 221, a CRISPR-Cas-gRNA combination as described in any one of claims 222-233, a polynucleotide as described in any one of claims 234-241, or a vector as described in any one of claims 242-249 into the lymphoid cells.

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

266. The method of claim 264 or 265, wherein the lymphoid cells are T cells.

267. The method of claim 264 or 265, wherein the lymphoid cells are natural killer (NK) cells.

268. The method of claim 264 or 265, wherein the lymphoid cells are derived from primary cells.

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

270. The method of any one of claims 264-269, wherein the lymphoid cells express engineered antigen receptors, optionally chimeric antigen receptors (CARs).

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

272. A method for increasing the production of IL-2 in T cells or by said lymphoid cells, the method comprising introducing an epigenetically modified DNA targeting system as described in any one of claims 1-196, a gRNA as described in any one of claims 197-219, a combination of gRNAs as described in claims 220 or 221, a CRISPR Cas-gRNA combination as described in any one of claims 222-233, or a polynucleotide as described in any one of claims 234-241, or a vector as described in any one of claims 242-249 into T cells.

273. The method of claim 271 or claim 272, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

274. The method of claim 271 or claim 272, wherein the T cell expresses an engineered antigen receptor, optionally a chimeric antigen receptor, or an eTCR.

275. A method for promoting the persistence of immune cells under repeated stimulation, the method comprising introducing an epigenetically modified DNA targeting system as described in any one of claims 1-196, a gRNA as described in any one of claims 197-219, a combination of gRNAs as described in claims 220 or 221, a CRISPR Cas-gRNA combination as described in any one of claims 222-233, or a polynucleotide as described in any one of claims 234-241, or a vector as described in any one of claims 242-249 into T cells, wherein, after the introduction, the T cells are subjected to repeated stimulation, the repeated stimulation initiating T cell activation signals.

276. The method of claim 275, wherein the stimulation employs anti-CD3 and anti-CD28 activating agents.

277. The method of claim 275 or claim 276, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

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

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

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

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

282. The method of any one of claims 271-281, wherein the T cells are T cells of the subject, and the method is performed in vivo.

283. The method of any one of claims 271-281, wherein the T cells are T cells of the subject or cells derived from the subject, and the method is performed in vitro.

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

285. The method of any one of claims 271-284, wherein the T cells are primary T cells.

286. The method of any one of claims 271-284, wherein the T cells are derived from T cell progenitor cells, pluripotent stem cells, or induced pluripotent stem cells.

287. The method of any one of claims 271-284, wherein the introduction is performed by transient delivery to the T cells.

288. The method of any one of claims 264-287, wherein the introduction is performed 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 as described in any one of claims 250-256 and 289.

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

293. The pharmaceutical composition of claim 291 or claim 292, wherein it comprises a pharmaceutically acceptable excipient.

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

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

296. The method of claim 295, wherein the modified T cell is an adoptive T cell therapy for treating the subject's disease or condition.

297. The method of claim 295 or claim 296, wherein the modified T cell is a tumor-infiltrating lymphocyte (TIL).

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

299. A method for treating a disease or symptom in a subject, the method comprising administering to the subject: Adoptive T-cell therapy for treating the disease or condition of the subject; and The epigenetic modified DNA targeting system as described in any one of claims 1-196, the gRNA as described in any one of claims 197-219, the combination of gRNAs as described in claim 220 or claim 221, the CRISPR Cas-gRNA combination as described in any one of claims 222-233, or the polynucleotide as described in any one of claims 234-241, or the vector as described in any one of claims 242-249.

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 that is specific to a target antigen associated with the disease or symptom.

302. The method of claim 298 or claim 301, wherein the recombinant receptor is an engineered T-cell receptor (eTCR) or a 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 symptom is cancer.

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

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

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

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

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