Methods and compositions for modifying nucleotide sequences

Abstract

Compositions and methods for modifying nucleotide sequences are disclosed. Aspects of the disclosure relate to compositions for reprogramming a cell comprising epigenetic effectors. In some embodiments, methods for reprogramming a cell at multiple locations of the cell's genome are provided.

Description

WSGR Ref.65120-702.601 METHODS AND COMPOSITIONS FOR MODIFYING NUCLEOTIDE SEQUENCES RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 487,538, filed on February 28, 2023, which is hereby incorporated by reference in its entirety herein. BACKGROUND

[0002] Modern therapeutic manipulations or biotechnological development entails effective techniques geared towards modifying nucleic acids, including epigenetic and genetic modifications. Introducing a combination of modifications at multiple target sites is desirable for effective therapeutic intervention but remains challenging, especially if different types of modifications (e.g., epigenetic and genetic, methylation and demethylation, transcriptional activation and repression) and / or simultaneous modifications at multiple target locations are desired. Tools for modification of the epigenome and / or genome can enable cellular reprogramming protocols to ensure that the desired combination of epigenetic and / or genetic modifications is obtained. These tools need to be accurate, capable of introducing modifications at the chosen epigenome or genome location with low rate of off-target modification. These tools also need to be customable; modulable; and programmable, suitable of making any modifications in any cells or organisms. Furthermore, these tools may also require a certain degree of control in targeting different areas of interest, especially when a combination of modifications is desired.

[0003] Precise, accurate, adaptable, robust nucleic acid editing techniques allows for customable gene editing. It also allows for effective therapeutic intervention. Currently available systems, however, can be limited due to a lack of accuracy, control, or reliability. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0004] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 65120-702601_SL.XML, created February 26, 2024, which is 179,011 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.WSGR Ref.65120-702.601 INCORPORATION BY REFERENCE

[0005] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. SUMMARY OF THE DISCLOSURE

[0006] In an aspect, the present disclosure provides a system, comprising: (a) a first epigenetic effector comprising: (i) a first CRISPR / Cas domain complexed with a first guide RNA comprising a first tracrRNA sequence, and (ii) a first effector moiety; and (b) a second epigenetic effector comprising: (i) a second CRISPR / Cas domain complexed with a second guide RNA comprising a second tracrRNA sequence, and (ii) a second effector moiety; wherein the first tracrRNA sequence and the second tracrRNA sequence have a phylogenetic distance of exceeding 0.09, when the phylogenetic distance is determined by DNAdist (v3.5c).

[0007] In some embodiments, the system further comprises a cell, wherein the cell comprises: (a) a target nucleic acid comprising one or more CpG islands; (b) the first epigenetic effector; and (c) the second epigenetic effector. In some embodiments, the first guide RNA further comprises a first spacer sequence that is complementary to a first sequence in a first CpG island of the one or more CpG islands in the target nucleic acid, and wherein the second guide RNA further comprises a second spacer sequence that is complementary to a second sequence in a second CpG island of the one or more CpG islands in the target nucleic acid. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain are Type II Cas domains. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain are Cas9 domains. In some embodiments, the first CRISPR / Cas domain is a Type II Cas domain, and wherein the second CRISPR / Cas domain is a Type V Cas domain. In some embodiments, the first CRISPR / Cas domain is a Cas9 domain and the second CRISPR / Cas domain is a Cas12 domain. In some embodiments, the first CRISPR / Cas domain and the secondWSGR Ref.65120-702.601 CRISPR / Cas domain are Type V Cas domains. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain are Cas12 domains.

[0008] In some embodiments, the first CRISPR / Cas domain is selected from the group consisting of AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas domain is selected from the group consisting of AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1.

[0009] In some embodiments, the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. In some embodiments, the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, and LmoCas9; and the second CRISPR / Cas domain is selected from the group consisting of Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a. In some embodiments, the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, and LmoCas9; and the second CRISPR-Cas domain is selected from the group consisting of CasPhi-2 and Cas12c-1. In some embodiments, the first CRISPR / Cas domain is selected from the group consisting of LanCas9, Sag1Cas9, and LmoCas9; and wherein the second CRISPR / Cas domain is a SpyCas9 domain. In some embodiments, the first CRISPR / Cas domain may be selected from the group consisting of Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a; and wherein the second CRISPR / Cas domain is selected from the group consisting of CasPhi-2 and Cas12c-1. In someWSGR Ref.65120-702.601 embodiments, the first CRISPR / Cas domain is a CasPhi-2 domain, and the second CRISPR / Cas domain is a Cas12c-1 domain. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain are catalytically inactive. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135.

[0010] In some embodiments, the first guide RNA or the second sgRNA, or the combination thereof, comprises a sequence selected from SEQ ID NOs.1-10. In some embodiments, the target nucleic acid comprises: (i) over 70,000 copies of a first PAM sequence associated with the first CRISPR / Cas domain in the one or more CpG islands in the target nucleic acid, and (ii) over 70,000 copies of a second PAM sequence associated with the second CRISPR / Cas domain in the one or more CpG islands in the target nucleic acid. In some embodiments, the over 70,000 copies of the first PAM sequence and the over 70,000 copies of the second PAM sequence are in a sense strand of the target nucleic acid. In some embodiments, the target nucleic acid comprises (i) over 1,000,000 copies of the first PAM sequence in the one or more CpG islands in the target nucleic acid, and (ii) over 1,000,000 copies of the second PAM sequence in the one or more CpG islands in the target nucleic acid. In some embodiments, the over 1,000,000 copies of the first PAM sequence and the over 1,000,000 copies of the second PAM sequence are in a sense strand of the target nucleic acid.

[0011] In some embodiments, the first PAM sequence is selected from the group consisting of NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; and the second PAM sequence is selected from the group consisting of NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA,WSGR Ref.65120-702.601 NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

[0012] In some embodiments, the first PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; and wherein the second PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. In some embodiments, the first PAM sequence is selected from the group consisting of NGG, NRG, and NBGG; and wherein the second PAM sequence is selected from the group consisting of TTTV, YYV, KKYV, YTV, and NYTV. In some embodiments, the first PAM sequence is selected from the group consisting of NGG, NRG, and NBGG; and wherein the second PAM sequence is selected from the group consisting of TBN and TG. In some embodiments, the first PAM sequence is selected from the group consisting of NGG, NRG, and NBGG; and wherein the second PAM sequence is NGG. In some embodiments, the first PAM sequence is selected from the group consisting of TTTV, YYV, KKYV, YTV, and NYTV; and wherein the second PAM sequence is selected from the group consisting of TBN and TG. In some embodiments, the first PAM sequence is selected from the group consisting of TBN and NYTV; and wherein the second PAM sequence is TG.

[0013] In some embodiments, the first effector moiety comprises a TET1 domain. In some embodiments, the first effector moiety comprises a domain selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the first effector moiety comprises two domains selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the first effector moiety comprises a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the second effector moiety comprises a TET1 domain. In some embodiments, the second effector moiety comprises a domain selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the second effector moiety comprises two domains selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the second effector moiety comprises a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the first effector moiety comprises a demethylase domain. In some embodiments, the second effector moiety comprises a methyltransferase domain. In some embodiments, the second effector moiety comprises a demethylase domain. In some embodiments, the firstWSGR Ref.65120-702.601 effector moiety comprises a methyltransferase domain. In some embodiments, the second effector moiety comprises a demethylase domain. In some embodiments, the second effector moiety comprises a methyltransferase domain. In some embodiments, the first effector moiety comprises a transcriptional activator domain. In some embodiments, the first effector moiety comprises a transcriptional repressor domain. In some embodiments, the first effector moiety comprises a transcriptional repressor domain. In some embodiments, the second effector moiety comprises a transcriptional activator domain. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell.

[0014] In another aspect, the present disclosure provides a method of reprogramming a cell, the method comprising: (a) providing in a cell comprising: (i) a first epigenetic effector comprising (I) a first CRISPR / Cas domain complexed with a first guide RNA and (II) a first effector moiety, wherein the first CRISPR / Cas domain complexed with the first guide RNA targets a first genomic locus in a genome of the cell; and (ii) a second epigenetic effector comprising a (I) second CRISPR / Cas domain complexed with a second guide RNA and (II) a second effector moiety, wherein the second CRISPR / Cas domain complexed with the second guide RNA targets a second genomic locus in the genome; wherein a first tracrRNA sequence of the first guide RNA and a second tracrRNA sequence of the second guide RNA have a phylogenetic distance of exceeding 0.09; (b) methylating a first target site in the first genomic locus using the first epigenetic effector; and (c) demethylating a second target site in the second genomic locus using the second epigenetic effector.

[0015] In some embodiments, the genome comprises one or more CpG islands. In some embodiments, the first epigenetic effector comprises a DNMT3A domain. In some embodiments, the first epigenetic effector further comprises a DNMT3L domain and a KRAB domain. In some embodiments, the second epigenetic effector comprises a TET1 domain. In some embodiments, the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain are catalytically inactive. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprise a sequence of any one of SEQ ID NOs: 130-135. In some embodiments, the first guide RNA or the second sgRNA comprises a sequence selected from SEQ ID NOs.1-10. In some embodiments, the first guideWSGR Ref.65120-702.601 RNA and the second sgRNA each comprises a sequence selected from SEQ ID NOs.1-10. In some embodiments, the first target site is in a first CpG island of the one or more CpG islands in the genome, and wherein the second target site is in a second CpG island of the one or more CpG islands in the genome. In some embodiments, the genome comprises (i) over 70,000 copies of a first PAM sequence associated with the first CRISPR / Cas domain in the one or more CpG islands in the genome, and (ii) over 70,000 copies of a second PAM sequence associated with the second CRISPR / Cas domain in the one or more CpG islands in the genome. In some embodiments, the genome comprises (i) over 1,000,000 copies of the first PAM sequence associated with the first CRISPR / Cas domain in the one or more CpG islands in the genome, and (ii) over 1,000,000 copies of the second PAM sequence associated with the second CRISPR / Cas domain in the one or more CpG islands in the genome. In some embodiments, the first PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; and wherein the second PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

[0016] In another aspect, the present disclosure provides a method of reprogramming a cell, the method comprising: (a) providing in a cell comprising a genome: (i) a first plurality of epigenetic effectors, each comprising (I) a first CRISPR / Cas domain and (II) a first effector moiety, wherein the first CRISPR / Cas domain is capable of specifically interacting with over 70,000 first targetable PAM sites in a first plurality of CpG islands in the genome; and (ii) a second plurality of epigenetic effectors, each comprising a (I) second CRISPR / Cas domain and (II) a second effector moiety, wherein the second CRISPR / Cas domain is capable of specifically interacting with over 70,000 second targetable PAM sites in a second plurality of CpG islands in the genome; (b) methylating a first plurality of target sites in the first plurality of CpG islands in the genome using the first plurality of epigenetic effectors; and (c) demethylating a second plurality of target sites in the second plurality of CpG islands in the genome using the second plurality of epigenetic effectors. In some embodiments, the first CRISPR / Cas domain is capable of specifically interacting with over 1,000,000 targetable PAM sites in the first plurality of CpG islands in the genome.

[0017] In some embodiments, the second CRISPR / Cas domain is capable of specifically interacting with over 1,000,000 targetable PAM sites in the second plurality of CpG islands in the genome. In some embodiments, (a) further comprises providing: (i) a first plurality of guide RNAs capable of binding to the first CRISPR / Cas domain in each epigenetic effector of the first plurality of epigenetic effectors, wherein the first plurality of guide RNAs target different CpGWSGR Ref.65120-702.601 islands of the first plurality of CpG islands in the genome; and (ii) a second plurality of guide RNAs capable of binding to the second CRISPR / Cas domain in each epigenetic effector of the second plurality of epigenetic effectors, wherein the second plurality of guide RNAs target different CpG islands of the second plurality of CpG islands in the genome. In some embodiments, in (b), the first plurality of target sites are in a same gene in the genome. In some embodiments, in (b), the first plurality of target sites are in different genes in the genome. In some embodiments, in (c), the second plurality of target sites are in a same gene in the genome. In some embodiments, in (c), the second plurality of target sites are in different genes in the genome.

[0018] In some embodiments, the first plurality of guide RNAs comprise a first common tracrRNA sequence, and wherein the second plurality of guide RNAs comprise a second common tracrRNA sequence, wherein the first common tracrRNA sequence and the second common tracrRNA sequence have a phylogenetic distance of exceeding 0.09. In some embodiments, the first plurality of guide RNAs each comprise a first common sequence selected from SEQ ID NOs.1-10. In some embodiments, the second plurality of guide RNAs each comprise a second common sequence selected from SEQ ID NOs.1-10. In some embodiments, the first CRISPR / Cas domain of each of the first plurality of epigenetic effectors is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas domain of each of the second plurality of epigenetic effectors is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. In some embodiments, the first CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135, and the second CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135. In some embodiments, the first targetable PAM sites each comprise a sequence selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; and wherein the second targetable PAM sites each comprise a sequence selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

[0019] In some embodiments, the first effector moiety of each of the first plurality of epigenetic effectors comprises a DNMT3A domain. In some embodiments, the first effector moiety of each of the first plurality of epigenetic effectors further comprises a DNMT3L domain and a KRAB domain. In some embodiments, the second effector moiety of each of the second plurality of epigenetic effectors comprises a TET1 domain. In some embodiments, methylating the firstWSGR Ref.65120-702.601 plurality of target sites thereby represses expression of a first gene. In some embodiments, demethylating the second plurality of target sites thereby enhances expression of a second gene.

[0020] In another aspect, the present disclosure provides a system, comprising: (a) a first epigenetic effector comprising: (i) a SpyCas9 domain complexed with a first guide RNA, and (ii) a demethylase; and (b) a second epigenetic effector comprising: (i) a SauCas9 domain complexed with a second guide RNA and (ii) a methylase. In some embodiments, the demethylase comprises a TET1 domain. In some embodiments, the methylase comprises a DNMT3A domain. In some embodiments, the second epigenetic effector further comprises a DNMT3L domain or a KRAB domain. In some embodiments, the system further comprises a cell, wherein the cell comprises: (a) a target nucleic acid comprising one or more CpG islands; (b) the first epigenetic effector; and (c) the second epigenetic effector.

[0021] In another aspect, the present disclosure provides a method of reprogramming a cell, the method comprising: (a) providing in a cell comprising: (i) a first epigenetic effector comprising (I) a SpyCas9 domain complexed with a first guide RNA and (II) a demethylase, wherein the SpyCas9 domain complexed with the first guide RNA targets a first genomic locus in a genome of the cell; and (ii) a second epigenetic effector comprising a (I) SauCas9 domain complexed with a second guide RNA and (II) a methylase, wherein the CasuCas9 domain complexed with the second guide RNA targets a second genomic locus in the genome; (b) demethylating a first target site in the first genomic locus using the first epigenetic effector; and (c) methylating a second target site in the second genomic locus using the second epigenetic effector. In some embodiments, the demethylase comprises a TET1 domain. In some embodiments, the methylase comprises a DNMT3A domain. In some embodiments, the second epigenetic effector further comprises a DNMT3L domain or a KRAB domain. In some embodiments, the genome comprises one or more CpG islands. In some embodiments, the first target site is in a first CpG island of the one or more CpG islands in the genome, and wherein the second target site is in a second CpG island of the one or more CpG islands in the genome.

[0022] Aspects of the disclosure provide a composition or a system for reprogramming a cell, the composition comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; and / or (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein and the second CRISPR / Cas protein do not cross-react substantially. In some embodiments, both the first CRISPR / Cas protein and the second CRISPR / Cas protein comprise one or more number of PAM sites in a CpG island. In some embodiments, the number of PAM sites in a CpG island isWSGR Ref.65120-702.601 optionally over 70,000, e.g., over 75, 000, over 80,000, over 90,000, over 100, 000. In some embodiments, the number of PAM sites in a CpG island is optionally over 100,000. In some embodiments, the first CRISPR / Cas protein comprises a higher number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein comprises an equivalent number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein comprises a lower number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein may be selected from of type I, type II, type III, type IV, type V Cas protein, and type VI Cas protein. In some embodiments, the first CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, Casl0d, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, and functional equivalents. In some embodiments, the second CRISPR / Cas protein may be selected from a type I, type II, type III, type IV, type V Cas protein, or type VI Cas protein. In some embodiments, the second CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, or a functional fragment thereof. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprise a Type II Cas protein. In some embodiments, the first CRISPR / Cas domain comprises a Type V Cas protein and the second CRISPR / Cas domain comprises a Type II Cas protein. In some embodiments, the first CRISPR / Cas domain comprises a Type II Cas protein and the second CRISPR / Cas domain comprises a Type V Cas protein. In some embodiments, the firstWSGR Ref.65120-702.601 CRISPR / Cas domain and the second CRISPR / Cas domain comprise a Type V Cas protein. In some embodiments, the first CRISPR / Cas protein may be a Cas9 protein and the second CRISPR / Cas protein may be a Cas12 protein. In some embodiments, the first CRISPR / Cas protein may be a Cas12 protein and the second CRISPR / Cas protein may be a Cas9 protein. In some embodiments, the first CRISPR / Cas protein may be a first Cas9 protein and the second CRISPR / Cas protein may be a second Cas9 protein. In some embodiments, the first Cas9 protein and the second Cas9 protein are orthologs. In some embodiments, the first CRISPR / Cas protein may be a first Cas12 protein and the second CRISPR / Cas protein may be a second Cas12 protein. In some embodiments, the first Cas9 protein and the second Cas12 protein are orthologs. In some embodiments, the first CRISPR / Cas protein may be selected AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1, Cpf1, FnCas12a, LbCas12a, LbCas12a, TsCas12a, SaCas12a, Pb2Cas12a, PgCas12a, MiCas12a, Mb2Cas12a, Mb3Cas12a, Lb4Cas12a, Lb5Cas12a, FbCas12a, CrbCas12a, CpbCas12a, CMaCas12a, BsCas12a, BfCas12a, BoCas12a; and the second CRISPR / Cas protein may be selected from AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1, Cpf1, FnCas12a, LbCas12a, LbCas12a, TsCas12a, SaCas12a, Pb2Cas12a, PgCas12a, MiCas12a, Mb2Cas12a, Mb3Cas12a, Lb4Cas12a, Lb5Cas12a, FbCas12a, CrbCas12a, CpbCas12a, CMaCas12a, BsCas12a, BfCas12a, BoCas12a. In some embodiments, the first CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. In some embodiments, the firstWSGR Ref.65120-702.601 CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, and LmoCas9, and the second CRISPR / Cas protein may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a. In some embodiments, the first CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, and LmoCas9, and the second CRISPR-Cas protein may be selected from Casphi-2 and Cas12c-1. In some embodiments, the first CRISPR / Cas protein may be selected from LanCas9, Sag1Cas9, and LmoCas9; and the second CRISPR / Cas protein may be a SpyCas9. In some embodiments, first CRISPR / Cas protein may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a. In some embodiments, the first CRISPR / Cas protein may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a and the second CRISPR / Cas protein may be selected from Casphi-2 and Cas12c-1. In some embodiments, the first CRISPR / Cas protein may be selected from CasPhi-2, and the second CRISPR / Cas protein may be Cas12c-1. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN, NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; and the second CRISPR / Cas protein comprises a PAM sequence selected from NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG and the second CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, NBGG, TTTV, YYV, KKYV,WSGR Ref.65120-702.601 YTV, NYTV, TBN, and TG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein comprises a PAM sequence selected from TTTV, YYV, KKYV, YTV, and NYTV. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein comprises a PAM sequence selected from TBN and TG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein comprises a PAM sequence NGG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from TTTV, YYV, KKYV, YTV, and NYTV and the second CRISPR / Cas protein comprises a PAM sequence selected from TBN and TG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence TBN and NYTV and the second CRISPR / Cas protein comprises a PAM sequence TG. In some embodiments, the first CRISPR / Cas protein is catalytically inactive. In some embodiments, the second CRISPR / Cas protein is catalytically inactive. In some embodiments, both the first CRISPR / Cas protein and the second CRISPR / Cas protein are catalytically inactive. In some embodiments, the composition comprises a first sgRNA and a second sgRNA. In some embodiments, the first sgRNA and the second sgRNA may be selected such that it does not cross react substantially with the other. In some embodiments, the first sgRNA or second sgRNA may comprise a sequence selected from SEQ ID NO: 1-4 at 3’ end. In some embodiments, the first sgRNA or second sgRNA may comprise a sequence selected from SEQ ID NO: 5-10 at 5’ end.

[0023] Aspects of the disclosure provide a composition or a system for reprogramming a cell, the composition comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; and / or (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein a first tracrRNA sequence interacting with the first CRISPR / Cas protein and the second tracrRNA sequence interacting with the second CRISPR / Cas protein have a phylogenetic distance of exceeding 0.09. In some embodiments the first tracrRNA sequence and the second tracrRNA sequence have a phylogenetic distance of exceeding 0.05, exceeding 0.1, exceeding 0.12, exceeding 0.14, exceeding 0.16, exceeding 0.18, exceeding 0.2, exceeding 0.22, exceeding 0.24, exceeding 0.26, exceeding 0.28, exceeding 0.3, exceeding 0.35, or exceeding 0.4. In some embodiments, both the first CRISPR / Cas protein and the second CRISPR / Cas protein comprise one or more number of PAM sites in a CpG island. In some embodiments, the number of PAM sites in a CpG island is optionally over 70,000, e.g., over 75, 000, over 80,000, over 90,000, over 100, 000. In some embodiments, the number of PAM sites in a CpG island is optionally over 100,000. In someWSGR Ref.65120-702.601 embodiments, the first CRISPR / Cas protein comprises a higher number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein comprises an equivalent number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein comprises a lower number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein may be selected from of type I, type II, type III, type IV, type V Cas protein, and type VI Cas protein. In some embodiments, the first CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, Casl0d, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, and functional equivalents. In some embodiments, the second CRISPR / Cas protein may be selected from a type I, type II, type III, type IV, type V Cas protein, or type VI Cas protein. In some embodiments, the second CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, or a functional fragment thereof. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprise a Type II Cas protein. In some embodiments, the first CRISPR / Cas domain comprises a Type V Cas protein and the second CRISPR / Cas domain comprises a Type II Cas protein. In some embodiments, the first CRISPR / Cas domain comprises a Type II Cas protein and the second CRISPR / Cas domain comprises a Type V Cas protein. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprise a Type V Cas protein. In some embodiments, the first CRISPR / Cas protein may be a Cas9 protein and the secondWSGR Ref.65120-702.601 CRISPR / Cas protein may be a Cas12 protein. In some embodiments, the first CRISPR / Cas protein may be a Cas12 protein and the second CRISPR / Cas protein may be a Cas9 protein. In some embodiments, the first CRISPR / Cas protein may be a first Cas9 protein and the second CRISPR / Cas protein may be a second Cas9 protein. In some embodiments, the first Cas9 protein and the second Cas9 protein are orthologs. In some embodiments, the first CRISPR / Cas protein may be a first Cas12 protein and the second CRISPR / Cas protein may be a second Cas12 protein. In some embodiments, the first Cas9 protein and the second Cas12 protein are orthologs. In some embodiments, the first CRISPR / Cas protein may be selected AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1, Cpf1, FnCas12a, LbCas12a, LbCas12a, TsCas12a, SaCas12a, Pb2Cas12a, PgCas12a, MiCas12a, Mb2Cas12a, Mb3Cas12a, Lb4Cas12a, Lb5Cas12a, FbCas12a, CrbCas12a, CpbCas12a, CMaCas12a, BsCas12a, BfCas12a, BoCas12a; and the second CRISPR / Cas protein may be selected from AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1, Cpf1, FnCas12a, LbCas12a, LbCas12a, TsCas12a, SaCas12a, Pb2Cas12a, PgCas12a, MiCas12a, Mb2Cas12a, Mb3Cas12a, Lb4Cas12a, Lb5Cas12a, FbCas12a, CrbCas12a, CpbCas12a, CMaCas12a, BsCas12a, BfCas12a, BoCas12a. In some embodiments, the first CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. In some embodiments, the first CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, and LmoCas9, and the second CRISPR / Cas protein may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a,WSGR Ref.65120-702.601 and Pb2Cas12a. In some embodiments, the first CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, and LmoCas9, and the second CRISPR-Cas protein may be selected from Casphi-2 and Cas12c-1. In some embodiments, the first CRISPR / Cas protein may be selected from LanCas9, Sag1Cas9, and LmoCas9; and the second CRISPR / Cas protein may be a SpyCas9. In some embodiments, first CRISPR / Cas protein may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a. In some embodiments, the first CRISPR / Cas protein may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a and the second CRISPR / Cas protein may be selected from Casphi-2 and Cas12c-1. In some embodiments, the first CRISPR / Cas protein may be selected from CasPhi-2, and the second CRISPR / Cas protein may be Cas12c-1. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; and the second CRISPR / Cas protein comprises a PAM sequence selected from NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG and the second CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas proteinWSGR Ref.65120-702.601 comprises a PAM sequence selected from TTTV, YYV, KKYV, YTV, and NYTV. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein comprises a PAM sequence selected from TBN and TG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein comprises a PAM sequence NGG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence selected from TTTV, YYV, KKYV, YTV, and NYTV and the second CRISPR / Cas protein comprises a PAM sequence selected from TBN and TG. In some embodiments, the first CRISPR / Cas protein comprises a PAM sequence TBN and NYTV and the second CRISPR / Cas protein comprises a PAM sequence TG. In some embodiments, the first CRISPR / Cas protein is catalytically inactive. In some embodiments, the second CRISPR / Cas protein is catalytically inactive. In some embodiments, both the first CRISPR / Cas protein and the second CRISPR / Cas protein are catalytically inactive. In some embodiments, the composition comprises a first sgRNA and a second sgRNA. In some embodiments, the first sgRNA and the second sgRNA may be selected such that it does not cross react substantially with the other. In some embodiments, the first sgRNA or second sgRNA may comprise a sequence selected from SEQ ID NO: 1-4 at 3’ end. In some embodiments, the first sgRNA or second sgRNA may comprise a sequence selected from SEQ ID NO: 5-10 at 5’ end.

[0024] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The disclosed methods and compositions for modifying nucleic acids are set forth with particularity in the appended claims. A better understanding of the features and advantages of the disclosure will be obtained by reference to the following detailed description that sets forthWSGR Ref.65120-702.601 illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (“FIG.” or “FIGs.” herein), of which:

[0026] FIG.1 shows a representation of clusters of Cas9 orthologs based on sequence and secondary structural homology of their associated tracrRNAs.

[0027] FIGs.2A and 2B shows the consensus PAM sequences of Cas9 orthologs from FIG.1. FIG.2A shows the consensus PAM sequences of Cas9 orthologs from Clusters 1-3. FIG.2B shows the consensus PAM sequences of Cas9 orthologs from Clusters 4-7.

[0028] FIGs.3A and 3B show the frequency of PAM sites in CpG islands for different Cas9 orthologs calculated from the custom script described in Example 1. The number of motifs listed represents the PAM sites located on one strand. FIG.3A shows frequency of PAM sites associated with Ain – Lrh Cas orthologs. FIG.3B shows frequency of PAM sites associated with Lsp1 – Cpf1 Cas orthologs.

[0029] FIG.4 shows the structural and sequence homology of the tracrRNAs of select Cas9 orthologs. The tracrRNA structures are grouped based on structural similarities of the nexus and 3’ hairpin regions. Representative nexus and 3’ hairpin region in each group is boxed. Sequence alignment of the tracrRNAs is shown on the bottom right.

[0030] FIG.5A-5C illustrates plasmid constructs of ExpON, ExpOFF, and MCP-VPR. FIG. 5A show plasmid constructs of ExpOFF. FIG.5B show plasmid constructs of ExpON. FIG.5C show plasmid constructs of MCP-VPR.

[0031] FIGs.6A and 6B show flow analysis for BFP reporter expression in cells transfected with ExpOFF plasmid and different sgRNAs. FIG.6A shows flow analysis for BFP reporter expression in cells transfected with ExpOFF plasmid and CD151 targeting sgRNA. FIG.6B shows flow analysis for BFP reporter expression in cells transfected with ExpOFF plasmid and CD81 targeting sgRNA or non-targeting sgRNA control.

[0032] FIGs.7A-7C illustrates flow analysis for CD81 and CD151 expression in cells transfected with ExpOFF plasmid and CD151 or CD81 targeting sgRNA. FIG.7A shows flow analysis for CD81 or CD151 expression in cells after 12 days post transfection. FIG.7B shows flow analysis for CD81 or CD151 expression in cells after 24 days post transfection. FIG.7C shows flow analysis for CD81 or CD151 expression in cells after 35 days post transfection.WSGR Ref.65120-702.601

[0033] FIG.8 illustrates a schematic representation of different constructs for OFF, ON, and ON with transactivation system, and sgRNA design (dCas = nuclease-deficient Cas; AD = Activation Domain; crRNA = CRISPR RNA; tracRNA = trans-activating CRISPR RNA; Rta = Epstein-Barr virus R transactivator).

[0034] FIG.9A-D illustrates flow analysis for CD81 and CD151 expression in cells targeted by SA_ExpOFF and CD151 guide RNA, SP_ExpOn and CD81 guide RNA, or a combination thereof. FIG.9A shows flow analysis for CD81 or CD151 expression in control K562_Native and K562_CD81 Silenced cells. FIG.9B shows flow analysis for CD81 or CD151 expression after 2 days post electroporation. FIG.9B shows flow analysis for CD81 or CD151 expression in cells after 5 days post electroporation. FIG.9C shows flow analysis for CD81 or CD151 expression in cells after 7 days post electroporation. FIG.9D shows flow analysis for CD81 or CD151 expression in cells after 9 days post electroporation.

[0035] FIGs.10A and 10B illustrate epigenetic maps generated of edited cells. FIG.10A illustrates an epigenetic map of the CD81 gene. FIG.10B illustrates an epigenetic map of the CD151 gene. DETAILED DESCRIPTION OF THE DISCLOSURE

[0036] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0037] The practice of some methods disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J.WSGR Ref.65120-702.601 MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)). Terminology

[0038] Singular forms “a,” “an,” “the,” and “said” include the plural forms as well, unless the context clearly indicates otherwise.

[0039] The terminology, “and / or,” used in a phrase such as “X and / or Y” includes both X and Y; X or Y; X (alone); and Y (alone).

[0040] The words “comprising,” “comprise,” “comprises,” “having,” “have,” “has,” “including,” “includes,” “include,” “containing,” “contains” and “contain” are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. Aspects and embodiments of the invention described include “comprising,” “consisting of,” and “consisting essentially of” (and variants thereof) aspects and embodiments.

[0041] Use of terminology like “some embodiments,” “an embodiment,” “one embodiment,” “other embodiments,” “various embodiments,” “another embodiment,” “some cases” and the like with reference to a particular feature or characteristic described in connection with the embodiment or case, means that the feature is included in one or more embodiments, but not necessarily all embodiments. Similarly, reference to “a method,” “the method,” “one method” and the like with reference to a particular feature or characteristic described in connection with the method, means that the feature is included in one or more methods, but not necessarily all methods.

[0042] “About” and “approximately” refer to the usual error range for the respective value readily known to the skilled person in this technical field. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Reference to “about” or “approximately” a value or parameter includes (and describes) embodiments directed to that value or parameter per se.

[0043] “Determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” refer to forms of measurement, include determining whether an element is present or not (for example, detection). can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can includeWSGR Ref.65120-702.601 determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0044] As used herein, the term “CpG” or a “CpG site” is used interchangeably and refer to a dinucleotide sequence containing an adjacent guanine and cytosine where the cytosine is located 5' of guanine.

[0045] “CpG Island” refers to a region that is at least 200 base pairs (bp) in length, with a GC percentage greater than 50%, and an observed-to-expected CpG ratio greater than 60%.

[0046] As used herein, a “cell” can generally refer to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an algal cell, a fungal cell an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a horse, a rodent, a rat, a mouse, a non-human primate, a human, etc.). A cell can be a somatic cell, e.g., a skin cell, a nerve cell, a muscle cell, a blood cell or a germ cell, e.g., an oocyte, a sperm. In some embodiments, the cell may be an adult cell, e.g., adult somatic cell, a sperm, an oocyte. In some embodiments, the cell may be a stem cell, e.g., an embryonic stem cell, an adult stem cell, an induce pluripotent stem cell (iPSC). In some embodiments, the stem cell may be a totipotent stem cell, a pluripotent stem cell, a multipotent stem cell, or an unipotent stem cell.

[0047] “Complementary” and “complementarity,” with reference to nucleic acids, refer to the association of double-stranded nucleic acids by base pairing through specific hydrogen bonds. The base paring may be standard Watson-Crick base pairing (e.g., 5'-A G T C-3' pairs with the complementary sequence 3'-T C A G-5') or other non-traditional type. Complementarity is typically measured with respect to a duplex region and thus excludes overhangs, for example. Complementarity between two strands of the duplex region may be partial and expressed as a percentage (e.g., 80%), if only some (e.g., 80%) of the bases are complementary.

[0048] “Domain” refers to a section or portion of a polypeptide or a nucleic acid sequence encoding the section or the portion of the polypeptide that contributes to a specified function toWSGR Ref.65120-702.601 the polypeptide. A domain may comprise a contiguous region or more than one distinct non- contiguous regions of a polypeptide.

[0049] “Edit” and “editing” with reference to a nucleic acid refers to any change in the nucleic acid. Changes in a nucleic acid may including an insertion, a deletion, or a substitution of one or more nucleotides or nucleobases in the nucleic acid; or an epigenetic change or epigenetic editing of the nucleic acid at, for example, one or more nucleotides of, or one or more proteins associated with, the nucleic acid. In some cases, “epigenetic editing” refers to the selective and reversible modification of DNA (e.g., methylation, demethylation) and histones (methylation, demethylation, acetylation, deacetylation). The changes can be in a genome of a cell. “Insertion,” “deletion,” and “substitution” have the following meanings: (A) “Insertion” refers to an addition of one or more nucleotides in a DNA sequence. Insertions can range from small insertions of a few nucleotides to insertions of large segments such as a cDNA or a gene. (B) “Deletion” refers to a loss or removal of one or more nucleotides in a DNA sequence or a loss or removal of the function of a gene. In some cases, a deletion can include, for example, a loss of a nucleotide, a few nucleotides, an exon, an intron, a gene segment, or the entire sequence of a gene. Deletion of a gene may include any deletion sufficient result in the elimination or reduction of the function or expression of the gene or its gene product. (C) “Substitution” refers to a mutation in which one nucleotide is replaced by a different nucleotide.

[0050] Editing may result in a gene knock-in, knock-out or knock-down, each defined as follows: (A) “Knock-in” refers to an addition of a DNA sequence, or fragment thereof into a genome. (B) “Knockout” refers to the elimination of a gene or the expression of a gene. (C) “Knock-down” refers to reduction in the expression of a gene or its gene product(s).

[0051] “Effector,” “epigenetic effector” and “effector polypeptide” refer to a polypeptide engineered to bind a specific target sequence in chromosomal DNA and modify the DNA or protein(s) associated with DNA at or near the target sequence and modify the target sequence. An epigenetic effector may, in some cases, include a nucleic acid binding moiety and one orWSGR Ref.65120-702.601 more effector moieties. “Effector moiety” refers to a domain that can alter the expression of a target gene when localized to an appropriate site in the nucleus of a cell, e.g., in a target nucleotide sequence.

[0052] “Gene” refers to a combination of polynucleotide elements, that when operatively linked in either a native or recombinant manner, provide some product or function. “Gene” is to be interpreted broadly and can encompass mRNA, cDNA, cRNA and genomic DNA forms of a gene. In some uses, “gene” encompasses the transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some cases, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some cases, a “gene” may not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, a “gene” may include not only the transcribed sequences, but in addition, also includes non- transcribed regions including upstream and downstream regulatory regions, enhancers, and promoters.

[0053] “Guide RNA,” “gRNA,” “single guide RNA,” and “sgRNA” refer to any RNA molecule (or a group of RNA molecules collectively) that facilitates binding of a polypeptide, such as a Cas protein, to a specific location of a target nucleic acid. A single guide RNA (sgRNA) can comprise a crRNA and tracrRNA that are fused together. A guide RNA (gRNA) can comprise a crRNA segment and / or a tracrRNA segment. Exemplary guide RNAs include, but are not limited to, crRNAs, pre-crRNAs (e.g., DR-spacer-DR), and mature crRNAs (e.g., mature JDR- spacer, mature DR-spacer-mature JDR). “Guide RNA” also encompasses an RNA molecule or suitable group of molecular segments that binds a Cas protein other than Cas9 (e.g., Cpfl protein) and that possesses a guide sequence within the single or segmented strand of RNA comprising the functions of a guide RNA which include Cas protein binding to form a gRNA:Cas protein complex capable of binding, nicking and / or cleaving a complementary target sequence in a target polynucleotide.

[0054] “Homolog” refers to a gene or a protein that is related to another gene or protein by a common ancestral DNA sequence and is functionally similar. Homologous proteins may but need not be structurally related or are only partially structurally related. “Ortholog” refers to a gene or protein that is related to another gene or protein by a speciation event. Orthologous proteins may in some cases be structurally related or only partially structurally related. In some cases, an ortholog may retain the same function as the gene or protein to which they areWSGR Ref.65120-702.601 orthologous. Non-limiting examples of Cas9 orthologs include: Akkermansia muciniphila Cas9 (AmCas9), Bifidobacterium longum Cas9 (BlCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9), Geobacillus stearothermophilus Cas9 (GeoCas9), Legionella pneumophila Cas9 (LpCas9), Neisseria lactamica Cas9 (NlCas9), Neisseria meningitidis Cas9 (NmCas9), Oscillospira luneus Cas9 (OlCas9), Staphylococcus aureus Cas9 (SaCas9 or SauCas9), Streptococcus pyogenes Cas9 (SpCas9 or SpyCas9), Streptococcus thermophilus CRISPR1 Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3 Cas9 (St3Cas9). Homologs and orthologs may be identified by homology modeling (e.g., see Filipek, S. (2023). Homology modeling: Methods and protocols. Humana Press.).

[0055] “Individual,” “patient,” and “subject” refer to any subject, e.g., a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. In particular embodiments, the patient is a human.

[0056] “Methylate” and “methylating” refer to (i) the addition one or more methyl groups to one or more cysteine residues, or (ii) the replacement of one or more unmethylated cysteine residues with one or more methylated cysteine residues, or (iii) the addition of one or more methyl to one or more sites to one or more histones. “Demethylate” and “demethylating” refer to (i) the removal of one or more methyl groups from one or more cysteine residues, or (ii) the replacement of one or more methylated cysteine residues with one or more unmethylated cysteine residues, or (iii) the removal of one or more methyl residues from one or more sites on one or more histones.

[0057] “Modifying,” “modification,” “modulate” and “modulating” refer to a change in the structure, expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. a modification (e.g., increase or decrease) includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels.

[0058] “Pharmaceutical composition” refers to an active agent (e.g., one or more epigenetic effectors or nucleic acid encoding the same, or one or more cells reprogrammed using the epigenetic effectors of the present disclosure), formulated together with one or more pharmaceutically acceptable carriers, excipient, or diluent. Non-limiting examples of excipients include buffers, salts, polymers, proteins, and preservatives.WSGR Ref.65120-702.601

[0059] “Polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid sequence” are used interchangeably to refer to a polymeric form of nucleotides, such as deoxyribonucleotides, ribonucleotides, NS analogs thereof. Polynucleotides may be provided in single-, double-, or multi-stranded form in a linear, branched, or circular conformation. A polynucleotide can be exogenous (e.g., a sequence that is not native to the cell, or a chromosomal sequence whose native location in the genome of the cell is in a different chromosomal location) or endogenous (e.g., a chromosomal sequence that is native to the cell) to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA, e.g., an mRNA. A polynucleotide can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). Non-limiting examples of modifications include addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7- deaza purines). Nucleotide analogs also include dideoxy nucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer.

[0060] The term “reprogram” or “reprogramming” as used herein, refers to a process that alters or reverses the differentiation state of a differentiated cell (e.g., a somatic cell). Reprogramming can encompass complete reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent state or a multipotent state. Reprogramming can encompass complete or partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in expression of particular genes by the cells, the expression of which further contributes to reprogramming. In certain examples described herein, reprogramming of a differentiated cell (e.g., a somatic cell) can cause the differentiated cell to assume an undifferentiated state (e.g., an undifferentiated cell).

[0061] “Reverse transcriptase” or “RT” refers to a class of enzymes that synthesize a DNA molecule from an RNA template. An RT may require the primer molecule with an exposed 3’ hydroxyl group. In some cases, the primer molecule of an RT may be a DNA molecule. In other cases, the primer molecule of an RT may be an RNA molecule. In some cases, an RT may comprise both DNA polymerase activity and RNase H activity. The two activities may reside in two separate domains in an RT.WSGR Ref.65120-702.601

[0062] “Linker” refers to a molecule linking two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two fusion proteins. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. In some cases, a linker may have various lengths, depending on the application of a linker or the sequences or molecules being linked by a linker.

[0063] “Protein”, “peptide”, and “polypeptide” refer to a molecule of two or more subunit amino acids, amino acid analogs or peptidomimetics. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0064] “Fusion protein” refers to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function. A domain may comprise a particular makeup of amino acids. A domain may also comprise a structure of proteins as described herein. In a fusion protein, the term “linker” refers to a protein fragment that is used to link these domains together.

[0065] “Sequence homology” or “sequence identity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. A degree of homology between sequences can be a function of the number of matching or homologous positions shared by the sequences. As a practical matter, whether any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein (which can correspond with a particular nucleic acid sequence described herein), such particular polypeptide sequence can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis.53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters can be set such that the percentage of identity can be calculated over the full length of the reference sequence and that gaps in sequence homology of up to 5% of the total reference sequence can be allowed.

[0066] “Sample” refers to a composition that is obtained or derived from a subject and / or individual of interest that contains or may contain a cellular and / or other molecular entity that isWSGR Ref.65120-702.601 to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof.

[0067] “Sequence homology” and “sequence identity” refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. A degree of homology between sequences can be a function of the number of matching or homologous positions shared by the sequences. As a practical matter, whether any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described he(which can correspond with a particular nucleic acid sequence described herein), such particular polypeptide sequence can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis.53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters can be set such that the percentage of identity can be calculated over the full length of the reference sequence and that gaps in sequence homology of up to 5% of the total reference sequence can be allowed. For an amino acid sequence, in some cases, the sequence identity between a reference sequence (query sequence, i.e., a sequence of the disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In some embodiments, parameters for a particular embodiment in which identity can be narrowly construed, used in a FASTDB amino acid alignment, can include: Scoring Scheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject sequence, whichever can be shorter. According to this embodiment, if the subject sequence can be shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manualWSGR Ref.65120-702.601 correction can be made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity can be corrected by calculating the number of residues of the query sequence that can be lateral to the N- and C-terminal of the subject sequence, which can be not matched / aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue can be matched / aligned can be determined by results of the FASTDB sequence alignment. This percentage can be then subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence, which can be not matched / aligned with the query sequence, can be considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence can be considered for this manual correction. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, and therefore, the FASTDB alignment does not show a matching / alignment of the first ten residues at the N-terminus. The ten unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched / total number of residues in the query sequence) so 10% can be subtracted from the percent identity score calculated by the FASTDB program. If the remaining ninety residues were perfectly matched, the final percent identity can be 90%. In another example, a 90-residue subject sequence can be compared with a 100-residue query sequence. This time the deletions can be internal deletions, so there can be no residues at the N- or C-termini of the subject sequence which can be not matched / aligned with the query. In this case, the percent identity calculated by FASTDB cannot be manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which can be not matched / aligned with the query sequence can be manually corrected for.

[0068] “Subject,” “host,” and “individual,” are used interchangeably to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method or composition described herein. A mammal can be administered a primer editor and / or a PEgRNA as described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g.,WSGR Ref.65120-702.601 dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some cases, a mammal is a human. A mammal may be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal may be male or female. A mammal can be a pregnant female. In some case, a subject may be a human. In some cases, a human may be more than about: 1 day to about 10 months old, from about 9 months to about 24 months old, from about 1 year to about 8 years old, from about 5 years to about 25 years old, from about 20 years to about 50 years old, from about 1 year old to about 130 years old or from about 30 years to about 100 years old. Humans can be more than about: 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 years of age. Humans can be less than about: 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 years of age.

[0069] “A promoter sequence of a target gene” refers to at least a portion of a non-coding DNA sequence which directs the expression of the target gene. The portion of the non-coding DNA sequence may be in the 5'-prime direction or in the 3 '-prime direction from the coding region of the target gene. The portion of the non-coding DNA sequence may be located in an intron of the target gene.

[0070] “Homolog” refers to a gene or a protein that is related to another gene or protein by a common ancestral DNA sequence and is functionally similar. Homologous proteins may but need not be structurally related, or are only partially structurally related. The term “ortholog” as used herein refers to a gene or protein that is related to another gene or protein by a speciation event. Orthologous proteins may but need not be structurally related, or are only partially structurally related. In some cases, an ortholog may or may not retain the same function as the gene or protein to which they are orthologous. Non-limiting examples of Cas9 orthologs include S. aureus Cas9 ("spCas9"), S. thermophiles Cas9, L. pneumophilia Cas9, N. lactamica Cas9, N. meningitides Cas9, B, longum Cas9, A. muciniphilu Cas>9, and O. luneus Cas9.Homologs and orthologs may be identified by homology modelling (see, e.g., Greer, Science vol.228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or "structural BLAST" (Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a "structural BLAST": using structural relationships to infer function. Protein Sci.2013 Apr;22(4):359-66. doi: 10.1002 / pro.2225.).

[0071] “Modifying”, “modification”, “modulate,” or “modulating” refers to a change in the structure, expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, a modification (e.g., increase or decrease) includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels.WSGR Ref.65120-702.601

[0072] “Methylate” or “methylating” refers to (i) the addition one or more methyl groups to one or more cysteine residues, or (ii) the replacement of one or more unmethylated cysteine residues with one or more methylated cysteine residues, or (iii) the addition of one or more methyl to one or more sites to one or more histones. The terms “demethylate” or “demethylating” as used herein are used interchangeably to refer to (i) the removal of one or more methyl groups from one or more cysteine residues, or (ii) the replacement of one or more methylated cysteine residues with one or more unmethylated cysteine residues, or (iii) the removal of one or more methyl residues from one or more sites on one or more histones.

[0073] “Simultaneous,” or “concurrent” with respect to simultaneous or concurrent modification of multiple genomic sequences in a cell, is intended to indicate that the modification of the multiple sequences overlaps, such that there is overlap in the time periods during which epigenetic effectors effectuating the modification of the multiple sequences are active in the cell. In some cases, the epigenetic effectors effectuating the modification of the multiple sequences are delivered to a cell or cell culture or organism at the same time or within a time period selected to permit temporal overlap in the operation of the epigenetic effectors in one or more of the cells.

[0074] “Subject,” “host,” and “individual” refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method or composition described herein. A mammal can be administered a primer editor and / or a PEgRNA as described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some cases, a mammal is a human. A mammal may be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal may be male or female. A mammal can be a pregnant female. In some case, a subject may be a human. In some cases, a human may be more than about: 1 day to about 10 months old, from about 9 months to about 24 months old, from about 1 year to about 8 years old, from about 5 years to about 25 years old, from about 20 years to about 50 years old, from about 1 year old to about 130 years old or from about 30 years to about 100 years old. Humans can be more than about: 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 years of age. Humans can be less than about: 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 years of age.

[0075] “Targeting” refers to a polynucleotide containing a target nucleic acid sequence. A target polynucleotide may be single-stranded or double- stranded, and, in some embodiments, isWSGR Ref.65120-702.601 double-stranded DNA. In some embodiments, the target polynucleotide is single-stranded RNA. A "target nucleic acid sequence" or "target sequence," as used herein, means a specific sequence or the complement thereof that one wishes to modify using an epigenetic effector and / or an editor described herein.

[0076] “Target nucleic acid” refers to a specific nucleic acid substrate that contains a nucleic acid sequence complementary to the entirety or a part of the spacer in an RNA guide. In some embodiments, the target nucleic acid comprises a gene or a sequence within a gene in some embodiments, the target nucleic acid comprises a non-coding region {e.g., a promoter) in some embodiments, the target nucleic acid is single- stranded. In some embodiments, the target nucleic acid is double-stranded.

[0077] “Transcription regulatory element” refers to a nucleotide sequences of a gene that are involved in regulation of genetic transcription. A transcription regulatory element may be a promoter, an enhancer, e.g., a super enhancer, a silencer, an insulator, or a transcription initiation site operably linked to a gene. The term "promoter" as used herein refers to a DNA region, typically but not exclusively 5' of the site of transcription initiation, sufficient to confer accurate transcription initiation. The promoter may be constitutively active or the promoter may be conditionally active (e.g., where transcription is initiated only under certain physiological conditions).

[0078] Disclosed herein are compositions and methods of modifying a nucleic acid, e.g., to modulate expression of a target gene through the use of an epigenetic effector, a composition comprising one or more epigenetic effectors or a system described herein. In some embodiments, the nucleic acid is a DNA, e.g., genomic DNA. In some embodiments, the nucleic acid is an RNA, e.g., messenger RNA(mRNA). A sequence agnostic orthologous Cas based editing system, methods of making such a system, and methods of a nucleic acid is also disclosed.

[0079] Many different diseases and syndromes, including cancer, immune disorder, e.g., autoimmune disorder, inflammatory diseases, cardiovascular disorder, neurological disorder, fibrotic disorder, eye disorder, skin disorder, kidney disorder, developmental disorder, or metabolic disorder, can be caused by dysregulation of gene expression. Studies have shown that aberrant expression (e.g., over-expression or under-expression) of transcription factors such as Yamanaka factors (e.g., OCT4, SOX2, KIF4, and c-MYC) are associated with such disorders.WSGR Ref.65120-702.601 Epigenetic modifying compositions and systems

[0080] Recognized herein is a need for the ability to introduce targeted, multiplexed epigenetic modifications for reprogramming a cell. Reprogramming a cell from a first cellular state (e.g., a diseased state) to a second cellular state (e.g., a healthy state) can require multiple epigenetic modifications and multiple sites. For example, reprogramming a cell can require methylation of multiple target sites in a cell genome. Reprogramming a cell can require a combination of methylation of certain target sites and demethylation of other target sites. In these applications, the use of orthologous epigenetic effectors can prevent undesired epigenetic modifications during simultaneous epigenetic editing by different epigenetic effectors, when different modifications are intended at different target sites.

[0081] In many applications for cellular reprogramming, CpG islands comprise important target sites for epigenetic modifications. CpG islands can be located in a promoter region of a gene or within a transcription regulatory element of a gene, and methylation or demethylation within a CpG island can result in changes in gene expression. The disclosure provides in part, compositions and systems for reprogramming a cell comprising a set of two or more epigenetic effectors. In some embodiments, the two or more epigenetic effectors are orthologous and do not crosstalk substantially. In some embodiments, the set of two or more epigenetic effectors are configured to target sites in CpG islands.

[0082] In one aspect, the present disclosure provide a system comprising a cell, wherein the cell comprises a target nucleic acid comprising one or more CpG islands. The cell can be a eukaryotic cell, for example, a mammalian or a plant cell. In some embodiments, the cell is a human cell. In some embodiments, the system further comprises a first epigenetic effector comprising: (i) a first CRISPR / Cas domain complexed with a first guide RNA comprising a first tracrRNA sequence, and (ii) a first effector moiety. In some embodiments, the system further comprises a second epigenetic effector comprising: (i) a second CRISPR / Cas domain complexed with a second guide RNA comprising a second tracrRNA sequence, and (ii) a second effector moiety. In some embodiments, the first tracrRNA sequence and the second tracrRNA sequence have a phylogenetic distance of exceeding 0.09, when the phylogenetic distance is determined by DNAdist (v3.5c). In some embodiments the first tracrRNA sequence and the second common tracrRNA sequence have a phylogenetic distance of exceeding 0.1, exceeding 0.12, exceeding 0.14, exceeding 0.16, exceeding 0.18, exceeding 0.2, exceeding 0.22, exceeding 0.24, exceeding 0.26, exceeding 0.28, exceeding 0.3, exceeding 0.35, or exceeding 0.4, when the phylogenetic distance is determined by DNAdist (v3.5c). In some embodiments, the first guide RNA or theWSGR Ref.65120-702.601 second guide RNA comprise a sequence selected from SEQ ID NOs.1-10. In some embodiments, the first guide RNA and the second guide RNA comprise a sequence selected from SEQ ID NOs.1-10. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135.

[0083] In some embodiments, the first guide RNA further comprises a first spacer sequence that is complementary to a first sequence in a first CpG island of the one or more CpG islands in the target nucleic acid, and the second guide RNA further comprises a second spacer sequence that is complementary to a second sequence in a second CpG island of the one or more CpG islands in the target nucleic acid.

[0084] In some embodiments, the target nucleic acid comprises over 1,000, over 2,000, over 3,000, over 4,000, over 5,000, over 6,000, over 7,000, over 8,000, over 10,000, over 20,000, over 30,000, over 40,000, over 50,000, over 60,000, over 70,000, over 80,000, over 90,000, over 100,000, over 150,000, over 200,000, over 250,000, over 300,000, over 350,000, over 400,000, over 450,000, over 500,000, over 550,000, over 600,000, over 650,000, over 700,000, over 750,000, over 800,000, over 850,000, over 900,000, over 1,000,000, over 1,100,000, over 1,200,000, over 1,300,000, over 1,400,000, over 1,500,000, over 1,750,000, or over 2,000,000 copies of a first PAM sequence associated with the first CRISPR / Cas domain in the one or more CpG islands in the target nucleic acid. In some embodiments, the target nucleic acid comprises over 1,000, over 2,000, over 3,000, over 4,000, over 5,000, over 6,000, over 7,000, over 8,000, over 10,000, over 20,000, over 30,000, over 40,000, over 50,000, over 60,000, over 70,000, over 80,000, over 90,000, over 100,000, over 150,000, over 200,000, over 250,000, over 300,000, over 350,000, over 400,000, over 450,000, over 500,000, over 550,000, over 600,000, over 650,000, over 700,000, over 750,000, over 800,000, over 850,000, over 900,000, over 1,000,000, over 1,100,000, over 1,200,000, over 1,300,000, over 1,400,000, over 1,500,000, over 1,750,000, or over 2,000,000 copies of a second PAM sequence associated with the second CRISPR / Cas domain in the one or more CpG islands in the target nucleic acid.

[0085] In some embodiments, the first PAM sequence and / or the second PAM sequence is selected from the group consisting of NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT,WSGR Ref.65120-702.601 NNAAAG, NGGDT, NHGYNANA, NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

[0086] The first CRISPR / Cas domain and the second CRISPR / Cas domain can be Type I, Type II, Type III, Type IV, Type V, or Type VI Cas domains. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain are both Type II Cas domains (e.g., Cas9 domains). In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain are both Type V Cas domains (e.g., Cas12 domains). In some embodiments, the system comprises a Type II Cas domain and a Type V Cas domain. In some embodiments, the first CRISPR / Cas domain is selected from the group consisting of AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas domain is selected from the group consisting of AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. The first CRISPR / Cas domain and / or the second CRISPR / Cas domain can be catalytically inactive (e.g., a dCas domain).

[0087] In some embodiments, the first effector moiety comprises a demethylase domain, for example, a TET1 domain. In some embodiments, the first effector moiety comprises a methyltransferase domain, for example, a DNMT3A domain. In some embodiments, the first effector moiety comprises a domain selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the first effector moiety comprises two domains selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the first effector moiety comprises aWSGR Ref.65120-702.601 DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the second effector moiety comprises a demethylase domain, for example, a TET1 domain. In some embodiments, the second effector moiety comprises a methyltransferase domain, for example, a DNMT3A domain. In some embodiments, the second effector moiety comprises a domain selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the second effector moiety comprises two domains selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain. In some embodiments, the second effector moiety comprises a DNMT3A domain, a DNMT3L domain, and a KRAB domain.

[0088] In some embodiments, the first and / or second effector moiety comprises a transcriptional activator domain. In some embodiments, the first and / or second effector moiety comprises a transcriptional repressor domain. In some embodiments, the first effector moiety comprises a transcriptional activator domain and the second effector moiety comprises a transcriptional repressor domain. In some embodiments, the first effector moiety comprises a transcriptional repressor domain and the second effector moiety comprises a transcriptional activator domain.

[0089] In some embodiments, the system comprises at least two epigenetic effectors. In some embodiments, the system comprises at least two epigenetic effectors, each comprising a nucleic acid binding moiety (e.g., a CRISPR / Cas domain) and an effector moiety, e.g., at least one effector moiety. The nucleic acid binding moiety can specifically interact or bind to a target nucleic sequence in a target nucleic acid, while the effector moiety can catalyze an epigenetic edit, e.g., methylation, demethylation, acetylation, and / or deacetylation at a target site in the target nucleic acid. In some embodiments, an epigenetic effector comprises a CRISPR / Cas domain complexed with a guide RNA.

[0090] In some embodiments, the first epigenetic effector comprises multiple effector moieties e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 effector moieties. In some embodiments, the second epigenetic effector comprises multiple effector moieties e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 effector moieties. In some embodiments, the system may be capable of methylation, demethylation, acetylation, and / or deacetylation. In some embodiments, the system may be capable of methylation and / or demethylation. In some embodiments, the first epigenetic effector is capable of adding or removing a methyl group in a nucleic acid, and the second epigenetic effector capable of adding or removing a methyl group in a nucleic acid. In some embodiments, the first epigenetic effector is capable of adding or removing a methyl group in a nucleic acid, and the second epigenetic effector capable of adding or removing an acetyl group in a histone. InWSGR Ref.65120-702.601 some embodiments, the first epigenetic effector is capable of adding or removing an acetyl group in a histone, and the second epigenetic effector capable of adding or removing an acetyl group in a histone. In some embodiments, the first epigenetic effector is capable of adding a methyl group in a nucleic acid, and the second epigenetic effector capable of removing a methyl group in a nucleic acid. In some embodiments, the first epigenetic effector is capable of adding a methyl group in a nucleic acid, and the second epigenetic effector capable of adding a methyl group in a nucleic acid. In some embodiments, the first epigenetic effector is capable of removing a methyl group in a nucleic acid, and the second epigenetic effector capable of removing a methyl group in a nucleic acid. In some embodiments, the first epigenetic effector is capable of adding a methyl group in a histone, and the second epigenetic effector capable of removing a methyl group in a histone. In some embodiments, the first epigenetic effector is capable of adding a methyl group in a histone, and the second epigenetic effector capable of adding a methyl group in a histone. In some embodiments, the first epigenetic effector is capable of removing a methyl group in a histone, and the second epigenetic effector capable of removing a methyl group in a histone. In some embodiments, the first epigenetic effector is capable of adding a methyl group in a histone, and the second epigenetic effector capable of removing a methyl group in a nucleic acid. In some embodiments, the first epigenetic effector is capable of adding a methyl group in a nucleic acid, and the second epigenetic effector capable of removing a methyl group in a histone. In some embodiments, the first epigenetic effector is capable of adding a methyl group in a histone, and the second epigenetic effector capable of adding a methyl group in a nucleic acid. In some embodiments, the first epigenetic effector is capable of removing a methyl group in a histone, and the second epigenetic effector capable of removing a methyl group in a nucleic acid.

[0091] In another aspect, the present disclosure provides a system, comprising: (a) a first epigenetic effector comprising: (i) a SpyCas9 domain complexed with a first guide RNA, and (ii) a demethylase; and (b) a second epigenetic effector comprising: (i) a SauCas9 domain complexed with a second guide RNA and (ii) a methylase. In some embodiments, the demethylase comprises a TET1 domain. In some embodiments, the methylase comprises a DNMT3A domain. In some embodiments, the second epigenetic effector further comprises a DNMT3L domain or a KRAB domain. In some embodiments, the system further comprises a cell, wherein the cell comprises: (a) a target nucleic acid comprising one or more CpG islands; (b) the first epigenetic effector; and (c) the second epigenetic effector.WSGR Ref.65120-702.601

[0092] In some aspects, the present disclosure provides a system comprising two epigenetic effectors that may increase or be capable of increasing methylation and / or acetylation at two target sites. In some embodiments, the first epigenetic effector may increase or be capable of increasing histone methylation, histone acetylation, or DNA methylation, or transcriptionally activate, or repress at a first target site, and the second epigenetic effector may increase or be capable of increasing histone methylation, histone acetylation, or DNA methylation, or transcriptionally activate, or repress at a second target site.

[0093] In some embodiments, the first epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) DNA methylation at a first target site, and the second epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) DNA methylation at a second target site. In some embodiments, the first epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) DNA methylation at a first target site, and the second epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) histone methylation at a second target site. In some embodiments, the first epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) DNA methylation at a first target site, and the second epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) histone acetylation at a second target site. In some embodiments, the first epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) histone methylation at a first target site, and the second epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) histone methylation at a second target site. In some embodiments, the first epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) histone methylation at a first target site, and the second epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) histone acetylation at a second target site. In some embodiments, the first epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) histone acetylation at a first target site, and the second epigenetic effector may modulate (e.g., increase or decrease) or be capable of modulating (e.g., increasing or decreasing) histone acetylation at a second target site. In some embodiments, the first epigenetic effector may further transcriptionally activate or repress at a first target site and / or the second epigenetic effector may further transcriptionally activate or repress at a second target site. In some embodiments, the first epigenetic effector comprises anWSGR Ref.65120-702.601 epigenetic effector selected from a DNA methylase, a DNA demethylation enzyme, a histone methyltransferase, a histone deacetylase, a histone demethylase, a protein-lysine N- methyltransferase, or a transcription repressor moiety and / or the second epigenetic effector comprises an epigenetic effector selected from a DNA methylase, a DNA demethylation enzyme, a histone methyltransferase, a histone deacetylase, a histone demethylase, a protein- lysine N-methyltransferase, or a transcription repressor moiety. In some embodiments, the DNA methyltransferase may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, or a functional equivalent thereof. In some embodiments, the DNA demethylase may be selected from TET1, TET2, and TET3 or a functional equivalent thereof. In some embodiments, the histone methyltransferase may be selected from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional equivalent thereof. In some embodiments, the histone demethylase may be selected from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional equivalent thereof. In some embodiments, the histone acetyltransferase may be selected from KAT1, KAT2A, KAT3A, KAT3B, KAT13C, or a functional equivalent thereof. In some embodiments, the histone deacetylase may be selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional equivalent of any thereof. In some embodiments, the transcriptional repressor moiety may be or selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional equivalent.

[0094] In some embodiments, the system comprises two different nucleic acid binding moieties. In some embodiments, the system comprises one CRISPR / Cas domain and one zinc finger domain. In some embodiments, the system comprises one CRISPR / Cas domain and one TAL domain.

[0095] In some embodiments, the system comprises two CRISPR / Cas domains. In some embodiments, the CRISPR / Cas domains in the system of any of the embodiments described are derived from, or have components derived from, two or more different bacteria species. In some embodiments, the CRISPR / Cas proteins in the system are derived from, or have components derived from, two or more different bacteria genera. In some embodiments, the CRISPR / Cas proteins in the system are derived from, or have components derived from, two or more different phage or viruses. In some embodiments, the CRISPR / Cas proteins in the system are derivedWSGR Ref.65120-702.601 from one or more bacteria and one or more phage or viruses. In some embodiments, the system comprises two CRISPR / Cas proteins selected such that a first tracrRNA sequence interacting with the first CRISPR / Cas protein and a second tracrRNA sequence interacting with the second CRISPR / Cas protein have a phylogenetic distance of exceeding 0.05, exceeding 0.09, exceeding 0.1, exceeding 0.12, exceeding 0.14, exceeding 0.16, exceeding 0.18, exceeding 0.2, exceeding 0.22, exceeding 0.24, exceeding 0.26, exceeding 0.28, exceeding 0.3, exceeding 0.35, or exceeding 0.4.

[0096] In some embodiments, a first epigenetic effector is an epigenetic effector described herein. In some embodiments, the second epigenetic effector, is an epigenetic effector described herein. In some embodiments, the first epigenetic effector optionally further comprises a transcriptional activator and / or a transcriptional repressor. In some embodiments, the second epigenetic effector optionally further comprises a transcriptional activator and / or a transcriptional repressor. In some embodiments, the first epigenetic effector comprises a transcriptional activator, and the second epigenetic effector comprises a transcriptional repressor. In some embodiments, the first epigenetic effector comprises a transcriptional activator, and the second epigenetic effector comprises a transcriptional activator. In some embodiments, the first epigenetic effector comprises a transcriptional repressor, and the second epigenetic effector comprises a transcriptional repressor. In some embodiments, the transcriptional activators of the system may be independently selected from the same category or different categories. In some embodiments, the transcriptional repressors of the system may be independently selected from the same category or different categories. In some embodiments, the epigenetic effectors may be independently selected from categories comprising a DNA demethylase, histone acetyltransferase, histone demethylase, or a transcription repressor moiety. In some embodiments, the epigenetic effectors may include one or more DNA demethylases and / or functional equivalents thereof. In some embodiments, the epigenetic effectors may include one or more histone acetyltransferases and / or functional equivalents thereof. In some embodiments, the epigenetic effectors may include one or more histone methyltransferases and / or functional equivalents thereof. In some embodiments, the epigenetic effectors may include one or more histone demethylases and / or functional equivalents thereof. In some embodiments, the epigenetic effectors may include one or more DNA methyltransferases and / or functional equivalents thereof. In some embodiments, the epigenetic effectors may include one or more histone deacetylases and / or functional equivalents thereof. In some embodiments, the epigenetic effectors may include one or more transcription repressor moieties and / or functional equivalents thereof.WSGR Ref.65120-702.601

[0097] In some embodiments, the first effector moiety is selected from a DNA methylase, a DNA demethylation enzyme, a histone methyltransferase, a histone deacetylase, a histone demethylase, a protein-lysine N-methyltransferase, or a transcription repressor moiety. In some embodiments, the second effector moiety is selected from a DNA methylase, a DNA demethylation enzyme, a histone methyltransferase, a histone deacetylase, a histone demethylase, a protein-lysine N-methyltransferase, or a transcription repressor moiety. In some embodiments, the first effector moiety is selected from a DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, or KAT13C, or a functional equivalent. In some embodiments, the second effector moiety is selected from a DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, or KAT13C, or a functional equivalent.

[0098] In some embodiments, the first effector moiety may be a DNA methyltransferase. In some embodiments, the second effector moiety may be a DNA methyltransferase. In some embodiments, the DNA methyltransferase may be selected from a m6A methyltransferase, an m4C methyltransferase, and an m5C methyltransferase. In some embodiments, the DNA methyltransferase may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, or a functional equivalent of any thereof.

[0099] In some embodiments, the first effector moiety may be capable of DNA demethylation. In some embodiments, the second effector moiety may be capable of DNA demethylation. In some embodiments, the first effector moiety may be selected from TET1, TET2, and TET3. In some embodiments, the second effector moiety may be selected from TET1, TET2, and TET3.

[0100] In some embodiments, the first effector moiety may be a DNA methyltransferase, and the second effector moiety may be capable of DNA demethylation. In some embodiments, the first effector moiety may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1,WSGR Ref.65120-702.601 DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, or a functional equivalent thereof, and the second effector moiety may be selected from TET1, TET2, and TET3 or a functional equivalent thereof. In some embodiments, the first effector moiety may be selected from TET1, TET2, and TET3 or a functional equivalent thereof, and the second effector moiety may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1, MET1, DRM2, CMT2, CMT3, or a functional equivalent thereof.

[0101] In some embodiments, the set of the first and the second epigenetic effectors further comprises additional epigenetic effectors, e.g., third, fourth, fifth, sixth or more epigenetic effectors. In some embodiments, the first epigenetic effector comprises one or more additional effector moieties. In some embodiments, the second epigenetic effector comprises one or more additional effector moieties. In some embodiments, the first epigenetic effector comprises a third effector moiety. In some embodiments, the second epigenetic effector comprises a fourth effector moiety. In some embodiments, the third effector moiety may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, or KAT13C,or a functional equivalent thereof. In some embodiments, the fourth effector moiety may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, or KAT13C, or a functional equivalent thereof.

[0102] In some embodiments, the first effector moiety may be a durable effector moiety. In some embodiments, the second effector moiety may be a durable effector moiety. In some embodiments, the first effector moiety may be a transient effector moiety. In some embodiments, the second effector moiety may be a transient effector moiety. In someWSGR Ref.65120-702.601 embodiments, the first effector moiety may be a durable effector moiety, and the second effector moiety may be a transient effector moiety.

[0103] In some embodiments, the first epigenetic effector binds to a first transcription regulatory element selected from a promoter, an enhancer, or a transcription start site operably linked to a gene, and the second epigenetic effector binds to a second transcription regulatory element selected from a promoter, an enhancer, or a transcription start site operably linked to a gene. In some embodiments, the first and the second transcription regulatory elements are identical. In some embodiments, the first and the second transcription regulatory elements are different.

[0104] In some embodiments, the first epigenetic effector may bind to a promoter and the second epigenetic effector may bind to an enhancer. In some embodiments, the first epigenetic effector may bind to a promoter and the second epigenetic effector may bind to a transcription start site operably linked to a gene. In some embodiments, the first epigenetic effector may bind to an enhancer and the second epigenetic effector may bind to a promoter. In some embodiments, the first epigenetic effector may bind to an enhancer and the second epigenetic effector may bind to a transcription start site operably linked to a gene. In some embodiments, the first epigenetic effector may bind to a transcription start site operably linked to a gene and the second epigenetic effector may bind to a promoter. In some embodiments, the first epigenetic effector may bind to a transcription start site operably linked to a gene and the second epigenetic effector may bind to an enhancer. In some embodiments, the first transcription regulatory element and the second epigenetic effector are associated with same target gene. In some embodiments, the first transcription regulatory element and the second epigenetic effector are associated with different target genes.

[0105] In some embodiments, the first epigenetic effector may bind to a first CpG island and the second epigenetic effector may bind to a second CpG island. In some embodiments, the first CpG island is located in a first transcription regulatory element, and the second CpG island is located in a second transcription regulatory element.

[0106] In some embodiments, the first epigenetic effector comprises a first nucleic acid binding moiety comprising a CRISPR / Cas domain, a zinc finger domain, or a TAL domain. In some embodiments, the second epigenetic effector comprises a first nucleic acid binding moiety comprising a CRISPR / Cas domain, a zinc finger domain, a TAL domain, or a functional equivalent of any of the foregoing. In some embodiments, the first epigenetic effector comprises a first nucleic acid binding moiety comprising a first CRISPR / Cas domain comprising a firstWSGR Ref.65120-702.601 CRISPR / Cas protein, and the second epigenetic effector comprises a first nucleic acid binding moiety comprising a second CRISPR / Cas domain comprising a second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein may be selected from of type I, type II, type III, type IV, type V Cas protein, and type VI Cas protein. In some embodiments, the first CRISPR / Cas protein may be selected from of type I, type II, type III, type IV, type V Cas protein, and type VI Cas protein. In some embodiments, the second CRISPR / Cas protein may be selected from a type I, type II, type III, type IV, type V Cas protein, or type VI Cas protein. In some embodiments, the first CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, and functional equivalents. In some embodiments, the second CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, or a functional fragment thereof. In some embodiments, the first CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1,WSGR Ref.65120-702.601 Cas12c / C2c3, Cas12b / C2c1, or a functional fragment thereof, and the second CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, or a functional fragment thereof.

[0107] In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprise a Type III Cas protein. In some embodiments, the first CRISPR / Cas domain comprises a Type V Cas protein and the second CRISPR / Cas domain comprises a Type III Cas protein. In some embodiments, the first CRISPR / Cas domain comprises a Type III Cas protein and the second CRISPR / Cas domain comprises a Type V Cas protein. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprise a Type V Cas protein. In some embodiments, the first CRISPR / Cas protein may be a Cas9 protein and the second CRISPR / Cas protein may be a Cas12 protein. In some embodiments, the first CRISPR / Cas protein may be a Cas12 protein and the second CRISPR / Cas protein may be a Cas9 protein. In some embodiments, the first CRISPR / Cas protein may be a first Cas9 protein and the second CRISPR / Cas protein may be a second Cas9 protein. In some embodiments, the first Cas9 protein and the second Cas9 protein are orthologs. In some embodiments, the first CRISPR / Cas protein may be a first Cas12 protein and the second CRISPR / Cas protein may be a second Cas12 protein. In some embodiments, the first Cas12 protein and the second Cas12 protein are orthologs. In some embodiments, the first nucleic acid binding moiety comprises a CRISPR / Cas domain and the second nucleic acid binding moiety comprises a zinc finger domain. In some embodiments, the first nucleic acid binding moiety comprises a zinc finger domain and the second nucleic acid binding moiety comprises a CRISPR / Cas domain. In some embodiments, the first nucleic acid binding moiety comprises a first zinc finger domain and the second nucleic acid binding moiety comprises a second zinc finger domain. In some embodiments, the first zinc finger domain and the second zinc finger domain bind to different target sequences. Epigenetic modifying systems with CRISPR / Cas domainsWSGR Ref.65120-702.601

[0108] Disclosed herein, in some embodiments, are compositions or systems comprising one or more epigenetic effectors or nucleic acid sequence encoding one or more epigenetic effectors. In some embodiments, the compositions or systems comprise two or more epigenetic effectors or nucleic acid sequence encoding the two or more epigenetic effectors. In some embodiments, the two or more epigenetic effectors comprise a first epigenetic effector comprises a first CRISPR / Cas domain, and a second epigenetic effector comprising a second CRISPR / Cas domain. In some embodiments, the first and second CRISPR / Cas domains are different. In some embodiments, the first and second CRISPR / Cas domains are derived from, or have components derived from, two or more different bacteria species. In some embodiments, the first and second CRISPR / Cas domains are derived from, or have components derived from, two or more different bacteria genera. In some embodiments, the first and second CRISPR / Cas domains are derived from, or have components derived from, two or more different phage or viruses. In some embodiments, the first and second CRISPR / Cas domains are derived from one or more bacteria and one or more phage or viruses. In some embodiments, the CRISPR / Cas domain comprises all or a portion of a CRISPR / Cas domain.

[0109] In some embodiments, the first CRISPR / Cas domain in the system may be selected from AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. In some embodiments, the first CRISPR / Cas domain in the system may be selected from SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas domain in the system may be selected from SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. In some embodiments, the first CRISPR / Cas domain in the system may be selected from SpyCas9, LanCas9, Sag1Cas9, and LmoCas9, and the second CRISPR / Cas domain in the system may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a. In some embodiments, the first CRISPR / Cas domain in the system may be selected from SpyCas9, LanCas9, Sag1Cas9, and LmoCas9, and the second CRISPR / Cas domain in the system may be selected from Casphi-2 and Cas12c-1. In some embodiments, the first CRISPR / Cas domain in the system may be selected from Cpf1, Hkcas12a, PiCas12a,WSGR Ref.65120-702.601 Fn3Cas12a, and Pb2Cas12a; and the second CRISPR / Cas domain in the system may be selected from Casphi-2 and Cas12c-1. In some embodiments, the first CRISPR / Cas domain in the system may be selected from CasPhi-2 and Pb2Cas12a, and the second CRISPR / Cas domain in the system may be selected from CasPhi-2. In some embodiments, the first CRISPR / Cas domain in the system may be selected from Lan, Lmo and Sag1 and the second CRISPR / Cas domain in the system may be a SpyCas9. In some embodiments the Cas domain is a catalytically inactive Cas domain. In some embodiments, CasPhi comprises SEQ ID NO: 130. In some embodiments, FN3Cas12a comprises SEQ ID NO: 131. In some embodiments, HKCas12a comprises SEQ ID NO: 132. In some embodiments, LanCas9 comprises SEQ ID NO: 133. In some embodiments, SauCas9 comprises SEQ ID NO: 134. In some embodiments, SpyCas9 comprises SEQ ID NO: 135.

[0110] In some embodiments, the system comprise two or more CRISPR / Cas proteins, wherein the first CRISPR / Cas protein comprises a PAM sequence selected from NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNGRRT, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG and the second CRISPR / Cas protein comprises a PAM sequence selected from NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNGRRT, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. In some embodiments, the first CRISPR / Cas protein in the system comprises a PAM sequence selected from NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG and the second CRISPR / Cas protein in the systemWSGR Ref.65120-702.601 comprises a PAM sequence selected from NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. In some embodiments, the first CRISPR / Cas protein in the system comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein in the system comprises a PAM sequence selected from TTTV, YYV, KKYV, YTV, and NYTV. In some embodiments, the first CRISPR / Cas protein in the system comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein in the system comprises a PAM sequence selected from TBN and TG. In some embodiments, the first CRISPR / Cas protein in the system comprises a PAM sequence selected from TTTV, YYV, KKYV, YTV, and NYTV and the second CRISPR / Cas protein in the system comprises a PAM sequence selected from TBN and TG. In some embodiments, the first CRISPR / Cas protein in the system comprises a PAM sequence selected from TBN and NYTV and the second CRISPR / Cas protein in the system comprises a PAM sequence TG.

[0111] In some embodiments, the system comprise two or more CRISPR / Cas proteins each comprising one or more number of PAM sites in a CpG island. In some embodiments, the first CRISPR / Cas protein comprises a higher number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein comprises an equivalent number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. In some embodiments, the first CRISPR / Cas protein comprises a lower number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein.

[0112] In some embodiments, the system comprise two or more CRISPR / Cas proteins that do not cross-react substantially.

[0113] In some embodiments, the system comprises two CRISPR / Cas proteins selected such that a first tracrRNA sequence interacting with the first CRISPR / Cas protein and a second tracrRNA sequence interacting with the second CRISPR / Cas protein have a phylogenetic distance of exceeding 0.2. In some embodiments, the system comprises a first tracrRNA sequence interacting with the first CRISPR / Cas protein and a second tracrRNA sequence interacting with the second CRISPR / Cas protein having a phylogenetic distance of exceeding 0.2. In some embodiments, predicted cross-reactivity negatively correlates with phylogenetic distance. Phylogenetic distance between protein sequences may be measured using the BLOSUM 62 matrix excluding indels.

[0114] In some embodiments, the system comprises two or more CRISPR / Cas proteins, further comprising a first sgRNA and a second sgRNA. In some embodiments, first sgRNA and secondWSGR Ref.65120-702.601 sgRNA in the system may be selected such that it does not cross react substantially with the other. In some embodiments, first sgRNA or second sgRNA in the system may comprise a sequence selected from SEQ ID NO: 1-4 at 3’ end (Table 1). In some embodiments, first sgRNA or second sgRNA in the system may comprise a sequence selected from SEQ ID NO: 5-10 at 5’ end (Table 1). Table 1—sgRNA SequencesWSGR Ref.65120-702.601

[0115] In some embodiments, the system comprises a first epigenetic effector comprising a first guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a first desired site of epigenetic modification and a second epigenetic effector comprising a second guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a second desired site of epigenetic modification. The first epigenetic effector may be any of the epigenetic effectors with guide RNAs described herein. The second epigenetic effector may be any of the epigenetic effectors with guide RNAs described herein. In some embodiments, the first epigenetic effector in the system comprises a methylase and a first guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a desired site of methylation, and the second epigenetic effector in the system comprises a demethylase and a second guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a desired site of demethylation. In some embodiments, the first epigenetic effector in the system comprises a transcriptional activator and a first guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a desired site of transcriptional activation, and the second epigenetic effector in the system comprise a transcriptional repressor and a second guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a desired site of transcriptional repression.

[0116] The disclosure provides in part, a system comprising (a) a first guide RNA, wherein the guide RNA comprises a direct repeat sequence and a first spacer sequence capable of hybridizing to a DNA sequence within 1 kb of a first target site, (b) a first CRISPR-Cas effector polypeptide or a nucleic acid encoding the effector polypeptide, wherein the first effector polypeptide may be capable of binding to a first target sequence complementary to the first spacer sequence, wherein binding of the first effector polypeptide to the first target sequence increases methylation at the first target site, (c) a second guide RNA, wherein the guide RNA comprises a direct repeat sequence and a second spacer sequence capable of hybridizing to a DNA sequence 1 kb of a second target site, and (d) a second CRISPR-Cas effector polypeptide or a nucleic acid encoding the effector polypeptide, wherein the second effector polypeptideWSGR Ref.65120-702.601 may be capable of binding to a second target sequence complementary to the second spacer sequence; and wherein binding of the second effector polypeptide to the second target sequence decreases methylation at the second target site.

[0117] In some embodiments, the system comprises: (a) a first guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a first desired site of epigenetic modification, and (b) a second guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a second desired site of epigenetic modification, wherein the first guide RNA interacts with the first epigenetic and the second guide RNA interacts with the second epigenetic effector. In some embodiments, the first effector and the second effector may act concurrently. In some embodiments, the first effector and the second effector may act substantially concurrently. In some embodiments, the first effector and the second effector may act sequentially. In some embodiments, the first effector and the second effector may act sequentially within a time period of about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 22 hours, 24 hours or less.

[0118] In some aspects, the disclosure provides in part, a system for simultaneous nucleic acid modification. In some embodiments, the system comprises a system comprising a first epigenetic effector and a second epigenetic effector for simultaneous epigenetic modification at one or more target sites. In some embodiments, the first epigenetic effector induces transcriptional activation at a first target site, and the second epigenetic effector induces transcriptional repression at a second target. In some embodiments, the first epigenetic effector may be capable of increasing methylation at a first target site, and a second epigenetic effector may be capable of decreasing methylation at a second target site. In some embodiments, the first epigenetic effector comprises methylation modifying activity, and the second epigenetic effector comprises methylation modifying activity, wherein the first and second epigenetic effector have different methylation modifying activity.

[0119] In some aspects, the disclosure provides a system comprising one or more epigenetic effector and one or more nucleic acid cutting editor. In some embodiments, an editor comprises a nucleic acid binding domain and a DNA polymerase domain. In some embodiments, the nucleic acid binding domain is selected from a CRISPR / Cas domain, a zinc finger domain, or a TAL domain. In some embodiments, the DNA polymerase domain is a reverse transcriptase, e.g., a MMLV-RT, TERT-RV, RT_nLTR, RT_Bac_Retron, or RVT2.WSGR Ref.65120-702.601

[0120] In some embodiments, the system for simultaneous nucleic acid modification comprises an epigenetic effector described herein and a nucleic acid cutting editor described herein (e.g., a CRISPR / Cas effector) for simultaneous epigenetic modification and nuclei acid cutting followed by editing of one or more genomic sequences in a cell. In some embodiments, the epigenetic effector modifies, e.g., modulates (e.g., increases or decreases DNA methylation, increases or decreases histone methylation, or increases or decreases histone acetylation),in a first target site and the editor edits, e.g., inserts one or more nucleotides, deletes one or more nucleotides, and / or substitutes one or more nucleotides a second target site.

[0121] In some aspects, the disclosure provides in part, a system comprising a set of effectors, e.g., a first and a second epigenetic effector described herein for modulating activity of a cell. In some embodiments, the set of effectors comprises two or more epigenetic effectors e.g., a first and a second epigenetic effector described herein to modulate the methylation profile of the genome of the cell. In some embodiments, the system comprises an epigenetic effector described herein and a nucleic acid cutting editor described herein.

[0122] In some aspects, the disclosure provides in part, a system, e.g., a system comprising a set of effectors e.g., a first and a second epigenetic effector described herein for treating or preventing a disease in a subject. In some embodiments, the set of effectors comprises two or more epigenetic effectors e.g., a first and a second epigenetic effector described herein. In some embodiments, the set of two or more epigenetic effectors e.g., a first and a second epigenetic effector described herein is capable of reprogramming a diseased cell into a healthy cell by modulating the methylation profile of the genome of the diseased cell. In some embodiments, the composition comprises an epigenetic effector described herein and an editor described herein.

[0123] In some aspects, the disclosure provides in part, a composition , e.g., a system comprising a set of effectors, e.g., a first and a second epigenetic effector described herein for altering developmental potency of a cell. In some embodiments, the set of effectors is capable of modulating one or more components of a cellular pathway associated with the developmental potency of the cell. In some embodiments, the set of effectors comprises two or more epigenetic effectors. In some embodiments, the composition comprises an epigenetic effector described herein and an editor described herein.WSGR Ref.65120-702.601

[0124] In some aspects, the present disclosure provides a system, e.g., comprising two epigenetic effectors, e.g., a first and a second epigenetic effector described herein that for simultaneous methylation and / or acetylation at two target sites. Epigenetic effector

[0125] As described herein, the present disclosure in part provides an epigenetic effector comprising a nucleic acid binding moiety and an effector moiety. In some embodiments, the effector moiety of the epigenetic effector may be or may comprise a moiety capable of modifying a nucleic acid. In some embodiments, the nucleic acid is a DNA, e.g., genomic DNA. In some embodiments, the nucleic acid is a RNA, e.g., mRNA. In some embodiments, the effector moiety is capable of altering methylation profile of a genome of a cell. In some cases, effector moiety can modify a nucleic acid by increasing or decreasing methylation in a target nucleic acid. In other cases, the effector moiety modifies the chromatin structure of a cell through histone modifications, e.g., via modulating histone methylation and / or acetylation profile. In some embodiments, the epigenetic effector comprises a nucleic acid binding moiety and multiple effector moieties (e.g., 1, 2, 3, 4, 5, 6.7.8.9. Or 10 effector moieties). In some embodiments, the nucleic acid binding moiety and the effector moiety are covalently linked, e.g., via a peptide bond. In some embodiments, the nucleic acid binding moiety and the effector moiety are not covalently linked.

[0126] In some embodiments, the nucleic acid binding moiety of the epigenetic effector determines the site of nucleic acid modification through specific binding with a target nucleic acid. In some embodiments, the nucleic acid binding moiety may be or comprise a CRISPR / Cas domain, a zinc finger domain, or a TAL domain. In some embodiments, the nucleic acid binding moiety of the epigenetic effector may be or may comprise a Cas9 protein or a functional equivalent. In some embodiments, the nucleic acid binding moiety of the epigenetic effector may be or may comprise a Cas12 protein or a functional equivalent. In some embodiments, the epigenetic effector may be capable of binding to a transcription regulatory element (e.g., a promoter, an enhancer, or a transcription start site operably linked to a gene) and facilitating an epigenetic modification at the desired target site. In some embodiments, the epigenetic effector may be capable of binding to a site in a CpG island of a target nucleic acid and introducing an epigenetic modification at a desired target site. In some embodiments, the epigenetic effector may be capable of methylating or demethylating at least one CpG site of a target nucleic acid.WSGR Ref.65120-702.601

[0127] In some embodiments, the epigenetic effector is capable of binding to a transcription regulatory element. In some embodiments, the epigenetic effector is capable of binding to a transcription regulatory element selected from a promoter, an enhancer, a silencer, an insulator, a locus control region, or a transcription start site operably linked to a gene. In some embodiments, the epigenetic effector is capable of binding to a promoter element. In some embodiments, the epigenetic effector is capable of binding to a promoter element selected from a TATA box, a CAAT box, a GC box, an INR, a DPE, an MTE, a DCE, or a BRE. In some embodiments, the epigenetic effector is capable of binding to a TATA box. In some embodiments, the epigenetic effector is capable of binding to a CAAT box. In some embodiments, the epigenetic effector is capable of binding to a GC box. In some embodiments, the epigenetic effector is capable of binding to an INR. In some embodiments, the epigenetic effector is capable of binding to a DPE. In some embodiments, the epigenetic effector is capable of binding to an MTE. In some embodiments, the epigenetic effector is capable of binding to a DCE. In some embodiments, the epigenetic effector is capable of binding to a BRE. The consensus sequences of exemplary promoter elements are provided in Table 2 below. In some embodiments, the promoter may be constitutively active. Alternatively, in some embodiments, the promoter may be conditionally active (e.g., where transcription is initiated only under certain physiological conditions). In some embodiments, the epigenetic effector is capable of binding to an enhancer. In some embodiments, the epigenetic effector is capable of binding to a silencer. In some embodiments, the epigenetic effector is capable of binding to an insulator. In some embodiments, the epigenetic effector is capable of binding to a locus control region. In some embodiments, the epigenetic effector is capable of binding to a transcription start site. Table 2—Exemplary Promoter ElementsWSGR Ref.65120-702.601

[0128] In some embodiments, a nucleic acid binding moiety binds to its target sequence with a KD of less than or equal to 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.003, 0.002, or 0.001M. In some embodiments, a nucleic acid binding moiety does not bind, e.g., does not detectably bind to a non-target sequence. In some embodiments, the nucleic acid binding moiety comprises a sequence complimentary, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% complimentary to the target sequence.

[0129] In some cases, an epigenetic effector may comprise a fusion protein comprising a nucleic acid binding domain and an effector domain. In some instances, the nucleic acid binding domain of an epigenetic effector may be located at the N-terminus or C-terminus of the effector domain. In some cases, the nucleic acid binding domain is located at the N-terminus of the effector domain. In other cases, the nucleic acid binding domain is located at the C-terminus of the effector domain. In some cases, the nucleic acid binding domain is located within the effector domain. In other cases, the effector domain is located within the nucleic acid binding domain. In some embodiments, the epigenetic effector comprises more than one effector domain. For an epigenetic effector comprising more than one effector domain, in some cases, the first effector domain may be located at the N-terminus or C-terminus of the second effector domain. In other cases, first effector domain may be located at the N-terminus of the nucleic acid binding domains, and the second effector domain may be located at the C-terminus of the nucleic acid binding domain. The epigenetic effector may comprise any combination of arrangements of the nucleic acid binding moiety and the effector moiety described in this disclosure.

[0130] In some embodiments, the epigenetic effector, e.g., an epigenetic effector described herein may be capable of methylation, demethylation, acetylation, and / or deacetylation. In some embodiments, the epigenetic effector is capable of adding or removing a methyl group in a nucleic acid. In some embodiments, the epigenetic effector is capable of adding or removing a methyl group in a histone. In some embodiments, the epigenetic effector is capable of adding or removing an acetyl group in a histone. In some embodiments, the epigenetic effector comprises an effector moiety selected from DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1, MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP),WSGR Ref.65120-702.601 SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, KAT13C, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional equivalent thereof.

[0131] In some embodiments, the epigenetic effector, e.g., an epigenetic effector described herein may comprise multiple effector moieties, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 effector moieties.

[0132] In some embodiments, the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10theffector moiety is selected from one or more of DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1, MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, KAT13C, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional equivalent thereof.

[0133] In some embodiments, the epigenetic effector, an epigenetic effector described herein may simultaneously methylate and transcriptionally repress a target site. In some embodiments, the epigenetic effector, an epigenetic effector described herein may simultaneously methylate and transcriptionally activate a target site. In some embodiments, the epigenetic effector, an epigenetic effector described herein may simultaneously demethylate and transcriptionally repress a target site. In some embodiments, the epigenetic effector, an epigenetic effector described herein may simultaneously demethylate and transcriptionally activate a target site. In some embodiments, the epigenetic effector, an epigenetic effector described herein may simultaneously acetylate and transcriptionally repress a target site. In some embodiments, the epigenetic effector, an epigenetic effector described herein may simultaneously deacetylate and transcriptionally activate a target site. Effector moietyWSGR Ref.65120-702.601

[0134] As described herein, the present disclosure in part provides an effector moiety of the epigenetic effector. In some embodiments, the effector moiety may be or may comprise a moiety capable of modifying a nucleic acid (e.g., DNA, e.g., genomic DNA). In some embodiments, the effector moiety may be or may comprise a moiety capable of modifying a nucleic acid (e.g., RNA, e.g., mRNA). In some embodiments, the effector moiety may be or may comprise a moiety capable of modifying a histone. In some embodiments, the effector moiety may be capable of altering methylation profile of a genome of a cell.

[0135] In some embodiments, the epigenetic effector comprises an effector moiety comprising a DNA methylation, DNA demethylation, histone methylation, or histone acetylation activity. In some embodiments, the epigenetic effector may be or comprise a methylase or a demethylase. In some embodiments, the effector moiety may be selected from a DNA methyltransferase, DNA demethylase, a histone methyltransferase, a histone demethylase, a histone acetyltransferase, or a histone deacetylase. In some embodiments, the effector moiety may be or comprise a transcriptional activator or a transcriptional repressor. In some embodiments, the effector moiety is selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1, MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, KAT13C, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional equivalent thereof.

[0136] In some embodiments, the effector moiety of the epigenetic effector may enhance or repress methylation in a target nucleic acid. The effector moiety of the epigenetic effector may be or comprise a DNA methyltransferase or a functional equivalent thereof. The DNA methyltransferase may be selected from a m6A methyltransferase, an m4C methyltransferase, and an m5C methyltransferase. The DNA methyltransferase may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1, MET1, DRM2, CMT2, CMT3, or a functional equivalent thereof.

[0137] In some embodiments, the effector moiety may be or may comprise a moiety capable of effecting DNA demethylation. The effector moiety may be or comprise a DNA demethylase.WSGR Ref.65120-702.601 The effector moiety may be selected from TET1, TET2, and TET3, or a functional equivalent thereof.

[0138] In other embodiments, the effector moiety of the epigenetic effector may increase or decrease methylation or acetylation in a histone. Increasing or decreasing methylation or acetylation in a histone can modify chromatin structure. In some embodiments, the effector moiety may be or comprise a histone methyltransferase or a functional equivalent thereof. The histone methyltransferase may be selected from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional equivalent thereof. The effector moiety of the epigenetic effector may be or comprise a histone demethylase or a functional equivalent thereof. The histone demethylase may be selected from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional equivalent thereof.

[0139] In some embodiments, the effector moiety of the epigenetic effector may be capable of adding or removing an acetyl group in a histone. In some embodiments, the effector moiety of the epigenetic effector may be or comprise a histone acetyltransferase or a functional equivalent thereof. The histone acetyltransferase may be selected from KAT1, KAT2A, KAT3A, KAT3B, KAT13C, or a functional equivalent thereof. The effector moiety of the epigenetic effector may be or comprise a histone deacetylase. The histone deacetylase may be selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional equivalent of any thereof.

[0140] In some embodiments, the effector moiety of the epigenetic effector may be or comprise a transcriptional activator moiety. In some embodiments, the transcriptional activator moiety may be selected from categories comprising a DNA demethylase, histone acetyltransferase, histone methyltransferase, and histone demethylase.

[0141] In other embodiments, the effector moiety of the epigenetic effector may be or comprise a transcription repressor moiety. In some embodiments, the transcriptional repressor moiety may be selected from the categories comprising a DNA methyltransferase, histone deacetylase, histone methyltransferase, and histone demethylase. In some embodiments, the effector moiety may comprise a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional equivalent.WSGR Ref.65120-702.601

[0142] In some embodiments, the epigenetic effector may comprise multiple, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 effector moieties. In some embodiments, the epigenetic effector may comprise a first effector moiety and a second effector moiety. The second effector moiety may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1, MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, KAT13C, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional equivalent thereof.

[0143] In some embodiments, the effector moiety may be a durable effector moiety. In some embodiments, the effector may be a transient effector moiety. In some embodiments, the epigenetic effector may comprise at least two durable effector moieties. In some embodiments, the epigenetic effector may comprise at least two transient effector moieties. In some embodiments, the epigenetic effector may comprise at least one durable effector moiety and at least one transient effector moiety. Nucleic acid binding moiety

[0144] As described herein, an epigenetic effector disclosed herein comprises a nucleic acid binding moiety. In some embodiments, the nucleic acid binding moiety may bind to a target nucleic acid, e.g., a DNA, e.g., a genomic RNA, e.g., a RNA, e.g., an mRNA. In some embodiments, the nucleic acid binding moiety may bind one or more genomic sequences in a cell. In some embodiments, the nucleic acid binding moiety may be or comprise a CRISPR / Cas domain, a zinc finger domain, a TAL domain, a tetR domain, a meganuclease, or an oligonucleotide. CRISPR / Cas domain

[0145] In some embodiments, a nucleic acid binding moiety may be or comprises a CRISPR / Cas domain. In some embodiments, the CRISPR / Cas domain comprises one or more RNA molecules, which can be a crRNA and / or a tracrRNA and / or optionally, an engineered guide RNA or sgRNA. In some embodiments, the CRISPR / Cas domain forms a complex withWSGR Ref.65120-702.601 its partner RNA or RNAs. In some embodiments, the CRISPR / Cas domain and RNA complex utilizes RNA-DNA base pairing to determine the binding site to a target nucleic acid. In some embodiments, the CRISPR / Cas domain optionally complexed with its partner sgRNA or sgRNAs binds to a CpG site in a target nucleic acid. In some embodiments, the CRISPR / Cas domain optionally complexed with its partner sgRNA or sgRNAs binds to a protospacer adjacent motif (PAM) sequence in the target nucleic acid. In some embodiments, the PAM sequence is located within a CpG Island in a target nucleic acid.

[0146] In some embodiments, the CRISPR / Cas domain may comprise a CRISPR / Cas protein. In some embodiments, a CRISPR / Cas domain may be derived from a protein involved in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system or have structural and / or functional similarities to a protein involved in the CRISPR system and optionally a guide RNA, e.g., a single guide RNA (sgRNA). Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “guide RNA”, typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence. crRNA also contains a region that binds to the tracrRNA to form a partially double- stranded structure which is cleaved by RNase III, resulting in a crRNA / tracrRNA hybrid. A crRNA / tracrRNA hybrid then directs Cas9 endonuclease to recognize and cleave a target DNA sequence. Another class II CRISPR system includes the type V endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpfl -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system requires only Cpfl nuclease and a crRNA to cleave a target DNA sequence. The CRISPR / Cas protein may be selected from a type I, type II, type III, type IV, type V Cas protein, and type VI Cas protein. The CRISPR / Cas protein may be selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, AsCas12a, Cas13a, Cas13b, Cas13c, Cas13d, Cas13X, Cas13Y, LbCas12a, HypaCas9, a Type I Cas effector protein, a Type II Cas effector protein, a Type III Cas effector protein, a Type IV Cas effector protein, a Type VWSGR Ref.65120-702.601 Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, and functional fragments and derivatives thereof. In some embodiments, the CRISPR / Cas protein may be or comprise a Cas9 ortholog. The Cas9 protein may be selected from SpCas9, SaCas9, ScCas9, StCas9, NmCas9, VRERCas9, VERCas9, xCas9, espCas91.0, espCas1.1, Cas9HF1, hypaCas9, evoCas9, HiFiCas9, and CjCas9. In some embodiments, the CRISPR / Cas protein may be or comprise a Cas12 ortholog. The Cas12 protein may be selected from Cpf1, FnCas12a, LbCas12a, AsCas12a, LbCas12a, TsCas12a, SaCas12a, Pb2Cas12a, PgCas12a, MiCas12a, Mb2Cas12a, Mb3Cas12a, Lb4Cas12a, Lb5Cas12a, FbCas12a, CrbCas12a, CpbCas12a, CMaCas12a, BsCas12a, BfCas12a, BoCas12a. In some embodiments, the CRISPR / Cas protein may be derived from a bacteria or has one or more components derived from a bacteria, and wherein the one or more components may optionally be derived from different bacteria. The bacteria origin of the CRISPR / Cas protein of each of the epigenetic effectors may be selected from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Bacillus niameyensis, Bacillus okhensis, Capnocytophaga canis, Chryseobacterium gallinarum, Coriobacterium_glomerans_PW2, Dechloromonas denitrificans, Enterococcus cecorum, Enterococcus faecium, Enterococcus italicus, Eubacterium dolichum, Eubacterium sp., Eggerthella sp. YY7918, Exiguobacterium sibiricum, Flavobacterium frigidarium, Facklamia hominis, Finegoldia_magna_ATCC_29328, Kingella kingae, Lactobacillus_rhamnosus_LOCK900, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus sp., Microscilla marina, Mycoplasma_gallisepticum_CA06, Neisseria meningitidis, Ornithobacterium rhinotracheale, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Pediococcus acidilactici, Prevotella histicola, Parabacteroides sp., Streptococcus_agalactiae_NEM316, Streptococcus_dysgalactiae_subsp._equisimilis_AC-2713, Streptococcus equinus, Streptococcus gallolyticus, Streptococcus gordonii, Streptococcus mutans GS-5, Streptococcus macedonicus, Streptococcus ratti, Streptococcus_salivarius_JIM8777, Streptococcus sinensis, Streptococcus suis D9, Streptococcus thermophilus LMG 18311, Tissierellia bacterium KA00581, Treponema denticola ATCC 35405, Treponema putidum, Turicibacter sp., Veillonella parvula ATCC 17745, Weeksella virosa, Streptococcus equi, Streptococcus agalactiae, Lactobacillus animalis KCTC 3501, Listeria monocytogenes, Lachnospiraceae bacterium ND2006, Acidaminococcus sp. BV3L6, Helcococcus kunzii, Prevotella ihumii, Prevotella bryantii B14, Compost_meta_-WSGR Ref.65120-702.601 _Ga0079224_100045232_-_CRISPR associated_protein,_Csn1_family_CDS_translation_Compost_meta, Geyser Hotspring_Yellowstone_Ga0078972_1022257_-_CRISPR- associated_protein,_Csn1_family_CDS_translation_Community_metagenome, Geyser- Hotspring_Yellowstone_Ga0078972_1010018_-_CRISPR- associated_protein,_Csn1_family_CDS_translation_Community_metagenome, Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Clostridium Tyrobutyricum, Clostridium beijerinckii, Clostridium perfringens, Clostridium autoethanogenum, Finegoldia magna, Natranaerobius thermophilus, Methanococcus maripaludis, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Lactobacillus crispatus, Acidithiobacillus ferrooxidans, Acidaminococcus intestine RyC-MR95, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Streptococcus thermophilus, Lactococcus lactis, Staphylococcus epidermidis Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Clostridium acetobutylicum , Synechococcus elongatus UTEX 2973, Actinoplanes sp., B. subtilis, Corynebacterium glutamicum, Streptomyces sp., Clostridium difficile, Clostridium saccharoperbutylacetonicum N1-4, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. Other suitable Cas proteins can be found in Gasiunas, G., Young, J.K., Karvelis, T. et al. A catalogue of biochemically diverse CRISPR-Cas9 orthologs. Nat Commun 11, 5512 (2020), which is hereby incorporated by reference in its entirety.

[0147] The CRISPR / Cas protein may be derived from a virus, e.g., a phage virus, e.g., a bacteriophage, e.g., a Biggievirus or has one or more components derived from a virus, e.g., a phage virus, e.g., a bacteriophage, e.g., a Biggievirus and wherein the one or more components may optionally be derived from different virus.

[0148] In some embodiments, the CRISPR / Cas domain comprises a modified form of a wild type Cas protein. The modified form of the wild type Cas protein can comprise one or more amino acid changes (e.g., deletion, insertion, or substitution). In some embodiments, the endonuclease domain may comprise one or more amino acid substitutions as compared to a wild type endonuclease domain. In some embodiments, the CRISPR / Cas domain comprises anWSGR Ref.65120-702.601 endonuclease domain that has modified or reduced nuclease activity as compared to a wild type protein. For example, the endonuclease domain can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% nuclease activity of the wild type Cas protein. In some embodiments, the CRISPR / Cas domain comprises a catalytically inactive CRISPR / Cas protein (e.g., dCas9) or a CRISPR / Cas protein with substantially reduced nuclease activity compared to a wild type CRISPR / Cas protein. Many catalytically inactive CRISPR / Cas proteins are known in the art. A catalytically inactive CRISPR / Cas protein or a CRISPR / Cas protein that has reduced DNA cleavage activity with respect to both strands of a double-stranded target DNA can result from deletion or mutation of all of the nuclease domains of a CRISPR / Cas protein (e.g., both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cpf1 protein). For example, a catalytically inactive S. pyogenes Cas9 can result from a D10A (aspartate to alanine at position 10) mutation in the RuvC domain and H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain. A catalytically inactive CRISPR / Cas protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. Examples of mutations in SpyCas9 (NCBI Reference Sequence: WP_038431314) include but are not limited to D10A, D11A, D16A, D17A, H557A, H558A, H588A, N611A, N612A, H589A, H820A, H821A, D839A, H840A, N863A, N864A, D917A, D918A, H969A, H970A, E993A,E994A, N995A, N996A, E1006A, E1007A, D1255A, D1256A, or any combination thereof. In some embodiments, a spCas9 mutation include e.g., D10A / H820A, D1OA, D10A / D839A / H840A, and D10A / D839A / H840A / N863A or any combination thereof.

[0149] In some embodiments, the CRISPR / Cas domain comprises a CRISPR / Cas domain that has single strand DNA cleavage activity when contacted with a double stranded DNA sequence. In some embodiments, the CRISPR / Cas domain comprises a CRISPR / Cas domain (i.e. a nickase) that can generate a single-strand break but not a double-strand break. Many CRISPR / Cas nickases are known in the art. A CRISPR / Cas nickase can result from deletion or mutation of one of the nuclease domains in a Cas protein comprising at least two nuclease domains (e.g., Cas9). For example, an S. pyogenes Cas9 nickase can result from a D10A (aspartate to alanine at position 10) mutation in the RuvC domain or a H839A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) mutation in the HNH domain.WSGR Ref.65120-702.601

[0150] In some embodiments, a Cas protein described herein is a mature Cas protein, e.g., lacking a N terminal methionine. A Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides. A Cas protein can be a chimera of various Cas proteins, for example, comprising domains of Cas proteins from different organisms. In some embodiments, a Cas9 is a chimeric Cas9, e.g., modified Cas9; e.g., synthetic RNA-guided nucleases (sRGNs), e.g., modified by DNA family shuffling, e.g., sRGN3.1, sRGN3.3. In some embodiments, the DNA family shuffling comprises, fragmentation and reassembly of parental Cas9 genes, e.g., one or more of Cas9s from Staphylococcus hyicus (Shy), Staphylococcus lugdunensis (Slu), Staphylococcus microti (Smi), and Staphylococcus pasteuri (Spa).

[0151] PAM sequences: A target DNA sequence is generally adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease; however, PAM sequences may appear throughout a given genome. In some embodiments, the PAM is required for target binding of the Cas protein. In some embodiments, the specific PAM sequence required for Cas protein recognition may depend on the specific type of the Cas protein. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In some embodiments, a PAM is between 2-6 nucleotides in length. In some embodiments, the PAM can be a 5’ PAM (e.g., located upstream of the 5’ end of the protospacer). In some embodiments, the PAM can be a 3’ PAM (e.g., located downstream of the 5’ end of the protospacer). In some embodiments, the Cas protein of a prime editor recognizes a canonical PAM, for example, a SpCas9 recognizes 5’-NGG-3’ PAM. In some embodiments, a Cas protein described herein has altered PAM specificity. In some embodiments, a Cas protein described herein may have one or mutations in a PAM recognition motif. Examples of specific PAM sequences are provided in Table 3 below. As used in PAM sequences in Table 3, “N” refers to any one of nucleotides A, G, C, and T, “R” refers to nucleotide A or G, “Y” refers to nucleotide C or T, “W” refers to nucleotide A or T, “K” refers to nucleotide G or T, “M” refers to nucleotide A or C, “B” refers to nucleotide C or G or T, “D” refers to nucleotide A or G or T, “H” refers to nucleotide A or C or T, and “V” refers to nucleotide A or C or G. Table 3— Exemplary PAM Sequences of CRISPR / Cas ProteinsWSGR Ref.65120-702.601WSGR Ref.65120-702.601WSGR Ref.65120-702.601Zinc finger domainWSGR Ref.65120-702.601

[0152] In some embodiments, a nucleic acid binding moiety may be or comprises a Zn finger domain. Zn finger proteins and methods for design and construction of fusion proteins are known to those of skill in the art. The Zn finger domain may comprise or consist essentially of or consist of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3- 10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6- 8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers. Zn finger proteins and / or multi fingered Zn finger proteins may be linked together, e.g., as a fusion protein, using any suitable linker sequences. The Zn finger domain may include any combination of suitable linkers between the individual Zn finger proteins and / or multi-fingered Zn finger proteins of the Zn finger molecule.

[0153] The Zn finger domain of an epigenetic effector may comprise a Zn finger molecule comprising an engineered zinc finger protein that binds (in a sequence- specific manner) to a DNA sequence in a target nucleic acid. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in their entireties. Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98 / 37186; WO 98 / 53057; WO 00 / 27878; and WO 01 / 88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02 / 077227.

[0154] In some cases, a Zn finger molecule may comprise a two-handed Zn finger protein. Two handed Zn finger proteins are those proteins in which two clusters of zinc finger proteins are separated by intervening amino acids so that the two Zn finger domains bind to two discontinuous target DNA sequences. An example of a two handed type of zinc finger binding protein is SIP1, where a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three Zn finger proteins is located at the carboxyl terminus (See Remacle, Jacques E., et al. “New mode of DNA binding of multi-zinc finger transcription factors: δEF1 family members bind with two hands to two target sites.” The EMBO journal 18.18 (1999): 5073-5084, which is herein incorporated by reference). Each cluster of zincWSGR Ref.65120-702.601 fingers in these proteins is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides. TAL domain

[0155] In some embodiments, a nucleic acid binding moiety is or comprises a Transcription Activator-like (TAL) domain. A TAL domain is derived from a TAL effector molecule that specifically binds a DNA sequence. TAL effectors typically comprise a plurality of TAL effector domains or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effectors (e.g., N- and / or C-terminal of the plurality of TAL effector domains). More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXalO and AvrBs3. Many TAL domains are known to those of skill in the art and are commercially available.

[0156] TAL effectors comprise a central repeat domain of tandemly arranged repeats (the repeat-variable di-residues, RVD domain) that determine the specific binding of TAL effectors. These repeats are typically 33 or 34 amino acids. Different TAL effectors may have a different number of repeats (typically ranging from 1.5 to 33.5 repeats) and a different order of their repeats. The C-terminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “half-repeat”. Each repeat of the TAL effector generally correlates to one base-pair in the target DNA sequence with different repeat types exhibiting different base- pair specificity. A smaller number of repeats generally results in weaker protein-DNA interactions. A number of 6.5 repeats in a TAL effector has been shown to be sufficient to activate transcription of a reporter gene (Scholze et ah, 2010).

[0157] Many variations between repeats occur at amino acid positions 12 and 13, which have been termed "hypervariable" and are responsible for the specificity of the interaction with the target DNA promoter sequence, as shown in Table 4 listing exemplary repeat variable di- residues (RVD) and their corresponding nucleic acid base targets. Table 4— RVDs and Nucleic Acid Base Specificity FNWSGR Ref.65120-702.601 RG

[0158] The RVD NK has also been shown to target G. Many target sites of TAL effectors also include a T flanking the 5' base targeted by the first repeat.

[0159] In some embodiments, the TAL domain described herein may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al.2011), Xanthomonas campestris pv. raphani strain 756C and Xanthomonas oryzae pv. oryzzco / a strain BLS256 (Bogdanove et al.2011). In some embodiments, the TAL domain comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and / or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. It may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector. The TAL domain can be designed to target a given nucleic acid sequence based on Table 4 and other nucleic acid base specificities known in the art. The TAL domain of an epigenetic effector can comprise a number of TAL effector domains (e.g., repeats (monomers or modules)) selected based on the desired binding site to a target nucleic acid. TAL effector domains, e.g., repeats, may be removed or added in order to suit a specific binding target sequence. In some cases, the TAL domain of an epigenetic effector may comprise between 6.5 and 33.5 TAL effector domains, e.g., repeats. In some cases, TAL domain of an epigenetic effector may comprise between 8 and 33.5 TAL effector domains, between 10 and 25 TAL effector domains, or between 10 and 14 TAL effector domains. In some cases, the TAL domain of an epigenetic effector may comprise TAL effector domains that correspond to a perfect match to the DNA target sequence. In some cases, the TAL domain of an epigenetic effector may comprise a mismatch between a repeat and a target base-pair in the target nucleic acid as along as it allows for the function of the epigenetic effector comprising the TAL effector molecule. In some cases, the TAL domain of an epigenetic effector comprises no more than 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, 2 mismatches, or 1 mismatch, and optionally no mismatch, with the target DNA sequence. In general, TAL binding is inversely correlated with the number of mismatches. Without wishing to be bound by theory, in general the smaller the number of TAL effector domains in the TAL domain of the epigenetic effector, the smaller the number of mismatches will be tolerated and still allow for the function of the epigenetic effector comprising the TAL domain. The binding affinity of the TAL domain to the target nucleic acid is thought to depend on the sum of matching repeat-DNAWSGR Ref.65120-702.601 combinations. For example, TAL effector molecules having 25 TAL effector domains or more may be able to tolerate up to 7 mismatches.

[0160] In addition to the TAL effector domains, the TAL domain of an epigenetic effector may comprise additional sequences derived from a naturally occurring TAL effector. The length of the C-terminal and / or N-terminal sequence(s) included on each side of the TAL effector domain portion of the TAL domain can vary and be selected by one skilled in the art. For example, a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins have been characterized (See Zhang, Feng, et al. "Programmable sequence-specific transcriptional regulation of mammalian genome using designer TAL effectors." Nature biotechnology 29.2 (2011): 149, which is herein incorporated by reference) and key elements have been identified that contribute to optimal binding to the target sequence and activation of transcription. Transcriptional activity was generally found to inversely correlate with the length of N-terminus. On the C-terminus side, an important element for DNA binding residues was identified within the first 68 amino acids of the Hax 3 sequence. Accordingly, in some cases, the first 68 amino acids on the C-terminal side of the TAL effector domains of the naturally occurring TAL effector may be included in the TAL domain of the epigenetic effector. In some cases, a TAL domain in an epigenetic effector comprises 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side of the TAL effector domains; and / or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from the naturally occurring TAL effector on the C-terminal side of the TAL effector domains.

[0161] It is possible to modify the repeats to target specific DNA sequences. In some embodiments, the TAL effector domain of an epigenetic effector can be engineered to carry the epigenetic effector to desired target sites. Linkers

[0162] In some embodiments, a linker may connect the domains or moieties of the effectors systems described herein. In some cases, a linker may connect a polypeptide to another polypeptide. In some cases, a linker may connect a polypeptide to a nucleic acid. In some cases, a linker may connect a nucleic acid to another nucleic acid. In some cases, a linker connects the nucleic acid binding domain and the effector domain of an epigenetic effector. A linker may be aWSGR Ref.65120-702.601 chemical bond. In some cases, a linker may be a covalent bond. In other cases, a linker may be a noncovalent bond. In some cases, a linker may be a peptide linker. In some cases, a peptide linker may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length. In some cases, a linker may be a rigid linker. Many suitable rigid linkers may be found in Vagner, Josef, et al. "Rigid linkers for bioactive peptides." Bioconjugate chemistry 17.6 (2006): 1545-1550, which is hereby incorporated by reference in its entirety. Rigid linkers may comprise an alpha helix structure or Pro-rich sequence. Rigid linkers maintain a substantially fixed spatial distance between domains. In other cases, a linker may be a flexible linker. Flexible linkers may comprise small amino acids (e.g. Gly, Ser, or Ala). Flexible linkers allow the domains they connect to have flexibility of movement relative to each other. In some cases, a linker may be a cleavable linker. Cleavable linkers may utilize the reversible nature of a disulfide bond. In some cases, a cleavable linker comprises a cleavage site motif for a protease. In some cases, a cleavable linker may be a self-cleaving linker. In vivo cleavage of linkers in compositions described herein may be cleaved in specific conditions. Suitable cleavable linkers can be found in Leriche, Geoffray, Louise Chisholm, and Alain Wagner. "Cleavable linkers in chemical biology." Bioorganic & medicinal chemistry 20.2 (2012): 571-582, which is hereby incorporated by reference in its entirety. NLS (nuclear localization signal)

[0163] In some instances, a polypeptide described herein may comprise one or more nuclear localization sequence (NLS) (e.g., an SV40 NLS). In some cases, the one or more NLS facilitates the import of the polypeptide comprising an NLS into the cell nucleus. In some cases, the polypeptide may comprise 1 NLS. In some cases, the polypeptide may comprise 2 NLSs. In some cases, the polypeptide may comprise 3 NLSs. In other cases, the polypeptide may comprise more than 3, 4, 5, 6, 7, 8, 9, or 10 NLSs. In some cases, the NLS is located at the N- terminus, C-terminus, or in an internal region of the polypeptide. In some cases, an NLS is fused to the N-terminus of the nucleic acid binding domain of an epigenetic effector described herein. In some cases, an NLS is fused to the C-terminus of the nucleic acid binding domain of an epigenetic effector. In some cases, an NLS is fused to the N-terminus of the effector domain of an epigenetic effector. In some cases, an NLS is fused to the C-terminus of the effector domain of an epigenetic effector. In some cases, the nucleic acid binding domain of the epigenetic effector does not comprise an NLS. In some cases, the effector domain of the epigenetic effector does not comprise an NLS. In some cases, an NLS is fused to the N-terminus of a CRISPR / CasWSGR Ref.65120-702.601 effector protein. In some cases, an NLS is fused to the C-terminus of a CRISPR / Cas effector protein. Examples of NLS sequences are provided in Table 5 below. Table 5—Exemplary NLS SequencesPolynucleotides

[0164] The disclosure provides in part, polynucleotides encoding all epigenetic effectors, effector domains, nucleic acid binding domains, polypeptides, and functional RNAs disclosed herein. Suitable methods for design and construction of polynucleotides can be found in Ashwini, Malla, et al. "Advances in molecular cloning." Molecular Biology 50 (2016): 1-6, which is hereby incorporated by reference in its entirety. Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil. It will be understood that when a nucleotide sequence is represented by a DNA sequence (e.g., comprising, A, T, G, C), this disclosure also provides the corresponding RNA sequence (e.g., comprising, A, U, G, C) in which "U" replaces "T." In some embodiments, the extended IUPAC nomenclature used herein may be found in Johnson AD. An extended IUPAC nomenclature code for polymorphic nucleic acids. Bioinformatics. 2010 May 15;26(10):1386-9, which is hereby incorporated by reference in its entirety. Non- limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides,WSGR Ref.65120-702.601 branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0165] In some embodiments, a nucleic acid encoding a CasPhi domain comprises SEQ ID NO: 136. In some embodiments, a nucleic acid encoding a FN3Cas12a domain comprises SEQ ID NO: 137. In some embodiments, a nucleic acid encoding a dHKCas12a domain comprises SEQ ID NO: 138. In some embodiments, a nucleic acid encoding a dLanCas9 domain comprises SEQ ID NO: 139. In some embodiments, a nucleic acid encoding a dSauCas9 domain comprises SEQ ID NO: 140. In some embodiments, a nucleic acid encoding a dSpyCas9 domain comprises SEQ ID NO: 141. Polynucleotide encoding the system

[0166] The disclosure provides in part, polynucleotides encoding all sets of epigenetic effectors, effector domains, nucleic acid binding domains, editors, DNA polymerase domains, proteins, and functional RNAs disclosed herein. Vectors

[0167] The present disclosure is further directed, in part, to vectors, e.g., a viral vector and / or a non-viral vector. In some embodiments, the vector is a viral vector. Examples of viral vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. An expression vector may be used to express natural or synthetic nucleic acids by operably linking a nucleic acid encoding the gene of interest to a promoter. Vectors can be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence. Viral vectors, including those derived from retroviruses such as lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. An expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art, and described in a variety of virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses (AAV), herpes viruses, and lentiviruses.WSGR Ref.65120-702.601

[0168] An AAV can be (a) AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAV 10, (b) a chimera thereof, (c) a hybrid thereof, or (d) any combination of (a) to (c). One can select the type of AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver.

[0169] In certain instances, recombinant AAV (rAAV) may be used. rAAVs utilizes the cis- acting 145-bp ITRs to flank vector transgene cassettes, providing up to 4.5 kb for packaging of foreign DNA.

[0170] In some embodiments, a vector comprises an expression cassette comprising the nucleic acid encoding a protein or functional RNA. In some embodiments, the protein or functional RNA in the expression cassette is operatively linked to a promoter sequence that controls the expression of the protein or functional RNA. The present disclosure should not be interpreted to be limited to use of any particular promoter or category of promoters. In some embodiments, the promoter may be an inducible promoter that is capable of turning on expression of a polynucleotide sequence to which it is operatively linked, when such expression is desired. In some embodiments, the inducible promoter is capable of turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0171] In some embodiments, the vector comprising an expression cassette may contain a selectable marker gene (e.g. antibiotic resistance gene) or a reporter gene (e.g. luciferase, beta- galactosidase, green fluorescent protein gene) to facilitate identification and selection of cells containing the vector. Suitable expression systems can be found in Chen, Rachel. "Bacterial expression systems for recombinant protein production: E. coli and beyond." Biotechnology advances 30.5 (2012): 1102-1107, which is hereby incorporated by reference in its entirety, and may be prepared using known techniques or obtained commercially.

[0172] In some embodiments, the present disclosure provides a composition of a vector or vector set encoding an epigenetic effector, a system, an epigenetic modifying composition comprising a system, e.g., a first epigenetic effector and a second epigenetic effector, a composition comprising an epigenetic effector and an editor, or a polypeptide portion thereof. In some such embodiments, provided vectors may be or include DNA, RNA, e.g., mRNA, or any other nucleic acid moiety or entity as described herein, and may be prepared by anyWSGR Ref.65120-702.601 technology described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription, etc.). In some embodiments, provided nucleic acids that encode an epigenetic effector or epigenetic modifying composition or polypeptide portion thereof may be operationally associated with one or more replication, integration, and / or expression signals appropriate and / or sufficient to achieve integration, replication, and / or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.). In some embodiments, the composition of vectors comprises a set of one or more nucleic acids comprising a first nucleic acid segment encoding a first epigenetic effector and a second nucleic acid segment encoding a second epigenetic effector. In some embodiments, the nucleic acid may be a DNA. In some embodiments, the nucleic acid may be an RNA, e.g., an mRNA. In some embodiments, the vector or vector set comprises the first and second nucleic acid segments.

[0173] In some embodiments, the vector is a non-viral vector, e.g., liposome, exosome, lipid nanoparticle. In some embodiments, the vector may be selected from a lipid nanoparticle, a liposome, an exosome, and a micro vesicle. In some embodiments, the viral vector may be derived from an adenovirus, a retrovirus, an adeno-associated virus, a vaccinia virus, a lentivirus, a phage virus, a herpes simplex virus, or a polio virus. In some embodiments, the lipid nanoparticle may comprise an ionizable lipid. In some embodiments, the lipid nanoparticle further comprises one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids, polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, or polymer conjugated lipids.

[0174] In some embodiments, the vector may be provided as a component of a reaction mixture.

[0175] In some embodiments, the vector may be provided as a component of a composition comprising the vector and a pharmaceutically acceptable carrier. In some embodiments, the vector may be provided as a component of a culture comprising a cell. In some embodiments, the vector may be provided as a component of a production vector. Cells

[0176] Disclosed herein, in some embodiments, is one or more cells. In some embodiments, the one or more cells comprises an epigenetic effector or an epigenetic modifying composition of the present disclosure. In some embodiments, the one or more cells comprises a target nucleic acid for site-specific editing using the methods and systems of the present disclosure. Any cell or cell line, e.g., a cell line suitable for expression of a recombinant polypeptide, is suitable toWSGR Ref.65120-702.601 comprise an epigenetic effector or an epigenetic modifying composition described herein. In some embodiments, a cell or cell line may be used to express an epigenetic effector or a composition described herein. In some embodiments, a cell or cell line may be used to express or amplify a nucleic acid. For example, a cell or cell line can expression or amplify a vector encoding an epigenetic effector or a composition described herein. In some embodiments, a cell comprises a nucleic acid encoding an epigenetic effector a composition described herein.

[0177] In some embodiments, a cell comprises a first nucleic acid encoding a first component (e.g., a first epigenetic effector) of an epigenetic modifying composition and a second nucleic acid encoding a second component (e.g., a second epigenetic effector) of an epigenetic modifying composition. In some embodiments, wherein a cell comprises nucleic acid encoding an epigenetic modifying composition comprising two or more epigenetic effectors, the sequences encoding each epigenetic effector are disposed on separate nucleic acid molecules, e.g., on different vectors, e.g., a first vector encoding a first epigenetic effector and a second vector encoding a second epigenetic effector. In some embodiments, the sequences encoding each epigenetic effector are disposed on the same nucleic acid molecule, e.g., on the same vector. In some embodiments, some or all of the nucleic acid encoding the epigenetic modifying composition is integrated into the genomic DNA of the cell. In some embodiments, the nucleic acid encoding a first epigenetic effector of an epigenetic modifying composition is integrated into the genomic DNA of a cell, and the nucleic acid encoding a second epigenetic effector of an epigenetic modifying composition is not integrated into the genomic DNA of a cell (e.g., is situated on a vector). In some embodiments, the nucleic acid(s) encoding a first and a second epigenetic effector of an epigenetic modifying composition are integrated into the genomic DNA of a cell, e.g., at the same (e.g., adjacent or colocalized) or different sites in the genomic DNA. In some embodiments, a cell comprises a first nucleic acid encoding a first component (e.g., an epigenetic effector) of a composition and a second nucleic acid encoding a second component (e.g., an editor) of a composition.

[0178] Non-limiting examples of cells that may comprise and / or express an expression repression composition or expression repressor described herein include, but are not limited to, hepatocytes, neuronal cells, endothelial cells, myocytes, and lymphocytes.

[0179] The one or more cells of the present disclosure may be made by a method or process described herein. In some embodiments, the disclosure provides a cell produced by: providing an epigenetic effector or an epigenetic modifying composition described herein, providing the cell, and contacting the cell with the epigenetic effector (or a nucleic acid encoding theWSGR Ref.65120-702.601 epigenetic effector, or a composition comprising said epigenetic effector or nucleic acid) or the epigenetic modifying composition (or a nucleic acid encoding the epigenetic modifying composition, or a composition comprising said epigenetic modifying composition or nucleic acid). In some embodiments, contacting a cell with an epigenetic effector comprises contacting the cell with a nucleic acid encoding the epigenetic effector under conditions that allow the cell to produce the epigenetic effector. In some embodiments, contacting a cell with an epigenetic effector comprises contacting an organism that comprises the cell with the epigenetic effector or nucleic acid encoding the expression repressor under conditions that allow the cell to produce the epigenetic effector.

[0180] Without wishing to be bound by theory, cell contacted with an epigenetic effector or an epigenetic modifying composition described herein may exhibit: an increase in expression of a target gene and / or a decrease in expression of the same or different target gene and / or a modification of epigenetic markers associated with the target gene. In some embodiments, a cell exhibiting said increase and / or decrease expression of a target gene and / or modification of epigenetic markers does not comprise the epigenetic effector or the epigenetic modifying composition. The increase and / or decrease in expression and / or modification of epigenetic markers may persist, e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely) after contact with the epigenetic effector or the epigenetic modifying composition.

[0181] In some embodiments, a cell previously contacted by an epigenetic effector or an epigenetic modifying composition retains the increase and / or decrease in expression and / or modification of epigenetic markers after the epigenetic effector or an epigenetic modifying composition is no longer present in the cell, e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely) after the epigenetic effector or an epigenetic modifying composition is no longer present in the cell. Pharmaceutical compositions

[0182] Disclosed herein, in some embodiments, is one or more pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises one or more epigenetic effectorsWSGR Ref.65120-702.601 or one or more nucleic acids encoding the one or more epigenetic effectors. In some embodiments, the pharmaceutical composition comprises two or more epigenetic effectors, comprising a first epigenetic effector and a second epigenetic effector. In some embodiments, the pharmaceutical composition comprises one or more nucleic acid binding domains. In some embodiments, the pharmaceutical composition can be formulated with one or more pharmaceutically acceptable carriers.

[0183] In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; trans-dermally; or nasally, pulmonary, and / or to other mucosal surfaces. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0184] In some embodiments, pharmaceutical compositions may be formulated for delivery to a cell and / or to a subject via any route of administration. Modes of administration to a subject may include injection, infusion, inhalation, intranasal, intraocular, topical delivery, inter- cannular delivery, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intra-orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intra-cerebrospinal, and 20 intra-sternal injection and infusion.

[0185] In some embodiments, administration may be a single dose. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, administrations may be given as needed, e.g., for as long as symptoms associated with the disease, disorder or condition persist.WSGR Ref.65120-702.601

[0186] The dosage of the administered agent or composition can vary based on, e.g., the condition being treated, the severity of the disease, the subject's individual parameters, including age, size and weight, physiological condition, the type of treatment to be performed (if any), duration of treatment, the particular route of administration and similar factors. Thus, the dose administered of the agents described herein can depend on such various parameters. The dosage of an administered composition may also vary depending upon other factors such as the subject's general medical condition, and severity of the disorder to be treated. n some embodiments, the compositions disclosed herein may be administered in combination with one or more therapeutic agents or methods known in the arts.

[0187] In some aspects, the present disclosure provides methods of delivering a therapeutic comprising administering a composition as described herein to a subject, wherein an epigenetic effector is a therapeutic and / or wherein delivery of a therapeutic causes changes in gene expression relative to gene expression in absence of a therapeutic. Targeting sites

[0188] In some aspects, the present disclosure provides a target site. The target site can comprise one or more nucleotides that are targeted to be modified by an epigenetic effector described herein. For example, the target site can comprise a nucleotide is that targeted to be methylated or demethylated by the epigenetic effector. The target site can be in a genomic locus of a cell. In some embodiments, the target site is in a CpG island. The target site can be in or near a region targeted by a spacer sequence of a guide RNA of the epigenetic effector. In some embodiments, the target site is within the target sequence that is complementary to the spacer sequence of the guide RNA. In some embodiments, the target site is at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 bp away from the target sequence that is complementary to the spacer sequence of the guide RNA.

[0189] In some aspects, the present disclosure provides a target sequence. The target sequence can be a sequence in a target nucleic acid that an epigenetic effector described herein is configured to specifically interact with. For example, in some embodiments, the target sequence is complementary to a spacer sequence of a guide RNA of an epigenetic effector described herein. In some embodiments, the target sequence comprises a target site or a nucleotide that isWSGR Ref.65120-702.601 targeted to be modified by an epigenetic effector described herein. For example, the target sequence can comprise a nucleotide that is targeted to be methylated or demethylated by an epigenetic effector described herein. In some embodiments, the target sequence may be a DNA segment. In some embodiments, the target sequence may be an RNA segment, e.g., an mRNA segment. In some embodiments, the mRNA may be expressed from the genome of an organism. In some embodiments, the organism may be a prokaryote. In some embodiments, the prokaryote may be a bacterium. In some embodiments, the organism may be a eukaryote. In some embodiments, the eukaryote may be a vertebrate. In some embodiments, the vertebrate may be a mammal. In some embodiments, the mammal may be a human.

[0190] In some embodiments, the target sequence may be associated with a disease or a disorder.

[0191] In some embodiments, the disease is selected from a cancer, an immune disorder, e.g., autoimmune disorder, inflammatory diseases, a cardiovascular disorder, a neurological disorder, a fibrotic disorder, an eye disorder, a skin disorder, a kidney disorder, a developmental disorder, or a metabolic disorder. In some embodiments, the disease or disorder is associated with misregulation of a target gene. In some embodiments, the target gene expresses a transcription factor. In some embodiments, the transcription factor is selected from Oct3 / 4, Sox2, Klf4, nanog, c-Myc, l-myc, SP1, AP1, C / EBP, Heat shock factor, ATF / CREB, NF1, CCAAT, GATA, HNF, PIT-1, MyoD, SV40, Myf5, Hox, Winged Helix, SREBP, p53, CREB, Mef2, STAT, R- SMAD, NF-kB, Notch, TUBBY, NFAT, or a nuclear receptor. In some embodiments, the target sequence may be associated with a germline gene. In some embodiments, the germline gene may be misregulated. In some embodiments, the misregulation may be associated with a developmental defect or disorder.

[0192] In some aspects, the present disclosure provides an epigenetic modifying composition. In some embodiments, the epigenetic modifying composition comprises a first effector and a second effector. In some embodiments, the first effector binds to a target sequence associated with a first target gene and the second effector binds to a target sequence associated with a second target gene. In some embodiments, the first target gene expresses a first transcription factor selected from Oct3 / 4, Sox2, Klf4, nanog, c-Myc, l-myc, SV40, SP1, AP1, C / EBP, Heat shock factor, ATF / CREB, NF1, CCAAT, GATA, HNF, PIT-1, MyoD, Myf5, Hox, Winged Helix, SREBP, p53, CREB, Mef2, STAT, R-SMAD, NF-κB, Notch, TUBBY, NFAT, or a nuclear receptor, and the second target gene expresses a second transcription factor selected from Oct3 / 4, Sox2, Klf4, nanog, c-Myc, l-myc, SV40, SP1, AP1, C / EBP, Heat shock factor,WSGR Ref.65120-702.601 ATF / CREB, NF1, CCAAT, GATA, HNF, PIT-1, MyoD, Myf5, Hox, Winged Helix, SREBP, p53, CREB, Mef2, STAT, R-SMAD, NF-kB, Notch, TUBBY, NFAT, or a nuclear receptor. In some embodiments, the first effector is a first epigenetic effector, and the second effector is a second epigenetic effector. In some embodiments, the first effector is a first epigenetic effector, and the second effector is an editor. Methods

[0193] Disclosed herein, in some embodiments, are methods of making and using the compositions and systems of the present disclosure. In some embodiments, methods comprise reprogramming a cell. In some embodiments, methods of reprogramming a cell comprise: providing in a cell: (i) a first epigenetic effector comprising (I) a first CRISPR / Cas domain complexed with a first guide RNA and (II) a first effector moiety, wherein the first CRISPR / Cas domain complexed with the first guide RNA targets a first genomic locus in a genome of the cell; and (ii) a second epigenetic effector comprising a (I) second CRISPR / Cas domain complexed with a second guide RNA and (II) a second effector moiety, wherein the second CRISPR / Cas domain complexed with the second guide RNA targets a second genomic locus in the genome. In some embodiments, a first tracrRNA sequence of the first guide RNA and a second tracrRNA sequence of the second guide RNA have a phylogenetic distance of exceeding 0.09.

[0194] In some embodiments, the method further comprises one or more CpG islands. In some embodiments, the method further comprises methylating a first target site in the first genomic locus using the first epigenetic effector. In some embodiments, the method further comprises demethylating a second target site in the second genomic locus using the second epigenetic effector.

[0195] In some embodiments, the first epigenetic effector comprises a DNMT3A domain. The first epigenetic effector can further comprise a DNMT3L domain and a KRAB domain. In some embodiments, the second epigenetic effector comprises a TET1 domain. In some embodiments, the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. The first CRISPR / Cas domain and / or the second CRISPR / Cas domainWSGR Ref.65120-702.601 can be catalytically inactive. In some embodiments, the first CRISPR / Cas domain and the second CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135.

[0196] In some embodiments, the first guide RNA and / or the second sgRNA comprises a sequence selected from SEQ ID NOs.1-10. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 1, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0197] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ IDWSGR Ref.65120-702.601 NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 2, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0198] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0199] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5.WSGR Ref.65120-702.601 In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 3, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0200] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 4, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0201] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ IDWSGR Ref.65120-702.601 NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 5, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0202] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 6, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0203] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ IDWSGR Ref.65120-702.601 NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 7, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0204] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 8, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.WSGR Ref.65120-702.601

[0205] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 9, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 10.

[0206] In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 1. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 2. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 3. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 4. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 5. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 6. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 7. In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 8.WSGR Ref.65120-702.601 In some embodiments, the first guide RNA of the first epigenetic effector comprises SEQ ID NO: 10, and the second guide RNA of the second epigenetic effector comprises SEQ ID NO: 9.

[0207] In some embodiments, the first target site is in a first CpG island of the one or more CpG islands in the genome, and the second target site is in a second CpG island of the one or more CpG islands in the genome. The genome can comprise over 1,000, over 2,000, over 3,000, over 4,000, over 5,000, over 6,000, over 7,000, over 8,000, over 10,000, over 20,000, over 30,000, over 40,000, over 50,000, over 60,000, over 70,000, over 80,000, over 90,000, over 100,000, over 150,000, over 200,000, over 250,000, over 300,000, over 350,000, over 400,000, over 450,000, over 500,000, over 550,000, over 600,000, over 650,000, over 700,000, over 750,000, over 800,000, over 850,000, over 900,000, over 1,000,000, over 1,100,000, over 1,200,000, over 1,300,000, over 1,400,000, over 1,500,000, over 1,750,000, or over 2,000,000 copies of a first PAM sequence associated with the first CRISPR / Cas domain in the one or more CpG islands in the genome. The genome can comprise over 1,000, over 2,000, over 3,000, over 4,000, over 5,000, over 6,000, over 7,000, over 8,000, over 10,000, over 20,000, over 30,000, over 40,000, over 50,000, over 60,000, over 70,000, over 80,000, over 90,000, over 100,000, over 150,000, over 200,000, over 250,000, over 300,000, over 350,000, over 400,000, over 450,000, over 500,000, over 550,000, over 600,000, over 650,000, over 700,000, over 750,000, over 800,000, over 850,000, over 900,000, over 1,000,000, over 1,100,000, over 1,200,000, over 1,300,000, over 1,400,000, over 1,500,000, over 1,750,000, or over 2,000,000 copies of a second PAM sequence associated with the second CRISPR / Cas domain in the one or more CpG islands in the genome. In some embodiments, the first PAM sequence and / or the second PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

[0208] In another aspect, the present disclosure provides a method of reprogramming a cell, the method comprising: (a) providing in a cell comprising a genome: (i) a first plurality of epigenetic effectors, each comprising (I) a first CRISPR / Cas domain and (II) a first effector moiety, wherein the first CRISPR / Cas domain is capable of specifically interacting with over 70,000 first targetable PAM sites in a first plurality of CpG islands in the genome. The method can further comprise providing in the cell: (ii) a second plurality of epigenetic effectors, each comprising a (I) second CRISPR / Cas domain and (II) a second effector moiety, wherein the second CRISPR / Cas domain is capable of specifically interacting with over 70,000 second targetable PAM sites in a second plurality of CpG islands in the genome.WSGR Ref.65120-702.601

[0209] In some embodiments, the first CRISPR / Cas domain of each of the first plurality of epigenetic effectors is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas domain of each of the second plurality of epigenetic effectors is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. In some embodiments, the first CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135, and the second CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135

[0210] In some embodiments, the first CRISPR / Cas domain is capable of specifically interacting with over 80,000, over 90,000, over 100,000, over 150,000, over 200,000, over 250,000, over 300,000, over 350,000, over 400,000, over 450,000, over 500,000, over 550,000, over 600,000, over 650,000, over 700,000, over 750,000, over 800,000, over 850,000, over 900,000, over 1,000,000, over 1,100,000, over 1,200,000, over 1,300,000, over 1,400,000, over 1,500,000, over 1,750,000, or over 2,000,000 first targetable PAM sites in the first plurality of CpG islands in the genome. In some embodiments, the second CRISPR / Cas domain is capable of specifically interacting with over 80,000, over 90,000, over 100,000, over 150,000, over 200,000, over 250,000, over 300,000, over 350,000, over 400,000, over 450,000, over 500,000, over 550,000, over 600,000, over 650,000, over 700,000, over 750,000, over 800,000, over 850,000, over 900,000, over 1,000,000, over 1,100,000, over 1,200,000, over 1,300,000, over 1,400,000, over 1,500,000, over 1,750,000, or over 2,000,000 second targetable PAM sites in the second plurality of CpG islands in the genome. In some embodiments, the first targetable PAM sites each comprise a sequence selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. In some embodiments, the second targetable PAM sites each comprise a sequence selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

[0211] In some embodiments, the method further comprises providing the cell: (iii) a first plurality of guide RNAs capable of binding to the first CRISPR / Cas domain in each epigenetic effector of the first plurality of epigenetic effectors, wherein the first plurality of guide RNAs target different CpG islands of the first plurality of CpG islands in the genome; and (iv) a second plurality of guide RNAs capable of binding to the second CRISPR / Cas domain in each epigenetic effector of the second plurality of epigenetic effectors, wherein the second plurality of guide RNAs target different CpG islands of the second plurality of CpG islands in the genome.WSGR Ref.65120-702.601

[0212] In some embodiments, the first plurality of guide RNAs comprise a first common tracrRNA sequence, and wherein the second plurality of guide RNAs comprise a second common tracrRNA sequence, wherein the first common tracrRNA sequence and the second common tracrRNA sequence have a phylogenetic distance of exceeding 0.09. In some embodiments the first common tracrRNA sequence and the second common tracrRNA sequence have a phylogenetic distance of exceeding 0.05, exceeding 0.1, exceeding 0.12, exceeding 0.14, exceeding 0.16, exceeding 0.18, exceeding 0.2, exceeding 0.22, exceeding 0.24, exceeding 0.26, exceeding 0.28, exceeding 0.3, exceeding 0.35, or exceeding 0.4. The first plurality of guide RNAs can each comprise a first common sequence selected from SEQ ID NOs.1-10. In some embodiments, the second plurality of guide RNAs each comprises a first common sequence selected from SEQ ID NOs.1-10.

[0213] The method can further comprise (b) methylating a first plurality of target sites in the first plurality of CpG islands in the genome using the first plurality of epigenetic effectors. In some embodiments, the first plurality of target sites are in a same gene in the genome. In other embodiments, the first plurality of target sites are in different genes in the genome. In some embodiments, methylating the first plurality of target sites represses expression of a first gene.

[0214] The method can further comprise (c) demethylating a second plurality of target sites in the second plurality of CpG islands in the genome using the second plurality of epigenetic effectors. In some embodiments, the second plurality of target sites are in a same gene in the genome. In other embodiments, the second plurality of target sites are in different gene in the genome. In some embodiments, demethylating the second plurality of target sites enhances expression of a second gene.

[0215] In some embodiments, the first effector moiety of each of the first plurality of epigenetic effectors comprises a DNMT3A domain. The first effector moiety of each of the first plurality of epigenetic effectors can further comprise a DNMT3L domain and a KRAB domain. In some embodiments, the second effector moiety of each of the second plurality of epigenetic effectors comprises a TET1 domain.

[0216] In another aspect, the present disclosure provides a method of reprogramming a cell, the method comprising: (a) providing in a cell comprising: (i) a first epigenetic effector comprising (I) a SpyCas9 domain complexed with a first guide RNA and (II) a demethylase, wherein the SpyCas9 domain complexed with the first guide RNA targets a first genomic locus in a genome of the cell; and (ii) a second epigenetic effector comprising a (I) SauCas9 domain complexedWSGR Ref.65120-702.601 with a second guide RNA and (II) a methylase, wherein the CasuCas9 domain complexed with the second guide RNA targets a second genomic locus in the genome; (b) demethylating a first target site in the first genomic locus using the first epigenetic effector; and (c) methylating a second target site in the second genomic locus using the second epigenetic effector. In some embodiments, the demethylase comprises a TET1 domain. In some embodiments, the methylase comprises a DNMT3A domain. In some embodiments, the second epigenetic effector further comprises a DNMT3L domain or a KRAB domain. In some embodiments, the genome comprises one or more CpG islands. In some embodiments, the first target site is in a first CpG island of the one or more CpG islands in the genome, and wherein the second target site is in a second CpG island of the one or more CpG islands in the genome.

[0217] In some aspects, the present disclosure provides methods of identifying a set of CRISPR / Cas proteins comprising a first CRISPR / Cas protein and a second CRISPR / Cas protein wherein the first CRISPR / Cas protein and the second CRISPR / Cas protein do not cross-react. In some embodiments, identifying the set of CRISPR / Cas proteins comprises (a) obtaining two or more distinct CRISPR / Cas proteins, (b) determining sequence homology of the CRISPR / Cas proteins of step (a), (c) selecting the first CRISPR / Cas protein and the second CRISPR / Cas protein wherein the sequence of a first tracrRNA associated with the first CRISPR / Cas protein and the sequence of a second tracrRNA associated with the second CRISPR / Cas protein have a phylogenetic distance above 0.2. In some embodiments, predicted cross-reactivity negatively correlates with phylogenetic distance. Phylogenetic distance between protein sequences may be measured using the BLOSUM 62 matrix excluding indels.

[0218] In some aspects, the present disclosure provides methods of modifying a cell. In some embodiments, a set of CRISPR / Cas proteins identified pursuant to any of the methods described herein or a composition of any of the compositions described herein is simultaneously delivered to the cell. In some embodiments, the set of CRISPR / Cas proteins or the composition of any of the compositions described herein is simultaneously delivered to an organism comprising the cell.

[0219] In some embodiments, one or more genomic sequences are modified in a cell. In some embodiments, the one or more genomic sequences are modified by simultaneously contacting the cell with a system described herein. In some embodiments, the one or more genomic sequences are modified by sequentially contacting the cell with the epigenetic effectors. In some embodiments, the cell is contacted with a first epigenetic effector with methylation activity and a second epigenetic effector with methylation activity. In some embodiments, the first and secondWSGR Ref.65120-702.601 epigenetic effectors have the same methylation activity. In some embodiments, the first and second epigenetic effectors different methylation activity.

[0220] In some embodiments, the cell is reprogrammed at multiple locations of the cell's genome. In some embodiments, the reprogramming comprises: (a) selecting a first CRISPR / Cas protein and a second CRISPR / Cas protein wherein the first CRISPR / Cas protein and the second CRISPR / Cas protein do not cross-react, (b) preparing a system, the preparing comprising: (c) preparing a first epigenetic effector capable of binding to a first site of the cell genome and comprising the first CRISPR / Cas protein and a first effector moiety; (d) preparing a second epigenetic effector capable of binding to a second site of the cell genome and comprising the second CRISPR / Cas protein and a second effector moiety; and (e) optionally, combining the first and second epigenetic effectors in a composition; and (f) contacting the cell with the first and the second epigenetic effector to reprogram the cell; wherein the first epigenetic effector and the second epigenetic effector bind to different locations of the cell genome.

[0221] In some embodiments, the cell is modified by modulating its activity. In some embodiments, the cell’s activity is modulated by modulating the methylation profile of the genome of the cell. In some embodiments, the methylation profile is modulated simultaneously. In some embodiments, the methylation profile is modulated sequentially. In some embodiments, methylation profile is modulated at multiple sites within the genome. In some embodiments, the modulation comprises demethylation at one or more sites and methylation at one or more other sites.

[0222] In some aspects, the present disclosure provides methods of treating or preventing a disease in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject. In some embodiments the pharmaceutical composition comprises a cell or a progeny of the cell, wherein the activity of the cell is modulated according to one or more methods described herein or using one or more compositions or epigenetic effectors described herein. In some embodiments, the activity of the cell is modulated simultaneously. In some embodiments, the activity of the cell is modulated sequentially.

[0223] In some embodiments, the epigenetic effector is capable of reprogramming a diseased cell into a healthy cell. In some embodiments, the epigenetic effector modulates the methylation profile of the genome of the diseased cell.WSGR Ref.65120-702.601

[0224] In some aspects, the present disclosure provides methods for directing DNA modification to two or more specific genetic loci in a cell. In some embodiments, at least two epigenetic effectors are administered to the cell. In some embodiments, the at least two epigenetic effectors are administered to an organism comprising the cell. In some embodiments, the at least two epigenetic effectors are administered to a composition of cells comprising the cell. In some embodiments, the at least two epigenetic effectors are administered simultaneously. In some embodiments, the at least two epigenetic effectors are administered sequentially. In some embodiments, the first and the second epigenetic effector are not identical. In some embodiments, the DNA modification comprises DNA methylation. In some embodiments, the DNA modification comprises DNA demethylation. In some embodiments, the DNA modification comprises histone methylation. In some embodiments, the DNA modification comprises histone demethylation. In some embodiments, the cell is contacted with one or more methylation agents. In some embodiments, the cell is contacted with one or more demethylation agents. In some embodiments, the cell is contacted with a combination of one or more methylation agents and one or more demethylation agents.

[0225] In some aspects, the present disclosure provides methods of modulating the expression level for two or more chronological markers in a cell. In some embodiments, the expression level is modulated by modulating the methylation profile of the cell in an amount and for a period of time sufficient to modulate the expression level of said at least two chronological markers. In some embodiments, modulating the methylation profile comprises modulating nucleic acid methylation. In some embodiments, modulating the methylation profile comprises modulating histone methylation. In some embodiments, the cell is contacted with one or more methylation agents. In some embodiments, the cell is contacted with one or more demethylation agents. In some embodiments, the cell is contacted with a combination of one or more methylation agents and one or more demethylation agents.

[0226] In some aspects, the present disclosure provides methods of transforming a cell from one cell type to another. In some embodiments, the cell is contacted with one or more methylation agents. In some embodiments, the cell is contacted with one or more demethylation agents. In some embodiments, the cell is contacted with a combination of one or more methylation agents and one or more demethylation agents. In some embodiments, the transformation or modulation may be from pluripotent stem cell to adult cell. In some embodiments, the transformation or modulation may be from adult cell to pluripotent stem cell. In some embodiments, the transformation or modulation may be from one stem cell type to another stem cell type. In someWSGR Ref.65120-702.601 embodiments, the transformation or modulation may be from one adult cell type to another adult cell type. In some embodiments, the transformation or modulation may be from one adult somatic cell type to another adult somatic cell type. In some embodiments, the transformation or modulation may be from one blood cell type to another blood cell type. In some embodiments, the transformation or modulation may be from one immune cell type to another immune cell type. In some embodiments, the transformation or modulation may comprise inducing a change in epigenetic state, chromatin structure, transcription, mRNA splicing, post-transcriptional modification, mRNA stability and / or half-life, translation, post-translational modification, protein stability and / or half-life and / or protein activity of the at least one component of the cell. In some embodiments, the somatic cell being transformed or modulated may be selected from a fibroblast cell, a liver cell, a cardiac cell, a CNS cell, a PNS cell, a kidney cell, a lung cell, a hematopoietic cell, a pancreatic beta cell, a bone cell, a skeletal muscle cell, a skin cell, an immune cell, a follicular cell, a vascular cell, a neural cell, an osteoblast cell, an osteoclast cell, and an endothelial cell. In some embodiments, the somatic cell being produced by the transformation or modulation may be selected from a fibroblast cell, a liver cell, a cardiac cell, a CNS cell, a PNS cell, a kidney cell, a lung cell, a hematopoietic cell, a pancreatic beta cell, a bone cell, a skeletal muscle cell, a skin cell, an immune cell, a follicular cell, a vascular cell, a neural cell, an osteoblast cell, an osteoclast cell, and an endothelial cell. In some embodiments, the cell may be isolated from an in-vivo tissue in a subject. In some embodiments, the cell may be isolated from a biopsy. In some embodiments, the cell may be isolated from a cancer biopsy. In some embodiments, the cell may be isolated from a blood sample. In some embodiments, the contacting step may be accomplished in-vitro. In some embodiments, the contacting step may be accomplished in-vivo. In some embodiments, the contacting step may be accomplished ex-vivo.

[0227] In some embodiments, the modified, transformed or modulated cell, e.g., the modified, transformed or modulated cell disclosed herein and / or their progeny may be administered to a subject. In some embodiments, the modified, transformed or modulated cells, e.g., the modified, transformed or modulated cell disclosed herein and / or their progeny may be used as input into processes for further modifying such cells. In some embodiments, the modified, transformed or modulated cells, e.g., the modified, transformed or modulated cell disclosed herein and / or their progeny may be used as input into processes for further modifying such cells to produce therapeutic cells. In some embodiments, the cell may be obtained from a subject having a disease. In some embodiments, the disease may be selected from a neurological disease, a cancer, a hormonal disease, an imprinting disease, an inflammatory disease, or anWSGR Ref.65120-702.601 infection. In some embodiments, the infection may be a chronic infection. In some embodiments, the chronic infection may be a viral or bacterial chronic infection.

[0228] In some embodiments, a method of epigenetically modifying a target nucleic acid sequence may modulate (e.g., increase or decrease) the level of the epigenetic modification by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100%) relative to the level of the epigenetic modification at that site in a cell not contacted by the epigenetic effector or the composition or treated with the method. In some embodiments, an epigenetic modification produced by a method described herein persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or longer or any time there between. In some embodiments, an epigenetic modification produced by a method described herein persists no longer than 1 hour to about 30 days or no longer than 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or longer or any time there between. Kits

[0229] In one aspect, a kit comprising an epigenetic effector or a composition, e.g., a composition comprising a first epigenetic effector and a second epigenetic effector, e.g., a composition comprising an epigenetic effector and an editor described herein, or nucleotides encoding the same. In some embodiments, the kit further comprises instructions for the use of said epigenetic effector or the composition.

[0230] In one aspect, the disclosure provides a system comprising multiple epigenetic effector reagents, robotics arranged to deliver multiple epigenetic effector reagents to one or more cells, and a computer system programmed to (i) control the robotics and (ii) select one or more sets of epigenetic effector reagents that do not substantially cross-react. ENUMERATED EMBODIMENTS 1. A system for reprogramming a cell, the composition comprising:WSGR Ref.65120-702.601 (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein and the second CRISPR / Cas protein do not cross- react substantially. 2. A system for reprogramming a cell, the composition comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein a first tracrRNA sequence interacting with the first CRISPR / Cas protein and the second tracrRNA sequence interacting with the second CRISPR / Cas protein have a phylogenetic distance of exceeding 0.2. 3. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein both the first CRISPR / Cas protein comprises one or more number of PAM sites in a CpG island. 4. The system of embodiment 3, wherein the first CRISPR / Cas protein comprises a higher number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. 5. The system of embodiment 3, wherein the first CRISPR / Cas protein comprises an equivalent number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. 6. The system of embodiment 3, wherein the first CRISPR / Cas protein comprises a lower number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein.WSGR Ref.65120-702.601 7. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein may be selected from AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1, Cpf1, FnCas12a, LbCas12a, LbCas12a, TsCas12a, SaCas12a, Pb2Cas12a, PgCas12a, MiCas12a, Mb2Cas12a, Mb3Cas12a, Lb4Cas12a, Lb5Cas12a, FbCas12a, CrbCas12a, CpbCas12a, CMaCas12a, BsCas12a, BfCas12a, BoCas12a and the second CRISPR / Cas protein may be selected from AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsaCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1, Cpf1, FnCas12a, LbCas12a, LbCas12a, TsCas12a, SaCas12a, Pb2Cas12a, PgCas12a, MiCas12a, Mb2Cas12a, Mb3Cas12a, Lb4Cas12a, Lb5Cas12a, FbCas12a, CrbCas12a, CpbCas12a, CMaCas12a, BsCas12a, BfCas12a, BoCas12a. 8. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety;WSGR Ref.65120-702.601 (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and the second CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1. 9. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, and LmoCas9, and the second CRISPR / Cas protein may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a. 10. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein may be selected from SpyCas9, LanCas9, Sag1Cas9, and LmoCas9, and the second CRISPR-Cas protein may be selected from Casphi-2 and Cas12c-1. 11. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety;WSGR Ref.65120-702.601 wherein the first CRISPR / Cas protein may be selected from Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a; and the second CRISPR / Cas protein may be selected from Casphi-2 and Cas12c-1. 12. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein may be selected from CasPhi-2 and Pb2Cas12a and the second CRISPR / Cas protein may be selected from CasPhi-2. 13. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein comprises a PAM sequence selected from NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG and the second CRISPR / Cas protein comprises a PAM sequence selected from NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT,WSGR Ref.65120-702.601 NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. 14. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG and the second CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG. 15. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein comprises a PAM sequence selected from TTTV, YYV, KKYV, YTV, and NYTV. 16. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein comprises a PAM sequence selected from NGG, NRG, and NBGG, and the second CRISPR / Cas protein comprises a PAM sequence selected from TBN and TG. 17. A system for reprogramming a cell, the system comprising:WSGR Ref.65120-702.601 (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein comprises a PAM sequence selected from TTTV, YYV, KKYV, YTV, and NYTV and the second CRISPR / Cas protein comprises a PAM sequence selected from TBN and TG. 18. A system for reprogramming a cell, the system comprising: (a) a first epigenetic effector comprising a first CRISPR / Cas protein and a first effector moiety; (b) a second epigenetic effector comprising a second CRISPR / Cas protein and a second effector moiety; wherein the first CRISPR / Cas protein comprises a PAM sequence TBN, and NYTV and the second CRISPR / Cas protein comprises a PAM sequence TG. 19. The system of any preceding embodiments, further comprising a first sgRNA and a second sgRNA. 20. The system of any preceding embodiments, wherein the first sgRNA and the second sgRNA may be selected such that it does not cross react substantially with the other. 21. The system of embodiments 19 or 20, wherein the first sgRNA or second sgRNA may comprise a sequence selected from SEQ ID NO: 1-4 at 3’ end. 22. The system of embodiments 19 or 20, wherein, the first sgRNA or second sgRNA may comprise a sequence selected from SEQ ID NO: 5-10 at 5’ end. 23. A system comprising a first tracrRNA sequence interacting with the first CRISPR / Cas protein and the second tracrRNA sequence interacting with the second CRISPR / Cas protein have a phylogenetic distance of exceeding 0.2. 24. A system comprising a first CRISPR / Cas protein and a second CRISPR / Cas protein wherein the first CRISPR / Cas protein comprises a higher number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein.WSGR Ref.65120-702.601 25. A system comprising a first CRISPR / Cas protein and a second CRISPR / Cas protein wherein the first CRISPR / Cas protein comprises a lower number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. 26. A system comprising a first CRISPR / Cas protein and a second CRISPR / Cas protein wherein the first CRISPR / Cas protein comprises an equivalent number of PAM sites in CpG islands compared to that of the second CRISPR / Cas protein. 27. A system comprising a first CRISPR / Cas protein and a second CRISPR / Cas protein wherein the first CRISPR / Cas protein may be selected from Lan, Lmo and Sag1 and the second CRISPR / Cas protein may be a Spy Cas9. 28. The system of any of the embodiments described herein further comprising a first guide RNA and a second guide RNA. 29. An epigenetic effector comprising a nucleic acid binding moiety and an effector moiety, wherein the effector moiety may be capable of altering methylation profile of a genome of a cell. 30. An epigenetic effector comprising a nucleic acid binding moiety and an effector moiety, wherein the effector moiety may be capable of adding or removing a methyl group in a target nucleic acid. 31. An epigenetic effector comprising a nucleic acid binding moiety and an effector moiety, wherein the effector moiety may be capable of enhancing or repressing methylation in a target nucleic acid. 32. An epigenetic effector comprising a nucleic acid binding moiety and an effector moiety, wherein the effector moiety may be capable of methylating or demethylating at least one CpG site of a target nucleic acid. 33. The epigenetic effector of any of embodiments 29-32, wherein the epigenetic effector targets one or more PAM sites in the CpG island. 34. The epigenetic effector of any of the embodiments 29-33, wherein the epigenetic effector may bind to the target nucleic acid. 35. The epigenetic effector of any of the embodiments 29-34, wherein the target nucleic acid is a CpG site, optionally located in a CpG island.WSGR Ref.65120-702.601 36. The epigenetic effector of any of the embodiments 29-34, wherein the CpG island is located in a transcription regulatory element. 37. The epigenetic effector of embodiment 36, wherein the transcription regulatory element is selected from a promoter, an enhancer, or a transcription start site operably linked to a gene. 38. The epigenetic effector of any of the embodiments 29-34, wherein the target nucleic acid may be a transcription regulatory element. 39. The epigenetic effector of any of the embodiments 29-34, wherein target nucleic acid may be a promoter, an enhancer, or a transcription start site operably linked to a gene. Nucleic acid binding moiety 40. The epigenetic effector of any of preceding embodiments, wherein the epigenetic effector may comprise a CRISPR / Cas domain, a zinc finger domain, a TAL domain, a tetR domain, a meganuclease, or an oligonucleotide. 41. The epigenetic effector of any of preceding embodiments, wherein the nucleic acid binding moiety of the epigenetic effector may comprise a CRISPR / Cas domain. 42. The epigenetic effector of any of preceding embodiments, wherein the CRISPR / Cas domain may comprise a CRISPR / Cas protein. 43. The epigenetic effector of embodiment 43, wherein the CRISPR / Cas protein may be selected from a type I, type II, type III, type IV, type V Cas protein, and type VI Cas protein. 44. The epigenetic effector of embodiment 42 or 43, wherein the CRISPR / Cas protein may be catalytically inactive. 45. The epigenetic effector of any of embodiments 42-44, wherein the CRISPR / Cas protein may be selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, AsCas12a, Cas13a, Cas13b, Cas13c, Cas13d, Cas13X, Cas13Y, LbCas12a, HypaCas9, a Type I CasWSGR Ref.65120-702.601 effector protein, a Type II Cas effector protein, a Type III Cas effector protein, a Type IV Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, Cas λ, and functional fragments and derivatives thereof. 46. The epigenetic effector of any of embodiments 42-45, wherein the CRISPR / Cas protein may be or comprise a Cas9 ortholog. 47. The epigenetic effector of any of embodiments 42-46, wherein the CRISPR / Cas may be or comprises a Cas9 protein, the Cas9 protein may be selected from SpCas9, SaCas9, ScCas9, StCas9, NmCas9, VRERCas9, VERCas9, xCas9, espCas91.0, espCas1.1, Cas9HF1, hypaCas9, evoCas9, HiFiCas9, and CjCas9. 48. The CRISPR / Cas protein of any of the epigenetic effectors of the embodiments may be or may comprise a Cas9 protein or a functional equivalent. 49. The CRISPR / Cas protein of any of the epigenetic effectors of the embodiments may be or may comprise a Cas12 protein or a functional equivalent. 50. The epigenetic effector of any preceding embodiments, wherein the CRISPR / Cas protein may be or may comprise a Cas12 ortholog. 51. The epigenetic effector of any preceding embodiments, wherein the Cas12 protein may be selected from Cpf1, FnCas12a, LbCas12a, AsCas12a, LbCas12a, TsCas12a, SaCas12a, Pb2Cas12a, PgCas12a, MiCas12a, Mb2Cas12a, Mb3Cas12a, Lb4Cas12a, Lb5Cas12a, FbCas12a, CrbCas12a, CpbCas12a, CMaCas12a, BsCas12a, BfCas12a, BoCas12a, Cas12j, or Cas12c. 52. The epigenetic effector of any preceding embodiments, wherein the CRISPR / Cas protein may be derived from a bacteria or has one or more components derived from a bacteria, and wherein the one or more components may optionally be derived from different bacteria. 53. The system of any of the embodiments described comprising CRISPR / Cas proteins derived from, or having components derived from, two or more different bacteria species. 54. The system of any of the embodiments described comprising CRISPR / Cas proteins derived from, or having components derived from, two or more different bacteria genera.WSGR Ref.65120-702.601 55. The epigenetic effector of any preceding embodiments, wherein the bacteria origin of the CRISPR / Cas protein of each of the epigenetic effectors may be selected from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Bacillus niameyensis, Bacillus okhensis, Capnocytophaga canis, Chryseobacterium gallinarum, Coriobacterium_glomerans_PW2, Dechloromonas denitrificans, Enterococcus cecorum, Enterococcus faecium, Enterococcus italicus, Eubacterium dolichum, Eubacterium sp., Eggerthella sp. YY7918, Exiguobacterium sibiricum, Flavobacterium frigidarium, Facklamia hominis, Finegoldia_magna_ATCC_29328, Kingella kingae, Lactobacillus_rhamnosus_LOCK900, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus sp., Microscilla marina, Mycoplasma_gallisepticum_CA06, Neisseria meningitidis, Ornithobacterium rhinotracheale, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Pediococcus acidilactici, Prevotella histicola, Parabacteroides sp., Streptococcus_agalactiae_NEM316, Streptococcus_dysgalactiae_subsp._equisimilis_AC-2713, Streptococcus equinus, Streptococcus gallolyticus, Streptococcus gordonii, Streptococcus mutans GS-5, Streptococcus macedonicus, Streptococcus ratti, Streptococcus_salivarius_JIM8777, Streptococcus sinensis, Streptococcus suis D9, Streptococcus thermophilus LMG 18311, Tissierellia bacterium KA00581, Treponema denticola ATCC 35405, Treponema putidum, Turicibacter sp., Veillonella parvula ATCC 17745, Weeksella virosa, Streptococcus equi, Streptococcus agalactiae, Lactobacillus animalis KCTC 3501, Listeria monocytogenes, Lachnospiraceae bacterium ND2006, Acidaminococcus sp. BV3L6, Helcococcus kunzii, Prevotella ihumii, Prevotella bryantii B14, Biggievirus strain Mos11, Compost_meta_-_Ga0079224_100045232_-_CRISPR- associated_protein,_Csn1_family_CDS_translation_Compost_meta, Geyser- Hotspring_Yellowstone_Ga0078972_1022257_-_CRISPR- associated_protein,_Csn1_family_CDS_translation_Community_metagenome, Geyser- Hotspring_Yellowstone_Ga0078972_1010018_-_CRISPR- associated_protein,_Csn1_family_CDS_translation_Community_metagenome, Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii,WSGR Ref.65120-702.601 Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Clostridium Tyrobutyricum, Clostridium beijerinckii, Clostridium perfringens, Clostridium autoethanogenum, Finegoldia magna, Natranaerobius thermophilus, Methanococcus maripaludis, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Lactobacillus crispatus, Acidithiobacillus ferrooxidans, Acidaminococcus intestine RyC-MR95, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Streptococcus thermophilus, Lactococcus lactis, Staphylococcus epidermidis Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Clostridium acetobutylicum , Synechococcus elongatus UTEX 2973, Actinoplanes sp., B. subtilis, Corynebacterium glutamicum, Streptomyces sp., Clostridium difficile, Clostridium saccharoperbutylacetonicum N1–4, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. 56. The epigenetic effector of any preceding embodiments, wherein the CRISPR / Cas protein may be derived from a virus or has one or more components derived from a virus, and wherein the one or more components may optionally be derived from different virus. 57. The system of any of the embodiments described comprising CRISPR / Cas proteins derived from, or having components derived from, two or more different virus. 58. The system of any of the embodiments described comprising CRISPR / Cas proteins derived from, or having components derived from, two or more different virus. 59. The epigenetic effector of any preceding embodiments, wherein the viral origin of the CRISPR / Cas protein of each of the epigenetic effectors may be selected from bacteriophages. 60. The nucleic acid binding moiety of the epigenetic effector may comprise a zinc finger domain. 61. The zinc finger domain may comprise or consist essentially of or consist of 1-10, 1-9, 1- 8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7- 10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers. 62. The nucleic acid binding moiety of the epigenetic effector may comprise a TAL domain.WSGR Ref.65120-702.601 63. The TAL domain of embodiment 60, wherein the TAL domain comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats. 64. The effector moiety may be a durable effector moiety. 65. The effector moiety may be a transient effector moiety. 66. The effector moiety of the epigenetic effector may be or may comprise a moiety capable of effecting methylation profile of a DNA or histone. 67. The effector moiety of the epigenetic effector of any preceding embodiments, wherein the effector moiety is capable of increasing or decreasing a gene expression. 68. The effector moiety of the epigenetic effector of any preceding embodiments, wherein the effector moiety is selected from a DNA methylase, a DNA demethylation enzyme, a histone methyltransferase, a histone deacetylase, a histone demethylase, a protein-lysine N-methyltransferase, or a transcription repressor moiety. 69. The effector moiety of the epigenetic effector of any preceding embodiments, wherein the effector moiety is selected from a DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, TET1, TET2, TET3, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, or KAT13C, or a functional equivalent. 70. The effector moiety of the epigenetic effector of embodiment 64, wherein the effector moiety is capable of DNA methylation. 71. The effector moiety of the epigenetic effector may be a DNA methyltransferase or a functional equivalent thereof. 72. The DNA methyltransferase may be selected from m6A methyltransferase, an m4C methyltransferase, and an m5C methyltransferase. 73. The DNA methyltransferase may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, or a functional equivalent thereof.WSGR Ref.65120-702.601 74. The effector moiety of the epigenetic effector of embodiment 64, wherein the effector moiety is capable of DNA demethylation. 75. The effector moiety may be selected from TET1, TET2, and TET3 or a functional equivalent thereof. 76. The effector moiety of the epigenetic effector may be or comprise a histone methyltransferase or a functional equivalent thereof. 77. The histone methyltransferase may be selected from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional equivalent thereof. 78. The effector moiety of the epigenetic effector may be or comprise a histone demethylase. 79. The histone demethylase may be selected from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional equivalent thereof. 80. The effector moiety of the epigenetic effector may be or comprise a histone acetyltransferase or a functional equivalent thereof. 81. The histone acetyltransferase may be selected from KAT1, KAT2A, KAT3A, KAT3B, KAT13C, or a functional equivalent thereof. 82. The effector moiety of the epigenetic effector may be or comprise a histone deacetylase. 83. The histone deacetylase may be selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional equivalent of any thereof. 84. The effector moiety of the epigenetic effector may be or comprise a transcription repressor moiety. 85. The effector moiety may comprise a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional equivalent. 86. The epigenetic effector of any preceding embodiments, further comprising a linker. 87. The linker of any preceding embodiments, wherein the linker is situated between the nucleic acid binding moiety and the effector moiety.WSGR Ref.65120-702.601 88. The linker of any preceding embodiments, wherein the linker is a cleavable linker. 89. The linker of any preceding embodiments, wherein the linker is a non-cleavable linker. 90. The linker of any preceding embodiments, wherein the linker is a peptide linker. 91. The epigenetic effector of any preceding embodiments, wherein the nucleic acid binding moiety is fused to the effector moiety. 92. The epigenetic effector of any preceding embodiments, wherein the 3’ end of nucleic acid binding moiety is connected to the 5’ end of the effector moiety. 93. The epigenetic effector of any preceding embodiments, wherein the 5’ end of nucleic acid binding moiety is connected to the 3’ end of the effector moiety. 94. The effector moiety of the epigenetic effector may be or comprise a second effector moiety. 95. The second effector moiety may be selected from DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, TRDMT1, MQ1,MET1, DRM2, CMT2, CMT3, TET1, TET2, and TET3 , SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, KAT13C , HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional equivalent thereof. 96. The epigenetic effector of any preceding embodiments further comprising a second linker. 97. The epigenetic effector of any preceding embodiments, wherein the first linker is situated between the first effector moiety and the nucleic acid binding moiety and the second linker is situated between the nucleic acid binding moiety and the second effector moiety. 98. The epigenetic effector of any preceding embodiments further comprising one or more nuclear localization signal (NLS).WSGR Ref.65120-702.601 99. The epigenetic effector of any preceding embodiments, wherein the NLS is selected from SV40, EGL-13, Tus-protein, polyoma large T-AG, Hepatitis D virus antigen, Rev protein, murine p53, or a nucleoplasmin. 100. The epigenetic effector of any preceding embodiments wherein the epigenetic effector comprises a first effector moiety and a second effector moiety, wherein the first effector moiety increases methylation at a target site and the second effector moiety represses transcription at the target site. 101. The epigenetic effector of any preceding embodiments wherein the epigenetic effector comprises a first effector moiety and a second effector moiety, wherein the first effector moiety increases methylation at a first target site and the second effector moiety increases methylation at a second target site. 102. The epigenetic effector of any preceding embodiments wherein the epigenetic effector comprises at least two effector moieties, wherein each moiety increase methylation at a target site. 103. A polynucleotide comprising a sequence encoding an epigenetic effector of any preceding embodiments. 104. The polynucleotide of any preceding embodiments, wherein the polynucleotide is a RNA, e.g., an mRNA. 105. An editor comprising a nucleic acid binding domain and a DNA polymerase domain. 106. The editor of embodiment 105, wherein the nucleic acid binding domain is selected from a CRISPR / Cas domain, a zinc finger domain, or a TAL domain. 107. The editor of embodiment 105, wherein the DNA polymerase domain is a reverse transcriptase. 108. The editor of embodiment 107, wherein the reverse transcriptase is selected from a MMLV-RT, TERT-RV, RT_nLTR, RT_Bac_HIV-RT, Retron, or RVT2. 109. A system comprising at least two epigenetic effectors wherein: (a) a first epigenetic effector increases methylation at a first target site; and (b) a second epigenetic effector that represses methylation at a second target site. 110. A system comprising at least two epigenetic effectors wherein:WSGR Ref.65120-702.601 (a) a first epigenetic effector increases methylation at a first target site; and (b) a second epigenetic effector increases methylation at a second target site. 111. A system comprising at least two epigenetic effectors wherein: (a) a first epigenetic effector represses methylation at a first target site; and (b) a second epigenetic effector represses methylation at a second target site. 112. A system for modifying a cell genome, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide may be capable of modifying DNA, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the first polypeptide may be capable of modifying DNA. 113. A system for modifying a cell genome, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide may be capable of modifying histone, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide may be capable of modifying DNA. 114. A system for modifying a cell genome, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide may be capable of modifying histone, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide may be capable of modifying histone. 115. A system for modifying a cell genome, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide increases histone methylation, histone acetylation, or DNA methylation at a first target site, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide increases histone methylation, histone acetylation, or DNA methylation at a second target site. 116. A system for modifying a cell genome, the system comprising:WSGR Ref.65120-702.601 (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide increases histone methylation, histone acetylation, or DNA methylation at a first target site, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide decreases histone methylation, histone acetylation, or DNA methylation at a second target site. 117. A system for modifying a cell genome, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide decreases histone methylation, histone acetylation, or DNA methylation at a first target site, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide decreases histone methylation, histone acetylation, or DNA methylation at a second target site. 118. A system for modifying a cell genome, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide may be capable of modifying DNA, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide may be capable of modifying DNA. 119. A system for modifying a cell genome, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide may be capable of modifying DNA methylation, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide may be capable of modifying DNA methylation. 120. A system for modifying DNA, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide increases DNA methylation at a first target site, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide increases DNA methylation at a second target site.WSGR Ref.65120-702.601 121. A system for modifying DNA, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide increases DNA methylation at a first target site, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide decreases DNA methylation at a second target site. 122. A system for modifying DNA, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide decreases DNA methylation at a first target site, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide decreases DNA methylation at a second target site. 123. A system for modifying histones, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide increases histone methylation at a first target site, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide increases histone methylation at a second target site. 124. A system for modifying histones, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide increases histone methylation at a first target site, and (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide decreases histone methylation at a second target site. 125. A system for modifying histones, the system comprising: (a) a first polypeptide or a first nucleotide encoding the first polypeptide, wherein the first polypeptide decreases histone methylation at a first target site, andWSGR Ref.65120-702.601 (b) a second polypeptide or a second nucleotide encoding the second polypeptide, wherein the second polypeptide decreases histone methylation at a second target site. 126. A system comprising a system, e.g., a first epigenetic effector and a second epigenetic effector, comprising: (a) a first guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a first desired site of epigenetic modification; and (b) a second guide RNA comprising a sequence complementary to a DNA sequence within 1 kb from a second desired site of epigenetic modification; wherein the first guide RNA interacts with the first epigenetic and the second guide RNA interacts with the second epigenetic effector. 127. The system of any preceding embodiments, wherein the first epigenetic effector is an epigenetic effector selected from any preceding embodiments. of p. 128. The system of any preceding embodiments, wherein the second epigenetic effector is an epigenetic effector selected from any preceding embodiments. 129. A system of any preceding embodiments, wherein: (a) the first epigenetic effector comprises a methylase, and (b) the second epigenetic effector comprises a demethylase. 130. A system of any preceding embodiments, wherein: (a) the first epigenetic effector comprises a transcriptional activator and; (b) the second epigenetic effector comprises a transcriptional repressor. 131. The system of any preceding embodiments, wherein the transcriptional repressors of the system may be independently selected from the same category or different categories. 132. The system of any preceding embodiments, wherein the transcriptional repressors of the system may be independently selected from TET1, TET2, TET3, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, KAT1, KAT2A, KAT3A, KAT3B, KAT13C, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, or SUZ12, or a functional equivalent.WSGR Ref.65120-702.601 133. The system of any preceding embodiments, wherein the epigenetic effectors may be independently selected from categories comprising a DNA demethylase, histone acetyltransferase, histone demethylase, or a transcription repressor moiety. 134. The system of any preceding embodiments, wherein the epigenetic effectors may include one or more DNA demethylases and / or functional equivalents thereof. 135. The system of any preceding embodiments, wherein the epigenetic effectors may include one or more histone acetyltransferases and / or functional equivalents thereof. 136. The system of any preceding embodiments, wherein the of the system may include one or more histone methyltransferases and / or functional equivalents thereof. 137. The system of any preceding embodiments, wherein the epigenetic effectors may include one or more histone demethylases and / or functional equivalents thereof. 138. The system of any preceding embodiments, wherein the epigenetic effectors may include one or more DNA methyltransferases and / or functional equivalents thereof. 139. The system of any preceding embodiments, wherein the epigenetic effectors may include one or more histone deacetylases and / or functional equivalents thereof. 140. Transcriptional repressors of the system may include one or more a transcription repressor moiety and / or functional equivalents thereof. 141. A system comprising: (a) a first guide RNA, wherein the guide RNA comprises a direct repeat sequence and a first spacer sequence capable of hybridizing to a DNA sequence within 1 kb of a first target site; (b) a first CRISPR-Cas effector polypeptide or a nucleic acid encoding the effector polypeptide, wherein the first effector polypeptide may be capable of binding to a first target sequence complementary to the first spacer sequence, wherein binding of the first effector polypeptide to the first target sequence increases methylation at the first target site; (c) a second guide RNA, wherein the guide RNA comprises a direct repeat sequence and a second spacer sequence capable of hybridizing to a DNA sequence 1 kb of a second target site; andWSGR Ref.65120-702.601 (d) a second CRISPR-Cas effector polypeptide or a nucleic acid encoding the effector polypeptide, wherein the second effector polypeptide may be capable of binding to a second target sequence complementary to the second spacer sequence; and wherein binding of the second effector polypeptide to the second target sequence decreases methylation at the second target site. 142. A system for simultaneous epigenetic modification at one or more target sites, the system comprising a system, the set comprising: (a) a first epigenetic effector that induces transcriptional activation at a first target site; and (b) a second epigenetic effector that induces transcriptional repression at a second target site. 143. A system for simultaneous epigenetic modification at one or more target sites, the system comprising a system, the set comprising: (a) a first epigenetic effector that may be capable of increasing methylation at a first target site; (b) a second epigenetic effector that may be capable of decreasing methylation at a second target site. 144. A system for simultaneous epigenetic modification of one or more genomic sequences in a cell, the system comprising a system, the set comprising: (a) a first epigenetic effector comprising methylation modifying activity; and (b) a second epigenetic effector comprising methylation modifying activity; wherein the first and second epigenetic effector have different methylation modifying activity. 145. A system comprising an epigenetic effector of any preceding embodiments and an editor of any preceding embodiments. 146. A system for simultaneous epigenetic modification and editing of one or more genomic sequences in a cell, the system comprising an epigenetic effector of any preceding embodiments and an editor of any preceding embodiments.WSGR Ref.65120-702.601 147. A system for modulating activity of a cell, the system comprising a set of two or more epigenetic effectors to modulate the methylation profile of the genome of the cell. 148. A system for treating or preventing a disease in a subject, the system comprising a set of two or more epigenetic effectors capable of reprogramming a diseased cell into a healthy cell by modulating the methylation profile of the genome of the diseased cell. 149. A system for altering developmental potency of a cell, the system comprising a set of two or more epigenetic effectors capable of modulating one or more components of a cellular pathway associated with the developmental potency of the cell. 150. The system of any preceding embodiments comprising: (a) a first epigenetic effector comprising a first nucleic acid binding moiety and a first effector moiety; and (b) a second epigenetic effector comprising a second nucleic acid binding moiety and a second effector moiety. 151. The system of any preceding embodiments comprising: (a) a first epigenetic effector capable of adding or removing a methyl group in a nucleic acid; and (b) a second epigenetic effector capable of adding or removing a methyl group in a nucleic acid. 152. The system of any preceding embodiments comprising: (a) a first epigenetic effector capable of adding a methyl group in a nucleic acid; and (b) a second epigenetic effector capable of removing a methyl group in a nucleic acid. 153. The system of any preceding embodiments comprising: (a) a first epigenetic effector capable of adding or removing a methyl group in a histone; and (b) a second epigenetic effector capable of adding or removing a methyl group in a histone. 154. The system of any preceding embodiments comprising:WSGR Ref.65120-702.601 (a) a first epigenetic effector capable of adding a methyl group in a histone; and (b) a second epigenetic effector capable of removing a methyl group in a histone. 155. The system of any preceding embodiments comprising: (a) a first epigenetic effector that binds to a first transcription regulatory element selected from a promoter, an enhancer, or a transcription start site operably linked to a gene; and (b) a second epigenetic effector that binds to a second transcription regulatory element selected from a promoter, an enhancer, or a transcription start site operably linked to a gene. 156. The system of any preceding embodiments, wherein the first and the second epigenetic effectors of further comprises additional epigenetic effectors, e.g., third, fourth, fifth, sixth or more epigenetic effectors. 157. The system of any preceding embodiments, wherein the first and the second transcription regulatory elements are identical. 158. The system of any preceding embodiments, wherein the first and the second transcription regulatory elements are different. 159. The system of any preceding embodiments, wherein the first epigenetic effector comprises a first nucleic acid binding moiety comprising a CRISPR / Cas domain, a zinc finger domain, or a TAL domain. 160. The system of any preceding embodiments, wherein the second epigenetic effector comprises a first nucleic acid binding moiety comprising a CRISPR / Cas domain, a zinc finger domain, a TAL domain, or a functional equivalent of any of the foregoing. 161. The system of any preceding embodiments, wherein the first epigenetic effector comprises a first nucleic acid binding moiety comprising a first CRISPR / Cas domain comprising a first CRISPR / Cas protein and the second epigenetic effector comprises a first nucleic acid binding moiety comprising a second CRISPR / Cas domain comprising a second CRISPR / Cas protein. 162. The system of any preceding embodiments, wherein the first CRISPR / Cas protein may be selected from of type I, type II, type III, type IV, type V Cas protein, and type VI Cas protein.WSGR Ref.65120-702.601 163. The system of any preceding embodiments, wherein the second CRISPR / Cas protein may be selected from a type I, type II, type III, type IV, type V Cas protein, or type VI Cas protein. 164. The first CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, and functional equivalents. 165. The second CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, or a functional fragment thereof. 166. The first CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, or a functional fragment thereof and theWSGR Ref.65120-702.601 second CRISPR / Cas protein may be selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csnl, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a Type II Cas effector protein, a Type V Cas effector protein, a Type VI Cas effector protein,...

Claims

WSGR Ref.65120-702.601 CLAIMS What is claimed is:

1. A system, comprising: (a) a first epigenetic effector comprising: (i) a first CRISPR / Cas domain complexed with a first guide RNA comprising a first tracrRNA sequence, and (ii) a first effector moiety; and (b) a second epigenetic effector comprising: (i) a second CRISPR / Cas domain complexed with a second guide RNA comprising a second tracrRNA sequence, and (ii) a second effector moiety; wherein the first tracrRNA sequence and the second tracrRNA sequence have a phylogenetic distance of exceeding 0.09, when the phylogenetic distance is determined by DNAdist (v3.5c).

2. The system of claim 1, further comprising a cell, wherein the cell comprises: (a) a target nucleic acid comprising one or more CpG islands; (b) the first epigenetic effector; and (c) the second epigenetic effector.

3. The system of claim 1 or 2, wherein the first guide RNA further comprises a first spacer sequence that is complementary to a first sequence in a first CpG island of the one or more CpG islands in the target nucleic acid, and wherein the second guide RNA further comprises a second spacer sequence that is complementary to a second sequence in a second CpG island of the one or more CpG islands in the target nucleic acid.

4. The system of any one of claims 1-3, wherein the first CRISPR / Cas domain and the second CRISPR / Cas domain are Type II Cas domains.

5. The system of claim 4, wherein the first CRISPR / Cas domain and the second CRISPR / Cas domain are Cas9 domains.

6. The system of any one of claims 1-3, wherein the first CRISPR / Cas domain is a Type II Cas domain, and wherein the second CRISPR / Cas domain is a Type V Cas domain.

7. The system of claim 6, wherein the first CRISPR / Cas domain is a Cas9 domain and the second CRISPR / Cas domain is a Cas12 domain.WSGR Ref.65120-702.601 8. The system of any one of claims 1-3, wherein the first CRISPR / Cas domain and the second CRISPR / Cas domain are Type V Cas domains.

9. The system of claim 8, wherein the first CRISPR / Cas domain and the second CRISPR / Cas domain are Cas12 domains.

10. The system of any one of claims 1-9, wherein the first CRISPR / Cas domain is selected from the group consisting of AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and wherein the second CRISPR / Cas domain is selected from the group consisting of AinCas9, BniCas9, BokCas9, Cca1Cas9, CgaCas9, CglCas9, Cme1Cas9, CpeCas9, DdeCas9, EceCas9, EdoCas9, EfaCas9, EitCasi, Esp1Cas9, Esp2Cas9, FfrCas9, FhoCas9, FmaCas9, Ghy2Cas9, Ghy4Cas9, KkiCas9, LanCas9, LmoCas9, LrhCas9, Lsp1Cas9, Lsp2Cas9, MgaCas9, Nme2Cas9, NspCas9, OrhCas9, PacCas9, PhiCas9, PspCas9, Sag1Cas9, Sag2Cas9, SdyCas9, Seq1Cas9, Seq2Cas9, SgaCas9, SgoCas9, SmuCas9, Sma2Cas9, SpyCas9, SraCas9, SsiCas9, SsuCas9, Sth1ACas9, TbaCas9, TdeCas9, TpuCas9, TspCas9, VpaCas9, WviCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1.

11. The system of any one of claims 1-10, wherein the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and wherein the second CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1.

12. The system of claim 11, wherein the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, and LmoCas9; andWSGR Ref.65120-702.601 wherein the second CRISPR / Cas domain is selected from the group consisting of Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a.

13. The system of claim 11, wherein the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, and LmoCas9; and wherein the second CRISPR-Cas domain is selected from the group consisting of CasPhi-2 and Cas12c-1.

14. The system of claim 11, wherein the first CRISPR / Cas domain is selected from the group consisting of LanCas9, Sag1Cas9, and LmoCas9; and wherein the second CRISPR / Cas domain is a SpyCas9 domain.

15. The system of claim 11, wherein the first CRISPR / Cas domain may be selected from the group consisting of Cpf1, Hkcas12a, PiCas12a, Fn3Cas12a, and Pb2Cas12a; and wherein the second CRISPR / Cas domain is selected from the group consisting of CasPhi-2 and Cas12c-1.

16. The system of claim 11, wherein the first CRISPR / Cas domain is a CasPhi-2 domain, and the second CRISPR / Cas domain is a Cas12c-1 domain.

17. The system of any one of claims 1-16, wherein the first CRISPR / Cas domain and the second CRISPR / Cas domain are catalytically inactive.

18. The system of any one of claims 1-17, wherein the first CRISPR / Cas domain and the second CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135.

19. The system of any one of claims 1-18, wherein the first guide RNA or the second sgRNA, or the combination thereof, comprises a sequence selected from SEQ ID NOs.1-10.

20. The system of any one of claims 1-19, wherein the target nucleic acid comprises: (i) over 70,000 copies of a first PAM sequence associated with the first CRISPR / Cas domain in the one or more CpG islands in the target nucleic acid, and (ii) over 70,000 copies of a second PAM sequence associated with the second CRISPR / Cas domain in the one or more CpG islands in the target nucleic acid.

21. The system of claim 20, wherein the over 70,000 copies of the first PAM sequence and the over 70,000 copies of the second PAM sequence are in a sense strand of the target nucleic acid.WSGR Ref.65120-702.601 22. The system of claim 20, wherein the target nucleic acid comprises (i) over 1,000,000 copies of the first PAM sequence in the one or more CpG islands in the target nucleic acid, and (ii) over 1,000,000 copies of the second PAM sequence in the one or more CpG islands in the target nucleic acid.

23. The system of claim 22, wherein the over 1,000,000 copies of the first PAM sequence and the over 1,000,000 copies of the second PAM sequence are in a sense strand of the target nucleic acid.

24. The system of any one of claims 20-23, wherein the first PAM sequence is selected from the group consisting of NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; and wherein the second PAM sequence is selected from the group consisting of NRTAW, NNGTAMAY, NNHDAGGDNA, BRTTTTT, BRRTTTW, NCAARC, NARNCCN, NNNNCVGAA, NNNNGYAA, NNGHWAAA, NTGARGNANY, BTGGDATNN, NGGGAHTNAN, NAAAG, NVARAACCN, NARATC, NNGAAAN , NNGAC, NAANARCN, YHHNGTH, NNNNCTAA , NTNTAAWA, NNGWAAYT, NCAAHYBY, NNGAD, NHDTCCA, NCNNTCCN, NNAAARG, NHTAAAA, NARGHWHAGNC, NRATTTT, NGGDAWT, NGGNG, NTAGANANN, NGGDAHT, NNAAAG, NGGDT, NHGYNANA,NGGDAGNN, NNNAGAAA, NHAAAAA, NHTAAAAA, NHGYRAA, NNNATTT, NAAAAY, NATAWNA, NNAAACN, NATARCH, HHAAATD, NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

25. The system of any one of claims 20-24, wherein the first PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG;WSGR Ref.65120-702.601 and wherein the second PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

26. The system of any one of claims 20-25, wherein the first PAM sequence is selected from the group consisting of NGG, NRG, and NBGG; and wherein the second PAM sequence is selected from the group consisting of TTTV, YYV, KKYV, YTV, and NYTV.

27. The system of any one of claims 20-25, wherein the first PAM sequence is selected from the group consisting of NGG, NRG, and NBGG; and wherein the second PAM sequence is selected from the group consisting of TBN and TG.

28. The system of any one of claims 20-25, wherein the first PAM sequence is selected from the group consisting of NGG, NRG, and NBGG; and wherein the second PAM sequence is NGG.

29. The system of any one of claims 20-25, wherein the first PAM sequence is selected from the group consisting of TTTV, YYV, KKYV, YTV, and NYTV; and wherein the second PAM sequence is selected from the group consisting of TBN and TG.

30. The system of any one of claims 20-25, wherein the first PAM sequence is selected from the group consisting of TBN and NYTV; and wherein the second PAM sequence is TG.

31. The system of any one of claims 1-30, wherein the first effector moiety comprises a TET1 domain.

32. The system of any one of claims 1-30, wherein the first effector moiety comprises a domain selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain.

33. The system of any one of claims 1-30 or 32, wherein the first effector moiety comprises two domains selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain.

34. The system of any one of claims 1-30, 32, or 33, wherein the first effector moiety comprises a DNMT3A domain, a DNMT3L domain, and a KRAB domain.

35. The system of any one of claims 1-34, wherein the second effector moiety comprises a TET1 domain.WSGR Ref.65120-702.601 36. The system of any one of claims 1-34, wherein the second effector moiety comprises a domain selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain.

37. The system of any one of claims 1-34 or 36, wherein the second effector moiety comprises two domains selected from the group consisting of: a DNMT3A domain, a DNMT3L domain, and a KRAB domain.

38. The system of any one of claims 1-34, 36 or 37, wherein the second effector moiety comprises a DNMT3A domain, a DNMT3L domain, and a KRAB domain.

39. The system of any one of claims 1-30, wherein the first effector moiety comprises a demethylase domain.

40. The system of claim 39, wherein the second effector moiety comprises a methyltransferase domain.

41. The system of claim 39, wherein the second effector moiety comprises a demethylase domain.

42. The system of any one of claims 1-30, wherein the first effector moiety comprises a methyltransferase domain.

43. The system of claim 42, wherein the second effector moiety comprises a demethylase domain.

44. The system of claim 42, wherein the second effector moiety comprises a methyltransferase domain.

45. The system of any one of claims 1-29, wherein the first effector moiety comprises a transcriptional activator domain.

46. The system of claim 45, wherein the first effector moiety comprises a transcriptional repressor domain.

47. The system of any one of claims 1-30, wherein the first effector moiety comprises a transcriptional repressor domain.WSGR Ref.65120-702.601 48. The system of claim 47, wherein the second effector moiety comprises a transcriptional activator domain.

49. The system of any one of claims 2-48, wherein the cell is a eukaryotic cell.

50. The system of any one of claims 2-49, wherein the cell is a human cell.

51. A method of reprogramming a cell, the method comprising: (a) providing in a cell comprising: (i) a first epigenetic effector comprising (I) a first CRISPR / Cas domain complexed with a first guide RNA and (II) a first effector moiety, wherein the first CRISPR / Cas domain complexed with the first guide RNA targets a first genomic locus in a genome of the cell; and (ii) a second epigenetic effector comprising a (I) second CRISPR / Cas domain complexed with a second guide RNA and (II) a second effector moiety, wherein the second CRISPR / Cas domain complexed with the second guide RNA targets a second genomic locus in the genome; wherein a first tracrRNA sequence of the first guide RNA and a second tracrRNA sequence of the second guide RNA have a phylogenetic distance of exceeding 0.09; (b) methylating a first target site in the first genomic locus using the first epigenetic effector; and (c) demethylating a second target site in the second genomic locus using the second epigenetic effector.

52. The method of claim 51, wherein the genome comprises one or more CpG islands.

53. The method of claim 52, wherein the first epigenetic effector comprises a DNMT3A domain.

54. The method of claim 53, wherein the first epigenetic effector further comprises a DNMT3L domain and a KRAB domain.

55. The method of any one of claims 51-54, wherein the second epigenetic effector comprises a TET1 domain.WSGR Ref.65120-702.601 56. The method of any one of claims 51-55, wherein the first CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and wherein the second CRISPR / Cas domain is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1.

57. The method of any one of claims 51-56, wherein the first CRISPR / Cas domain and the second CRISPR / Cas domain are catalytically inactive.

58. The method of any one of claims 51-57, wherein the first CRISPR / Cas domain and the second CRISPR / Cas domain comprise a sequence of any one of SEQ ID NOs: 130-135.

59. The method of any one of claims 51-58, wherein the first guide RNA or the second sgRNA comprises a sequence selected from SEQ ID NOs.1-10.

60. The method of any one of claims 51-59, wherein the first guide RNA and the second sgRNA each comprises a sequence selected from SEQ ID NOs.1-10.

61. The method of any one of claims 52-60, wherein the first target site is in a first CpG island of the one or more CpG islands in the genome, and wherein the second target site is in a second CpG island of the one or more CpG islands in the genome.

62. The method of any one of claims 52-61, wherein the genome comprises (i) over 70,000 copies of a first PAM sequence associated with the first CRISPR / Cas domain in the one or more CpG islands in the genome, and (ii) over 70,000 copies of a second PAM sequence associated with the second CRISPR / Cas domain in the one or more CpG islands in the genome.

63. The method of any one of claims 52-62, wherein the genome comprises (i) over 1,000,000 copies of the first PAM sequence associated with the first CRISPR / Cas domain in the one or more CpG islands in the genome, and (ii) over 1,000,000 copies of the second PAM sequence associated with the second CRISPR / Cas domain in the one or more CpG islands in the genome.

64. The method of claim 62 or 63, wherein the first PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; andWSGR Ref.65120-702.601 wherein the second PAM sequence is selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

65. A method of reprogramming a cell, the method comprising: (a) providing in a cell comprising a genome: (i) a first plurality of epigenetic effectors, each comprising (I) a first CRISPR / Cas domain and (II) a first effector moiety, wherein the first CRISPR / Cas domain is capable of specifically interacting with over 70,000 first targetable PAM sites in a first plurality of CpG islands in the genome; and (ii) a second plurality of epigenetic effectors, each comprising a (I) second CRISPR / Cas domain and (II) a second effector moiety, wherein the second CRISPR / Cas domain is capable of specifically interacting with over 70,000 second targetable PAM sites in a second plurality of CpG islands in the genome; (b) methylating a first plurality of target sites in the first plurality of CpG islands in the genome using the first plurality of epigenetic effectors; and (c) demethylating a second plurality of target sites in the second plurality of CpG islands in the genome using the second plurality of epigenetic effectors.

66. The method of claim 65, wherein the first CRISPR / Cas domain is capable of specifically interacting with over 1,000,000 targetable PAM sites in the first plurality of CpG islands in the genome.

67. The method of claim 65 or 66, wherein the second CRISPR / Cas domain is capable of specifically interacting with over 1,000,000 targetable PAM sites in the second plurality of CpG islands in the genome.

68. The method of any one of claims 65-67, wherein (a) further comprises providing: (iii) a first plurality of guide RNAs capable of binding to the first CRISPR / Cas domain in each epigenetic effector of the first plurality of epigenetic effectors, wherein the first plurality of guide RNAs target different CpG islands of the first plurality of CpG islands in the genome; andWSGR Ref.65120-702.601 (iv) a second plurality of guide RNAs capable of binding to the second CRISPR / Cas domain in each epigenetic effector of the second plurality of epigenetic effectors, wherein the second plurality of guide RNAs target different CpG islands of the second plurality of CpG islands in the genome.

69. The method of any one of claims 65-68, wherein, in (b), the first plurality of target sites are in a same gene in the genome.

70. The method of any one of claims 65-68, wherein, in (b), the first plurality of target sites are in different genes in the genome.

71. The method of any one of claims 65-70, wherein, in (c), the second plurality of target sites are in a same gene in the genome.

72. The method of any one of claims 65-70, wherein, in (c), the second plurality of target sites are in different genes in the genome.

73. The method of any one of claims 68-72, wherein the first plurality of guide RNAs comprise a first common tracrRNA sequence, and wherein the second plurality of guide RNAs comprise a second common tracrRNA sequence, wherein the first common tracrRNA sequence and the second common tracrRNA sequence have a phylogenetic distance of exceeding 0.

09.

74. The method of any one of claims 68-73, wherein the first plurality of guide RNAs each comprise a first common sequence selected from SEQ ID NOs.1-10.

75. The method of any one of claims 68-74, wherein the second plurality of guide RNAs each comprise a second common sequence selected from SEQ ID NOs.1-10.

76. The method of any one of claims 65-75, wherein the first CRISPR / Cas domain of each of the first plurality of epigenetic effectors is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1; and wherein the second CRISPR / Cas domain of each of the second plurality of epigenetic effectors is selected from the group consisting of SpyCas9, LanCas9, Sag1Cas9, LmoCas9, Cpf1, HkCas12a, PiCas12a, Fn3Cas12a, Pb2Cas12a, CasPhi-2, and Cas12c-1.WSGR Ref.65120-702.601 77. The method of any one of claims 65-76, wherein the first CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135, and the second CRISPR / Cas domain comprises a sequence of any one of SEQ ID NOs: 130-135.

78. The method of any one of claims 65-77, wherein the first targetable PAM sites each comprise a sequence selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG; and wherein the second targetable PAM sites each comprise a sequence selected from the group consisting of NGG, NRG, NBGG, TTTV, YYV, KKYV, YTV, NYTV, TBN, and TG.

79. The method of any one of claims 65-78, wherein the first effector moiety of each of the first plurality of epigenetic effectors comprises a DNMT3A domain.

80. The method of claim 79, wherein the first effector moiety of each of the first plurality of epigenetic effectors further comprises a DNMT3L domain and a KRAB domain.

81. The method of any one of claims 65-80, wherein the second effector moiety of each of the second plurality of epigenetic effectors comprises a TET1 domain.

82. The method of any one of claims 75-81, wherein methylating the first plurality of target sites thereby represses expression of a first gene.

83. The method of any one of claims 75-82, wherein demethylating the second plurality of target sites thereby enhances expression of a second gene.

84. A system, comprising: (a) a first epigenetic effector comprising: (i) a SpyCas9 domain complexed with a first guide RNA, and (ii) a demethylase; and (b) a second epigenetic effector comprising: (i) a SauCas9 domain complexed with a second guide RNA and (ii) a methylase.

85. The system of claim 84, wherein the demethylase comprises a TET1 domain.

86. The system of claim 84, wherein the methylase comprises a DNMT3A domain.

87. The system of claim 86, wherein the second epigenetic effector further comprises a DNMT3L domain or a KRAB domain.WSGR Ref.65120-702.601 88. The system of claim 84, further comprising a cell, wherein the cell comprises: (a) a target nucleic acid comprising one or more CpG islands; (b) the first epigenetic effector; and (c) the second epigenetic effector.

89. A method of reprogramming a cell, the method comprising: (a) providing in a cell comprising: (i) a first epigenetic effector comprising (I) a SpyCas9 domain complexed with a first guide RNA and (II) a demethylase, wherein the SpyCas9 domain complexed with the first guide RNA targets a first genomic locus in a genome of the cell; and (ii) a second epigenetic effector comprising a (I) SauCas9 domain complexed with a second guide RNA and (II) a methylase, wherein the CasuCas9 domain complexed with the second guide RNA targets a second genomic locus in the genome; (b) demethylating a first target site in the first genomic locus using the first epigenetic effector; and (c) methylating a second target site in the second genomic locus using the second epigenetic effector.

90. The method of claim 89, wherein the demethylase comprises a TET1 domain.

91. The method of claim 89, wherein the methylase comprises a DNMT3A domain.

92. The method of claim 89, wherein the second epigenetic effector further comprises a DNMT3L domain or a KRAB domain.

93. The method of any one of claims 89-92, wherein the genome comprises one or more CpG islands.

94. The method of claim 93, wherein the first target site is in a first CpG island of the one or more CpG islands in the genome, and wherein the second target site is in a second CpG island of the one or more CpG islands in the genome.

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