Compositions and methods for modulating chromatin state

Abstract

Aspects of the present disclosure are directed to at least methods and compositions for diagnosis and / or treatment of diseases associated with aberrant levels of m5C RNA, including but not limited chromatin associate RNA (caRNA). The disease may comprise cancer and / or pre-cancerous cells. The disease may be associated with mutations in a TET2 pathway, including but not limited to mutations in TET2, IDH1, IDH2, and / or ASXL1 encoding genes. Treatment of a disease may comprises administration of one or more inhibitors of MBD6, TET2, and / or NSUN2. Also provided herein are methods of treatment of a disease in an individual comprising administering one or more inhibitors of MBD6, TET2, and / or NSUN2 to the individual determined to have aberrant m5C RNA modifications.

Description

COMPOSITIONS AND METHODS FOR MODULATING CHROMATIN STATE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No.63 / 587,365, filed October 2, 2023, which is incorporated by reference herein in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on October 2, 2023, is named ARCD_P0795USP1_Sequence_Listing.xml and is 2,726,784 bytes in size. BACKGROUND I. Field

[0003] Aspects of this invention relate to at least the field of molecular biology and medicine. More particularly, aspects concern at least compositions and methods for modifying, detecting, mapping, and / or evaluating chromatin state for therapeutic purposes. II. Background

[0004] The gene Ten-eleven translocation enzyme 2 (TET2) and TET2 protein pathway associated genes are frequently disrupted in various diseases (1-3), such as human cancers, and have been shown to drive myeloid malignancy initation and progression. TET2 deficiency has been shown to result in globally opened chromatin and activation of genes contributing to aberrant hematopoietic stem cell self-renewal (4,5). However, the open chromatin consistently observed in TET2-deficient mouse embryonic stem cells, leukemic cells, and hematopoietic progenitor and stem cells is inconsistent with TET2’s described role of DNA 5mC oxidation. Additionally, suitable biomarkers for determining efficacy of treatment options associated with diseases comprising TET2 mutations and / or TET pathway associated mutations are lacking. Finally, there exists a need for methods and compositions for treating TET2 and / or TET2 pathway deficient diseases, such as cancer and / or pre-cancers. SUMMARY

[0005] The present disclosure addresses certain needs outlined above by providing at least methods, compositions, and kits for treatment of diseases associated with chromatin statemisregulation, in particular, chromatin state misregulation associated with mutations in TET2 and / or TET2 pathway associated genes.

[0006] TET2 and associated pathways are frequently disturbed in various diseases, such as human cancers. As reported herein, the inventors have identified a novel pathway through which TET2 acts to alter chromatin state and gene expression through oxidative demethylation of m5C on chromatin-associated RNAs (caRNA), including but not limited to chromatin associated repeat RNA and chromatin associated regulatory RNA (carRNA). As described herein, the inventors have characterized upstream writers (e.g., NSUN1 and / or NSUN2), readers (e.g., MBD5 and / or MBD6), and erasers (e.g., TET2) involved in m5C regulation in caRNA. In addition, genes associated with TET2 pathways, such as isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) are mutated in ~77% low-grade glioma and ~30% of cholangiocarcinoma. IDH1 / 2 mutations are known to produce oncometabolite R- 2HG, which can inhibit TET2 activities. TET2 and IDH1 / 2 mutants are known to be mutually exclusive in leukemia indicating they work along the same pathway in disease pathogenesis (e.g., TET pathways). Thus, biological systems with TET2 pathway deficiencies are prevalent and highly problematic, for example, oncogenic. Collectively, as described herein, the inventors have identified a new mode of chromatin-associated RNA m5C mediated chromatin regulation. For example, in certain cases, NSUN2 can install m5C in caRNA, and TET2 can mediate oxidative demethylation thereof. MBD5 and / or MBD6 can read m5C-modified RNA on chromatin and can mediate active deubiquitylation of major repressive marks, such as but not limited to H2AK119ub. H2AK119ub can be installed by the Polycomb Repressive Complex 1 (PRC1) to silence chromatin (e.g., promote heterochromatin). Perturbations of TET2 pathway, and therapeutic interventions to mitigate the same, can be broadly applied in contexts including but not limited to: 1) human diseases (e.g., cancers, etc.) with TET2 mutation and / or low expression; 2) human diseases (e.g., cancers, etc.) comprising IDH1 / 2 mutations; and / or 3) human diseases (e.g., cancers, etc.) comprising PRC1 or PR-DUB mutations and / or altered expression.

[0007] In some aspects, are methods for treating a disease in an individual, comprising the step of administering one or more inhibitors of methyl-CpG-binding domain protein 6 (MBD6) to an individual in need thereof. In some aspects, the disease comprises cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, and / or cervix. In some aspects, the disease comprises clonal hematopoiesis of indeterminate potential (CHIP). In some aspects, the disease is characterized by atherosclerosis, myocardial fibrosis, and / orheart failure. In some aspects, the disease comprises a blood cancer. In some aspects, the disease comprises a leukemia. In some aspects, the disease comprises a myeloid malignancy. In some aspects, the disease comprises acute myeloid leukemia. In some aspects, the disease comprises chronic myelomonocytic leukemia. In some aspects, the disease comprises a glioma. In some aspects, the disease comprises glioblastoma.

[0008] In some aspects, methods provided herein comprise reducing proliferation of a cancer and / or pre-cancerous cell. In some aspects, diseases for treatment utilizing methods provided here comprise cells with one or more mutations in one or more genes encoding a ten- eleven translocation (tet) methylcytosine dioxygenase 2 (TET2), ASXL transcriptional regulator 1 (ASXL1), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), tumor protein p53 (p53), DNA (cytosine-5-)-methyltransferase 3A (DNMT3A), Janus kinase 2 (JAK2), Protein Phosphatase Mn2+ / Mg2+-Dependent 1D (PPM1D), Spliceosome Factor 3b1 (SF3B1), and / or Serine and Arginine Rich Splicing Factor 2 (SRSF2).

[0009] In some aspects, methods of treatment described herein comprise treatment of a disease associated with diseased cells with one or more mutations in one or more genes encoding components of a canonical and / or non-canonical Polycomb Repressive Complex (PRC). In some aspects, one or more mutations in one or more genes encoding components of PRC can comprise one or more loss of function mutations. In some aspects, one or more mutations in one or more genes encoding components of PRC comprises, or expressly does not comprise, one or more mutations in E3 Ubiquitin Ligase RING1A / B, Polycomb Group Ring Finger 1 (PCGF1), Polycomb Group Ring Finger 2 (PCGF2), Polycomb Group Ring Finger 3 (PCGF3), Polycomb Group Ring Finger 4 (PCGF4), Polycomb Group Ring Finger 5 (PCGF5), and / or Polycomb Group Ring Finger 6 (PCGF6). In some aspects, the disease is associated with cells with one or more mutations in one or more genes encoding Polycomb Repressive- Deubiquitinase (PR-DUB) complex associated components O-linked N-acetylglucosamine Transferase (OGT), Lysine Demethylase 1B (KDM1B), Forkhead Box K1 (FOXK1), Forkhead Box K2 (FOXK2), BRCA1 Associated Protein 1 (BAP1), ASXL Transcriptional Regulator 1 (ASXL1), ASXL Transcriptional Regulator 2 (ASXL2), ASXL Transcription Regulator 3 (ASXL3), and / or Host Cell Factor C1 (HCFC1). In some aspects, the one or more mutations in PR-DUB complex associated components OGT, KDM1B, FOXK1, FOXK2, BAP1, ASXL1, ASXL2, ASXL3, and / or HCFC1 comprises, or expressly does not comprise, one or more gain of function mutations. In some aspects, the disease is associated with cells with one or more mutations in a TET2 encoding gene. In some aspects, the one or more mutations in a TET2 encoding gene comprises, or expressly does not comprise, one or moreloss of function mutations. In some aspects, the disease is associated with cells comprising one or more mutations in one or more genes encoding ASXL1, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2.

[0010] In some aspects, also provided herein are methods comprising administration of one or more additional therapies to an individual in need thereof. In some aspects, an additional therapy is surgery, radiation, chemotherapy, hormone therapy, and / or immunotherapy. In some aspects, an additional therapy comprises administration of an inhibitor of TET2. In some aspects, the inhibitor of TET2 comprises C35 and / or TETi76. In some aspects, an additional therapy comprises administration of an inhibitor of NSUN1 and / or NSUN2. In some aspects, an additional therapy comprises administration of an inhibitor of NSUN2. In some aspects, an additional therapy comprises 5-AzaC.

[0011] In some aspects, methods described herein further comprise a step of diagnosing the disease in the individual. In some aspects, a disease is associated with diseased cells characterized as comprising an open chromatin state relative to non-diseased cells of the same developmental lineage.

[0012] In some aspects, administering the one or more MBD6 inhibitors to the individual results in promotion of a closed chromatin state in one or more diseased cells in the individual. In some aspects, a disease is characterized as pro-inflammatory. In some aspects, methods provided herein comprise inhibition of diseased cell proliferation.

[0013] In some aspects, administering the one or more MBD6 inhibitors decreases m5C levels in one or more chromatin associated RNA (caRNA) andor decreasing association of the one or more caRNA with a PR-DUB complex in one or more diseased cells in the individual, relative to a control non-diseased cell. In some aspects, administering the one or more MBD6 inhibitors increases oxidation of m5C in a caRNA and / or inhibits installation of m5C in the one or more caRNA in one or more diseased cells in the individual. In some aspects, one or more caRNA comprises or consists essentially Long Terminal Repeat (LTR) RNAs. In some aspects, one or more caRNA comprises one or more of the caRNAs described in Table 1. In some aspects, one or more caRNA comprises one or more of the caRNAs described in Table 2. In some aspects, one or more caRNA comprise a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 100-614. In some aspects, one or more caRNA comprise a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 104 or 107. In some aspects, methods described herein comprise utilization of between at least 20 to 50 caRNAs. In some aspects, one or more caRNAs comprise a chromatin associated regulatoryRNA (carRNA) and / or a chromatin associated repeat RNA sequence. In some aspects, one or more caRNAs comprise or consist essentially of endogenous retroviral RNAs. In some aspects, an endogenous retroviral RNAs comprises or consists essentially of endogenous retrovirus-K (ERVK).

[0014] Also provided herein are methods of treatment, wherein histone ubiquitination is increased (e.g., in one or more diseased cells in an individual following administration of one or more MBD6 inhibitors). In some aspects, the histone ubiquitination comprises or consists of ubiquitination on H2A. In some aspects, the histone ubiquitination occurs at a site comprising or consisting essentially of H2AK119. In some aspects, methods described herein further comprise modifying histone methylation (e.g., in one or more diseased cells in an individual following administration of one or more MBD6 inhibitors). In some aspects, a histone that comprises modified methlaytion comprises or consists essentially of H3. In some aspects, the histone comprises modified methylation on a methylation site comprising or consisting essentially of H3K27me3. In some aspects, the histone that comprises modified methylation is localized near or at (e.g. within less than 4.5 to 5.5 kb, for example 5 kb) a genetic locus overlapping with a histone that comprises modified histone ubiquitination.

[0015] In some aspects, one or more inhibitors of MBD6 comprise a polynucleotide at least partially complementary to a gene encoding MBD6. In some aspects, the at least partially complementary polynucleotide comprises a short hairpin RNA and / or small interfering RNA. In some aspects, the at least partially complementary polynucleotide comprises a sequence at least 80% complementary to at least 15, 20, 25, or more than 25 contiguous nucleotides of any one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or an anti-sense sequence thereof. In some aspects, the polynucleotide comprises modified RNA phosphoramidites. In some aspects, the polynucleotide comprises RNA with one or more 2ʹ-O-Methyl (2ʹ-OMe) or 2ʹ-O-Methoxyethyl (2ʹ-MOE) modifications. In some aspects, the polynucleotide comprises RNA with every nucleotide comprising a 2ʹ-MOE modification. In some aspects, the polynucleotide comprises RNA with one or more phosphorothioate bonds. In some aspects, the polynucleotide is comprised within a lentiviral particle and / or nanoparticle. In some aspects, the inhibitor of MBD6 comprises more than one polynucleotide. In some aspects, one or more inhibitors of MBD6 comprise a proteolysis targeting chimera.

[0016] Also provided herein are methods of promoting histone ubiquitination in a cell comprising contacting the cell with one or more inhibitors of methyl-CpG-binding domain protein 6 (MBD6). In some aspects, promoting histone ubiquitination comprises or consists of promoting ubiquitination on H2A. In some aspects, the histone ubiquitination site comprisesor consists essentially of H2AK119. In some aspects, the cell targeted for promotion of histone ubiquitination comprises, or expressly does not comprise, one or more mutations in one or more genes encoding TET2, ASXL1, IDH1, IDH2, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2. In some aspects, the cell comprises, or expressly does not comprise, one or more mutations in one or more genes encoding RING1A / B, PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, PCGF6, OGT, KDM1B, FOXK1, FOXK2, BAP1, ASXL1, ASXL2, ASXL3, and / or HCFC1.

[0017] Also provided herein are methods of decreasing m5C levels in chromatin associated RNA (caRNA) in a cell, comprising contacting the cell with one or more inhibitors of MBD6. In some aspects, the caRNA comprise or consist essentially of LTR RNAs. In some aspects, the caRNA comprise or consist essentially of endogenous retroviral RNAs. In some aspects, the endogenous retroviral RNAs comprise or consist essentially of endogenous retrovirus-K (ERVK).

[0018] Also provided herein are manufactured articles for use in performing any method disclosed herein. Also provided are kits comprising means for performing any method disclosed herein.

[0019] Also provided herein are methods of treating a disease in an individual, wherein the disease is characterized by cells comprising one or more mutations in TET2, ASXL1, IDH1, IDH2, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2 encoding genes, the method comprising administering one or more inhibitors of MBD6, TET2, and / or NSUN2 to the individual in need thereof.

[0020] Also provided herein are compositions including polynucleotides and / or proteins. In some aspects, provided herein are fusion proteins comprising a sequence at least 80%, 85%, 90%, 95%, or 100% identical to any one or more of SEQ ID NOs: 1-4, 11-14, or 15-20. In some aspects, a fusion protein comprises a sequence at least 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 19-20.

[0021] Also provided herein are compositions and / or kits that include one or more components associated with a method described herein. In some aspects, such compositions and / or ktis can be utilized in a method of treating a disease associated with aberrant m5C RNA methylation in an individual, for example, by administering a fusion protein and / or composition described herein.

[0022] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0023] Aspect 1 is a method of treating a disease in an individual, comprising the step of administering one or more inhibitors of methyl-CpG-binding domain protein 6 (MBD6) to an individual in need thereof.

[0024] Aspect 2 is the method of aspect 1, wherein the disease comprises cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, and / or cervix.

[0025] Aspect 3 is the method of aspect 1, wherein the disease comprises clonal hematopoiesis of indeterminate potential (CHIP).

[0026] Aspect 4 is the method of aspect 3, wherein the disease is characterized by atherosclerosis, myocardial fibrosis, and / or heart failure.

[0027] Aspect 5 is the method of aspect 2, wherein the cancer comprises a blood cancer.

[0028] Aspect 6 is the method of aspect 5, wherein the blood cancer comprises a leukemia.

[0029] Aspect 7 is the method of aspect 5, wherein the blood cancer comprises a myeloid malignancy.

[0030] Aspect 8 is the method of aspect 7, wherein the myeloid malignancy comprises acute myeloid leukemia.

[0031] Aspect 9 is the method of aspect 7, wherein the myeloid malignancy comprises chronic myelomonocytic leukemia.

[0032] Aspect 10 is the method of aspect 2, wherein the cancer comprises a glioma.

[0033] Aspect 11 is the method of aspect 10, wherein the glioma comprises glioblastoma.

[0034] Aspect 12 is the method of any one of aspects 1 to 11, comprising reducing proliferation of a cancer and / or pre-cancerous cell.

[0035] Aspect 13 is the method of any one of aspects 1 to 12, wherein the disease is associated with diseased cells comprising one or more mutations in one or more genes encoding a ten-eleven translocation (tet) methylcytosine dioxygenase 2 (TET2), ASXL transcriptional regulator 1 (ASXL1), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), tumor protein p53 (p53), DNA (cytosine-5-)-methyltransferase 3A (DNMT3A), Janus kinase 2 (JAK2), Protein Phosphatase Mn2+ / Mg2+-Dependent 1D (PPM1D), Spliceosome Factor 3b1 (SF3B1), and / or Serine and Arginine Rich Splicing Factor 2 (SRSF2).

[0036] Aspect 14 is the method of any one of aspects 1 to 13, wherein the disease is associated with diseased cells with one or more mutations in one or more genes encoding components of a canonical and / or non-canonical Polycomb Repressive Complex (PRC).

[0037] Aspect 15 is the method of aspect 14, wherein the one or more mutations in one or more genes encoding components of PRC comprises one or more loss of function mutations.

[0038] Aspect 16 is the method of aspect 14 or 15, wherein the one or more mutations in one or more genes encoding components of PRC comprises one or more mutations in E3 Ubiquitin Ligase RING1A / B, Polycomb Group Ring Finger 1 (PCGF1), Polycomb Group Ring Finger 2 (PCGF2), Polycomb Group Ring Finger 3 (PCGF3), Polycomb Group Ring Finger 4 (PCGF4), Polycomb Group Ring Finger 5 (PCGF5), and / or Polycomb Group Ring Finger 6 (PCGF6).

[0039] Aspect 17 is the method of any one of aspects 1 to 16, wherein the disease is associated with diseased cells with one or more mutations in one or more genes encoding Polycomb Repressive-Deubiquitinase (PR-DUB) complex associated components O-linked N- acetylglucosamine Transferase (OGT), Lysine Demethylase 1B (KDM1B), Forkhead Box K1 (FOXK1), Forkhead Box K2 (FOXK2), BRCA1 Associated Protein 1 (BAP1), ASXL Transcriptional Regulator 1 (ASXL1), ASXL Transcriptional Regulator 2 (ASXL2), ASXL Transcription Regulator 3 (ASXL3), and / or Host Cell Factor C1 (HCFC1).

[0040] Aspect 18 is the method of aspect 17, wherein the one or more mutations in PR- DUB complex associated components OGT, KDM1B, FOXK1, FOXK2, BAP1, ASXL1, ASXL2, ASXL3, and / or HCFC1 comprises one or more gain of function mutations.

[0041] Aspect 19 is the method of aspect 13, wherein the disease is associated with diseased cells with one or more mutations in a TET2 encoding gene.

[0042] Aspect 20 is the method of aspect 19, wherein the one or more mutations in a TET2 encoding gene comprises one or more loss of function mutations.

[0043] Aspect 21 is the method of aspect 20, wherein the disease is associated with diseased cells comprising one or more mutations in one or more genes encoding ASXL1, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2.

[0044] Aspect 22 is the method of any one of aspects 1 to 21, wherein the individual is administered an additional therapy.

[0045] Aspect 23 is the method of aspect 22, wherein the additional therapy is surgery, radiation, chemotherapy, hormone therapy, and / or immunotherapy.

[0046] Aspect 24 is the method of aspect 22 or 23, wherein the additional therapy comprises administration of an inhibitor of TET2.

[0047] Aspect 25 is the method of aspect 24, wherein the inhibitor of TET2 comprises C35 and / or TETi76.

[0048] Aspect 26 is the method of any one of aspects 22 to 25, wherein the additional therapy comprises administration of an inhibitor of NSUN1 and / or NSUN2.

[0049] Aspect 27 is the method of any one of aspects 22 to 26, wherein the additional therapy comprises administration of an inhibitor of NSUN2.

[0050] Aspect 28 is the method of any one of aspects 22 to 27, further comprising administration of 5-AzaC.

[0051] Aspect 29 is the method of any one of aspects 1 to 27, further comprising a step of diagnosing the disease in the individual.

[0052] Aspect 30 is the method of any one of aspects 1 to 29, wherein the disease is is associated with diseased cells characterized as comprising an open chromatin state relative to non-diseased cells of the same developmental lineage.

[0053] Aspect 31 is the method of any one of aspects 1 to 30, wherein administering the one or more MBD6 inhibitors results in promotion of a closed chromatin state in one or more diseased cells in the individual.

[0054] Aspect 32 is the method of any one of aspects 1 to 31, wherein the disease is characterized as pro-inflammatory.

[0055] Aspect 33 is the method of any one of aspects 1 to 32, comprising inhibition of diseased cell proliferation.

[0056] Aspect 34 is the method of any one of aspects 1 to 33, wherein administering the one or more MBD6 inhibitors decreases m5C levels in one or more chromatin associated RNA (caRNA) and / or decreases association of one or more caRNA with a PR-DUB complex in one or more diseased cells in the individual.

[0057] Aspect 35 is the method of aspect 34, wherein administering the one or more MBD6 inhibitors increases oxidation of m5C in the one or more caRNA and / or inhibits installation of m5C in the one or more caRNA in one or more diseased cells in the individual.

[0058] Aspect 36 is the method of aspect 34 or 35, wherein the one or more caRNA comprises or consists essentially Long Terminal Repeat (LTR) RNAs.

[0059] Aspect 37 is the method of any one of aspects 34 to 36, wherein the one or more caRNA comprises one or more of the caRNAs described in Table 1.

[0060] Aspect 38 is the method of any one of aspects 34 to 37, wherein the one or more caRNA comprises one or more of the caRNAs described in Table 2.

[0061] Aspect 39 is the method of any one of aspects 34 to 38, wherein the one or more caRNA comprise a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 100-614.

[0062] Aspect 40 is the method of any one of aspects 34 to 39, wherein the one or more caRNA comprise a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs 104 or 107.

[0063] Aspect 41 is the method of any one of aspects 34 to 40, wherein the one or more caRNAs comprise between 20-50 caRNAs.

[0064] Aspect 42 is the method of any one of aspects 34 to 41, wherein the one or more caRNAs comprise a chromatin associated regulatory RNA (carRNA) and / or a chromatin associated repeat RNA sequence.

[0065] Aspect 43 is the method of any one of aspects 34 to 42, wherein the one or more caRNAs comprise or consist essentially of endogenous retroviral RNAs.

[0066] Aspect 44 is the method of aspect 43, wherein the endogenous retroviral RNAs comprise or consists essentially of endogenous retrovirus-K (ERVK).

[0067] Aspect 45 is the method of any one of aspects 1 to 44, wherein histone ubiquitination is increased in one or more diseased cells after administration of the one or more MBD6 inhibitors.

[0068] Aspect 46 is the method of aspect 45, wherein the histone ubiquitination comprises or consists of ubiquitination on H2A.

[0069] Aspect 47 is the method of aspect 46, wherein the histone ubiquitination occurs at a site comprising or consisting essentially of H2AK119.

[0070] Aspect 48 is the method of any one of aspects 1 to 46, wherein administering the one or more MBD6 inhibitors modifies histone methylation in one or more diseased cells in the individual.

[0071] Aspect 49 is the method of aspect 48, wherein a histone that comprises modified methylation comprises or consists essentially of H3.

[0072] Aspect 50 is the method of aspect 49, wherein the histone comprises modified methylation on a methylation site comprising or consisting essentially of H3K27me3.

[0073] Aspect 51 is the method of any one of aspects 48 to 50, wherein the histone that comprises modified methylation is localized near or at a genetic locus overlapping with a histone that comprises modified histone ubiquitination.

[0074] Aspect 52 is the method of any one of aspects 1 to 51, wherein the one or more inhibitors of MBD6 comprise a polynucleotide at least partially complementary to a gene encoding MBD6.

[0075] Aspect 53 is the method of aspect 52, wherein the polynucleotide comprises a short hairpin RNA and / or small interfering RNA.

[0076] Aspect 54 is the method of aspect 52, wherein the polynucleotide comprises a sequence at least 80% complementary to at least 15, 20, 25, or more than 25 contiguous nucleotides of any one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.

[0077] Aspect 55 is the method of any one of aspects 52 to 54, wherein the polynucleotide comprises modified RNA phosphoramidites.

[0078] Aspect 56 is the method of any one of aspects 52 to 55, wherein the polynucleotide comprises RNA with one or more 2ʹ-O-Methyl (2ʹ-OMe) or 2ʹ-O-Methoxyethyl (2ʹ-MOE) modifications.

[0079] Aspect 57 is the method of aspect 56, wherein the polynucleotide comprises RNA with every nucleotide comprising a 2ʹ-MOE modification.

[0080] Aspect 58 is the method of any one of aspects 52 to 57, wherein the polynucleotide comprises RNA with one or more phosphorothioate bonds.

[0081] Aspect 59 is the method of any one of aspects 52 to 58, wherein the polynucleotide is comprised within a lentiviral particle and / or nanoparticle.

[0082] Aspect 60 is the method of any one of aspects 52 to 59, wherein the inhibitor of MBD6 comprises more than one polynucleotide.

[0083] Aspect 61 is the method of any one of aspects 1 to 51, wherein the one or more inhibitors of MBD6 comprise a proteolysis targeting chimera.

[0084] Aspect 62 is a method of promoting histone ubiquitination in a cell comprising contacting the cell with one or more inhibitors of methyl-CpG-binding domain protein 6 (MBD6).

[0085] Aspect 63 is the method of aspect 62, wherein promoting histone ubiquitination comprises or consists of promoting ubiquitination on H2A.

[0086] Aspect 64 is the method of aspect 63, wherein the histone ubiquitination site comprises or consists essentially of H2AK119.

[0087] Aspect 65 is the method of any one of aspects 62 to 64, wherein the cell comprises one or more mutations in one or more genes encoding TET2, ASXL1, IDH1, IDH2, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2.

[0088] Aspect 66 is the method of any one of aspects 62 to 65, wherein the cell comprises one or more mutations in one or more genes encoding RING1A / B, PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, PCGF6, OGT, KDM1B, FOXK1, FOXK2, BAP1, ASXL1, ASXL2, ASXL3, and / or HCFC1.

[0089] Aspect 67 is a method of decreasing m5C levels in chromatin associated RNA (caRNA) in a cell, comprising contacting the cell with one or more inhibitors of MBD6.

[0090] Aspect 68 is the method of aspect 67, wherein the caRNA comprise or consists essentially of LTR RNAs.

[0091] Aspect 69 is the method of aspect 67 or 68, wherein the caRNA comprise or consists essentially of endogenous retroviral RNAs.

[0092] Aspect 70 is the method of aspect 69, wherein the endogenous retroviral RNAs comprise or consists essentially of endogenous retrovirus-K (ERVK).

[0093] Aspect 71 is a method of treating a disease in an individual, wherein the disease is characterized by cells comprising one or more mutations in TET2, ASXL1, IDH1, IDH2, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2 encoding genes, the method comprising administering one or more inhibitors of MBD6, TET2, and / or NSUN2 to the individual in need thereof.

[0094] Aspect 72 is a manufactured article for use in performing the method of any one of aspects 1 to 71.

[0095] Aspect 73 is a kit comprising means for performing the method according to any one of aspects 1 to 71.

[0096] Aspect 74 is a fusion protein comprising a sequence at least 80%, 85%, 90%, 95%, or 100% identical to any one or more of SEQ ID NOs: 1-4, 11-14, or 15-20.

[0097] Aspect 75 is the fusion protein of aspect 74, wherein the fusion protein comprises a sequence at least 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 19- 20.

[0098] Aspect 76 is a composition comprising the fusion protein of aspect 74 or 75.

[0099] Aspect 77 is a method of treating a disease associated with aberrant m5C RNA methylation in an individual, comprising administering the fusion protein and / or composition of any one of aspects 74 to 76.

[0100] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. Inventions described herein maybe better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0102] FIGs. 1A-1E, Elevated chromatin accessibility following Tet methylcytosine dioxygenase 2 (TET2) depletion was facilitated by Paraspeckle Component 1 (PSPC1) through its RNA binding activity in mouse embryonic stem cells (mESCs). FIGs.1A and 1B: Comparisons of chromatin accessibility (FIG.1A) and global nascent RNA synthesis rate (FIG.1B) of WT and Tet2 knockout (KO) mESCs. Representative images were selected from three independent experiments. FIG.1A, Left: assay of transposase-accessible chromatin with visualization (ATAC-see) visualizing global chromatin accessibility. Scale bar: 20 µm. The nucleus was counterstained with Hoechst 33342 (Hoechst). Right: quantification of relative fluorescence intensity of ATAC-see signal. FIG. 1B, Left: 5-ethynyl uridine (EU) incorporation followed by fluorescence imaging visualizing RNA synthesis rates. Scale bar: 20 µm. The nucleus was counterstained with Hoechst 33342 (Hoechst). Right: quantification of relative fluorescence intensity of EU signal. P values were determined using a Mann Whitney Wilcoxon Test. FIG. 1C: PSPC1 facilitated RNA binding of TET2 to repress DNA transcription. RNA synthesis rates of WT and Pspc1 KO mESCs were assessed by EU incorporation followed by fluorescence imaging. RNA synthesis rates of Pspc1 KO mESCs transfected with empty vector (EV), or expression vectors encoding wild-type mouse PSPC1, or PSPC1 mutant with impaired RNA binding abilities were measured with EU incorporation. P values were determined using a Mann Whitney Wilcoxon Test. FIG.1D: GSEA enrichment analysis was performed with genes upregulated (upDEGs) upon Pspc1 depletion in mESCs as the gene list and expression log2foldchange (log2FC) upon Tet2 knockout in mESCs cells as the rank list. Top: gene expression was quantified with total RNA-seq (GSE103269 and GSE48518), Bottom: gene expression was quantified with chromatin associated RNA-seq (caRNA-seq; this study). FIG. 1E: Box plot illustrating gene expression log2FC (quantified with caRNA-seq, this study) upon Pspc1 knockout or Tet2 knockout. Genes were categorized into two groups based on whether or not they were targeted by CXXC5 or PSPC1 in mESCs. P values were calculated by a nonparametric two-tailed Wilcoxon-Mann-Whitney test.

[0103] FIGs. 2A-2G, TET2 depletion led to elevated chromatin associated RNA (caRNA) m5C methylation and abundance, resulting in an upregulation of downstream gene expression in mESCs. FIG. 2A: TET2 oxidized RNA 5-methylcytosine to 5- hydroxylmethylcytosine. RNA 5-methylcytosine levels (m5C / C) (left) and 5- hydroxylmethylcytosine levels (hm5C / C) (right) in the chromatin-associated fraction were measured by mass spectrometry. P values were determined using unpaired Student’s t test withWelch’s correction. *P = 0.038 and **P = 0.0033 for comparison between WT and Tet2 KO for m5C and hm5C, respectively. Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG.2B: Proportion of m5C-marked peaks on repeat RNA and non- repeat RNA regions in WT mESCs (2,951 in non-Repeat RNAs; and 2,673 (47.5%) in Repeat RNAs). FIG.2C: Average profile and heatmap depicting the m5C level in WT mESCs, along with the corresponding ATAC-seq signal in WT and Tet2 KO mESC cells. The regions analyzed were m5C-marked peaks located within repeat RNA regions. FIG. 2D: Dot plot displaying m5C enrichment in various repeat RNA families (e.g., Long Terminal Repeat (LTR), Long Interspersed Nuclear Element (LINE), and Short Interspersed Nuclear Element (SINE)). The size of each dot corresponds to the number of loci that were m5C methylated in WT mESCs. The results showed both L1 in LINE and ERVK in the LTR class exhibited a significant enrichment of m5C methylation and that these m5C-marked repeat RNAs were associated with increased local chromatin accessibility. FIG.2E: The relative m5C methylation level of the Intracisternal A-particle (IAP) and L1 RNAs in WT, Tet2 KO and Pspc1 KO mESCs. P values were determined using unpaired Student’s t test with Welch’s correction. *P = 0.04 and *P = 0.01 for comparison between WT and Tet2 KO for LINE-1 and IAP, respectively; *P = 0.04 and **P = 0.003 for comparison between WT and Pspc1 KO for LINE- 1 and IAP, respectively; Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG. 2F: Cumulative curve showing the log2FC in the caRNA abundance of IAPEz-int repeats family between Tet2 KO and WT mESCs. Loci within the IAPEz-int repeats family were categorized into groups according to whether or not they were m5C methylated. P values were calculated by a nonparametric two-tailed Wilcoxon-Mann- Whitney test. FIG.2G: Tethering of the catalytic domain (CD) of TET2 (SEQ ID NO: 21) to the IAP RNAs led to RNA m5C hypomethylation and DNA 5mC hypermethylation. mESCs stably expressing dCas13b-TET2(CD) (shown in the figure as WT; SEQ ID NO: 27) or a catalytically dead (HxD mutation) version (dCas13b-TET2(CD)HxD (SEQ ID NO: 29), shown in the figure as HxD) fusion protein and guide RNA sequence downstream of a Tet operator (TetO)-controlled H1 promotor (H1-2O2) were used for IAP RNA targeting. DNA 5mC methylation at IAP loci (bottom) and RNA m5C methylation (top) on IAP transcripts were measured using MeDIP and MeRIP, respectively, followed by qPCR at indicated time points post doxycycline (DOX) treatment in each cell line. P values were determined using paired Student’s t test by comparing values at corresponding time points with values at “0 hour”, individually. DNA methylation: for dCas13b-TET2(CD)-HxD, **P = 0.0087, *P = 0.0164, *P = 0.0346, *P = 0.0338, **P = 0.0048, *P = 0.027 for 8h, 12h, 16h, 20h, 36h, and 72h versus0h, respectively; for dCas13b-TET2(CD), *P = 0.037, *P = 0.016 and **P = 0.0012 for 36h, 48h and 72h versus 0h, respectively. RNA methylation: for dCas13b-TET2(CD)-HxD, **P = 0.0029 for 12h versus 0h; for dCas13b-TET2(CD), ***P = 0.0003, ***P = 0.0010, **P = 0.0022, **P = 0.0026, **P = 0.0023, **P = 0.0019, **P = 0.0017, **P = 0.0017, and **P = 0.0022 for 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h and 72h versus 0h, respectively. NS denotes not significant (P > 0.05). Error bars represent mean ± standard deviation (S.D.) for three independent experiments.

[0104] FIGs.3A-3G, The activity of TET2 on RNA dictated the increased chromatin accessibility caused by TET2 depletion in mESCs. FIG. 3A: Bar graphs illustrating the overlapping ratios (Jaccard index) between enhancer, promoter, or repeats regions, respectively, with DNA hypermethylated or hypomethylated regions in Tet2 knockout mESCs. FIG.3B: Measurements of the average histone modification levels, as well as input signals, in WT and Tet2 KO mESCs at DNA hypermethylated regions (detected in Tet2 KO mESCs). FIG. 3C: Depicts the average ATAC signals in WT versus Tet2 KO, and WT versus Pspc1 KO mESCs at DNA hypermethylated regions (detected in Tet2 KO mESCs). FIG. 3D: Displays bar graphs illustrating the overlapping ratios (Jaccard index) of the genomic binding sites of PSPC1, TET2, and CXXC5 with DNA hypomethylated regions (detected in Tet2 knockout mESCs). FIG.3E: Measurements of the average histone modification levels, as well as input signals, in WT and Tet2 KO mESCs at DNA hypomethylated regions (detected in Tet2 KO mESCs. FIG. 3F: Depicts the average ATAC signals in WT versus Tet2 KO, and WT versus Pspc1 KO mESCs at DNA hypomethylated regions (detected in Tet2 KO mESCs). FIG. 3G: Displays bar graphs illustrating the overlapping ratios (Jaccard index) of RNA m5C peaks with the genomic binding sites of PSPC1, TET2, and CXXC5 in WT mESCs. DNA hypermethylated and hypomethylated regions were defined by Hon, Gary C et al. (GSE48519). PSPC1 ChIP-seq (GSE150399), TET2 ChIP-seq (GSE115964) and CXXC5 ChIP-seq (GSE132025).

[0105] FIGs. 4A-4L, Tet2 knockout enhanced MBD6 binding and induced H2AK119ub deubiquitylation, leading to elevated downstream gene expression in mESCs and mLK cells (mouse Hematopoietic Progenitor Cells (HPCs) that can give rise to myeloid or erythroid lineages, e.g., Lin-c-kit+). FIG. 4A: Bar graphs illustrating the overlapping ratios (Jaccard index) of various histone modification marked regions with DNA hypomethylated regions (detected with Tet2 KO) in mESCs. FIG. 4B: TET2 tethering increased the H2AK119ub level at IAP loci. H2AK119ub levels at IAP loci were measured by CUT&Tag followed by qPCR analysis in mESCs. Tethering of the catalytic domain of TET2(TET2(CD)) fusion protein with dCas13b (dCas13b-TET2(CD), shown in the figure as WT; SEQ ID NO: 27) or a catalytically dead (HxD mutation) version (dCas13b-TET2(CD)-HxD, shown in the figure as HxD; SEQ ID NO: 29) to IAP RNAs was induced by doxycycline (DOX) addition to the culture medium. Cells were harvested at eight hours post DOX treatment. P values were determined using unpaired Student’s t test with Welch’s correction by comparing values with values under condition “-Dox”, individually. *P = 0.010 for IAP +Dox for dCas13b-TET2(CD). NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG.4C: MBD6 enriched m5C- modified RNA in mESCs. rRNA-depleted RNAs crosslinked (CLIP) to Flag-tagged MBD6 with ultraviolet light (254 nm) were purified and digested to single nucleosides prior to mass spectrometry analysis. P values were determined using unpaired Student’s t test with Welch’s correction. ***P = 0.0008. Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG. 4D: MBD6 preferentially bound m5C-modified oligonucleotide probe. EMSA showing the binding preferences of the purified MBD domain of MBD6 fused to maltose binding protein (MBP) with C, m5C, or hm5C-containing single stranded oligonucleotide probes (100 nM). Sequences of the oligonucleotide probes (SEQ ID NOs: 95-99) were designed according to the motif identified by CLIP-Seq in mESCs, with X representing C, m5C, or hm5C. Gradient for MBP-MBD protein was 0, 0.1, 0.5, 2.5, 12, 50, and 100 μM. FIG. 4E: Mbd6 or Nsun2 knockdown rescued global chromatin accessibility caused by TET2 deficiency. DNase-TUNEL experiments were performed with accessible chromatin sites labeled by Alexa Fluor 488 fluorophore and quantified by flow cytometry. FIG. 4F: Volcano plot depicting the gene expression log2FC comparing Tet2- / -with WT mLK cells. Gene expression levels were quantified using RNA-seq in WT and Tet2- / -mLK cells. FIG.4G: Volcano plot depicting the log2FC of the H2AK119ub levels comparing Tet2- / -with WT mLK cells at repeat regions, with IAPEz-int regions showing an overall decreased H2AK119ub level upon Tet2 KO in mLK cells. FIG.4H: Depicts results of colony formation of replating assays of WT and Tet2- / -mLK cells transfected with control (shNC) or Mbd6 shRNA (shMbd6) plasmids, and then cultured in the presence of indicated cytokines (see Methods). Colony counts were scored every 7 days. Numbers of colonies after each replating were quantified. Statistical analyses were conducting by comparing number of colonies between shNC versus shMbd6 in WT or Tet2- / -groups, respectively. P values were determined using unpaired Student’s t test with Welch’s correction. ****P < 0.0001 for 2ndreplating, ****P < 0.0001 for 3rdreplating, and ****P < 0.0001 for 4threplating. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG.4I:Flow cytometry analyses of WT and Tet2- / -mLKs transfected with shNC or shMbd6 plasmids. mLKs were isolated from total BM cells of 6–8 weeks old mice and transfected with shNC or shMbd6 by electroporation 7 days before analyses. Frequency of hematopoietic stem and progenitor cells (HSPCs) was quantified by fractions of LK cells in the pool. P values were determined using unpaired Student’s t test with Welch’s correction by comparing values between shNC and shMbd6 groups. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. Differentiation towards monocyte / macrophages and granulocytes was quantified by fractions of CD11b+cells in the pool. P values were determined using unpaired Student’s t test with Welch’s correction. *P = 0.021 and *P = 0.023 for Tet2- / -shNC versus shMbd6, respectively. Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG. 4J: Venn diagram illustrating the overlap between the upregulated genes (upDEGs) by Tet2 knockout and the downregulated genes (downDEGs) by the treatment of IAP-targeting anti-sense oligonucleotide (ASO; SEQ ID NO: 89) in mLK cells. Two-tailed P values were calculated using the Fisher’s Exact Test. FIG.4K: GSEA enrichment analysis was conducted using genes downregulated (downDEGs) upon the treatment of ASO in mLKs as the gene list and expression log2FC upon Tet2 knockout in mLK cells as the rank list. The gene expression levels were quantified using total RNA-seq in WT and Tet2- / -mLK cells. FIG.4L: The correlation of gene expression log2FC between Tet2- / -versus WT and ASO targeting IAP versus control ASO (NC ASO; SEQ ID NO: 90) in mLK cells. Altered genes were associated with transcription misregulation in cancer.

[0106] FIGs. 5A-5K, MBD6 exhibited synergistic effects with TET2 deficiency in transcriptional regulation through the m5C-H2AK119ub axis in leukemia cells. FIG.5A: Proliferation curves showing that MBD6 knockdown significantly attenuated the proliferation of the TET2 mutant cell line SKM-1. MBD6 knockdown by two individual siRNAs (siMBD6- 1 and siMBD6-2, respectively) in three different human leukemia cells (THP-1, K-562, and SKM-1) were performed. Non-targeting siRNA (siNC) was used as a control. P values were determined using unpaired Student’s t test with Welch’s correction by comparing MTS signals at 96 hours of siMBD6-1 or siMBD6-2 versus siNC groups, respectively. ****P < 0.0001 and ***P = 0.0002 for THP-1; **P = 0.0044 and ***P = 0.0004 for K-562; ****P < 0.0001 and ****P < 0.0001 for SKM-1. Line plots represent mean ± standard deviation (S.D.) for six independent experiments. FIG. 5B: Bar plots summarizing proliferation of WT or TET2 KO K-562 or THP-1 cells with or without MBD6 knockdown. Cells stably expressing non-targeting shRNA (shNC) or shRNA targeting MBD6 RNA (shMBD6) were constructed via lentiviraltransduction. Cell proliferation was monitored with MTS assay at 96-hour post dilution. P values were determined using unpaired Student’s t test with Welch’s correction by comparing MTS signals at 96 hours of WT versus TET2 KO groups with MBD6 knockdown. ***P = 0.0008 for THP-1 cells and ****P < 0.0001 for K-562 cells. Line plots represent mean ± standard deviation (S.D.) for four independent experiments. FIG.5C: NOD scid gamma (NSG) mice were transplanted with K-562 (left) or THP-1 (right) cells as indicated, and the animals overall survival was measured and is depicted with the Kaplan-Meier estimator (WT shNC, n = 5; WT shMBD6, n = 5; TET2 KO shNC, n = 5; TET2 KO shMBD6, n = 5). **P = 0.0054 for WT, shMBD6 versus TET2 KO, shMBD6 K-562 cells and *P = 0.013 for WT, shMBD6 versus TET2 KO, shMBD6 THP-1 cells were determined by a log-rank Mantel–Cox test. FIG.5D: A negative correlation in repeat RNA abundance log2FC between TET2 KO versus WT and between MBD6 KD versus control in TET2 KO K-562 cells was observed. FIG.5E: Boxplot displaying the log2FC of repeat RNA abundance between TET2 KO versus WT, and between MBD6 KD versus control in TET2 KO K-562 cells. The repeat RNAs were categorized into two groups based on whether or not they were m5C methylated. P values were calculated by a nonparametric two-tailed Wilcoxon-Mann-Whitney test. FIG. 5F: The average profile of H2AK119ub around H2AK119ub peak center and flanking 2.5 kb regions in control versus MBD6 KD, and WT versus TET2 KO K-562 cells. FIG.5G: Depicts a cumulative curve of the log2FC of H2AK119ub between TET2 KO versus WT, and between MBD6 KD versus control K-562 cells at repeat loci. P values were calculated by a nonparametric two-tailed Wilcoxon- Mann-Whitney test. FIG. 5H: Dot plot displaying the m5C enrichment at various repeat families. The size of each dot corresponded to the number of loci that exhibited m5C methylation in WT K-562 cells. FIG.5I: Depicts average H2AK119ub modification levels in control versus MBD6 KD, and WT versus TET2 KO K-562 cells around LTR12C / ERV1 / LTR loci. FIG.5J: Correlation of gene expression log2FC between TET2 KO versus WT, and MBD6 KD versus control in TET2 KO K-562 cells. The genes were associated with various signaling pathways, including apoptosis and cell cycle. FIG. 5K: Schematic representations showing how the RNA m5C oxidation activity of TET2 on chromatin-associated RNA regulated the chromatin state through H2AK119ub deubiquitylation and its contribution to the progression of leukemia.

[0107] FIGs. 6A-6E, TET2 depletion led to global transcriptional activation in mESCs. FIG.6A: The mutation profiles of TET family proteins (TET1, TET2, and TET3) in different diseases were obtained from cbioportal.org. MDS denotes Myelodysplastic Syndromes (MDS IWG, IPSSM, NEJM Evidence 2022); MPN denotes MyeloproliferativeNeoplasms (CIMR, NEJM 2013); AML denotes Acute Myeloid Leukemia (OHSU, Nature 2018). FIG. 6B: TET2 protein is not covalently linked to a DNA-binding CXXC domain. Canonical amino acid sequences of human TET1-3 proteins and annotated domain structures were plotted (individual UniProt IDs: TET1 (Q8NFU7), TET2 (Q6N021) and TET3 (O43151)). The CXXC DNA binding domains and catalytic domains near the C-terminus were annotated. FIG.6C: Cumulative curve showing the nascent RNA transcription rate in WT and Tet2 KO mESCs. P values were calculated by a nonparametric two-tailed Wilcoxon-Mann- Whitney test. FIG.6D: Purities of different subcellular fractions of mESCs were validated with western blot. GAPDH was used as cytosolic (Cyt) protein marker, SNRP70 was used as nuclear soluble (Nuc) protein marker, and H3 was used as chromatin (Chr) marker. Equal aliquots of each isolated fractions were loaded onto each lane to enable direct comparison. The image shown is representative of three independent experiments. FIG. 6E: GSEA enrichment analysis was performed with genes downregulated (downDEGs) upon Pspc1 depletion in mESCs as the gene list and expression log2FC upon Tet2 KO in mESCs cells as the rank list. Left: gene expression was quantified with total RNA-seq (GSE103269 and GSE48518), Right: gene expression was quantified with caRNA-seq (this study).

[0108] FIGs.7A-7C, TET2 depletion activated caRNA expression in mESCs. FIG.7A: Volcano plot illustrating the chromatin associated regulatory RNA (carRNA) abundance log2FC comparing Tet2 KO with WT mESCs, along with the statistical significance of their changes. carRNAs were categorized into eRNAs, paRNAs, and repeat RNAs. P values for the comparison of expression levels between two groups were obtained using the Wald test by DESeq2. FIG.7B: Boxplots displaying the log2FC of carRNA abundance comparing Tet2 KO with WT, and Pspc1 KO with WT mESCs. The different groups of carRNAs, including eRNAs, paRNAs, and repeat RNAs, were further categorized into two subgroups based on whether or not they were marked with RNA m5C methylation. P values were calculated by a nonparametric two-tailed Wilcoxon-Mann-Whitney test. FIG. 7C: Average profile and heatmap depicting the m5C levels in WT mESCs, along with the ATAC-seq signal in WT and Tet2 KO mESC cells. The regions analyzed were m5C-marked peaks.

[0109] FIGs. 8A-8D, TET2 and PSPC1 regulated chromatin-associated LTR RNA m5C methylation and local chromatin state. FIG.8: Average profile and heatmap illustrating the m5C levels of different families of repeat RNAs in WT mESCs, along with the corresponding ATAC-seq signals in Tet2 KO versus WT, and Pspc1 KO versus WT mESC cells. The regions analyzed belong to the IAPEz-int (FIG.8A), RLTR10 (FIG.8B), L1MdA_I (FIG.8C), and L1MdA_II (FIG.8D) repeats families, respectively.

[0110] FIGs.9A-9B, NSUN2 installed m5C in caRNAs. FIG.9A: NSUN2 was the main methyltransferase for chromatin-associated RNA m5C installation. Chromatin-associated RNAs from WT mESCs transfected with non-targeting siRNA (siNC), and siRNAs targeting Nsun2, or Trdmt1 were measured by mass spectrometry. Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. ***P = 0.0005 by comparing values between siNsun2 and siNC. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG.9B: Volcano plots illustrating the gene expression level log2FC comparing Tet2 KO versus WT, Pspc1 KO versus WT, and Nsun2 KD versus control in mESCs, along with the statistical significance of their changes. The data presented were obtained using caRNA-seq. P values for the comparison of expression levels between two groups were obtained using the Wald test by DESeq2.

[0111] FIGs. 10A-10E, shows the results of targeted blockade and demethylation of IAP RNAs. FIG. 10A: qPCR analysis of IAP RNA fragments revealed main m5C sites near the 5′ end. Metagene profiles of IAP RNA m5C landscape in WT mESCs and qPCR primers for different regions were designed and individual amplicons were indicated. Provided is a zoom-in showing the 5′ end sequence (SEQ ID NO: 615) that contained multiple m5C sites as revealed by bisulfite sequencing (m5C sites indicated by asterisks). IAP-targeting ASO (SEQ ID NO: 89) was designed to interfere with m5C deposition near the IAP 5′ end (left). MeRIP- qPCR with primers targeting different amplicons enabled region-specific analysis of the IAP m5C level upon IAP ASO treatment in mESCs (see Methods, Table 2, for listing of qPCR primers). A non-targeting ASO (NC ASO; SEQ ID NO: 90) was used as control. P values were determined using unpaired Student’s t test with Welch’s correction by comparing values of IAP-targeting ASO with NC ASO by individual amplicons. ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ***P = 0.00086, and **P = 0.0046 for amplicon 1, 2, 3, 4, and 5, respectively. FIG.10B: IAP ASO treatment slightly downregulated IAP RNA levels in mESCs. P value was determined using unpaired Student’s t test with Welch’s correction by comparing values of IAP ASO with NC ASO, **P < 0.01. Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG. 10C: qPCR analysis of chromatin accessibility at IAP loci following ASO treatment. Hoxd13 loci was used as a control. P values were determined using unpaired Student’s t test with Welch’s correction by comparing values of IAP ASO with NC ASO for different loci. *P = 0.018 for IAP loci, IAP ASO versus NC ASO. NS denotes not significant (P > 0.05). FIG.10D: Presents a schematic of experiments comprising tethering of the catalytic domain of TET2 to the IAP RNA with the Tet-On system. Provided are schematics showing the design of CRISPR tethering system of the TET2 catalytic domain (TET2(CD)) toIAP RNA (e.g., as denotated in FIG.10A). For RNA targeting, mESCs stably expressed fusion proteins dCas13b-TET2(CD) or a catalytically dead version (CD with mutations in the HxD motif) (dCas13b-TET2(CD)-HxDmut) thereof, and a guide RNA sequence downstream of a Tet operator (TetO)-controlled H1 promotor (H1-2O2). Validation of the tethering system of dCas13b-TET2(CD)-HxDmut (bottom left) or dCas13b-TET2(CD) (bottom right) to IAP. Doxycycline (DOX)-induced expression of the tethered TET2(CD) fusion protein to IAP RNAs could be observed starting at 4 hours of DOX treatment. Bindings of the TET2(CD) fusion protein to IAP RNAs were measured by RIP-qPCR at different time points after DOX treatment. Relative enrichment values were obtained by calculating recovery rate by dividing IP to input and normalizing all time points to 0 h. P values were determined using paired Student’s t test by comparing values at the indicated time points with values at “0 hour”, individually. Left: **P = 0.0049, **P = 0.0028, **P = 0.0055, *P = 0.011, *P = 0.024, **P = 0.0083, ***P = 0.0003, **P = 0.0016, and **P = 0.0087 for 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h and 72h versus 0h, respectively. Right: ***P = 0.00025, ***P < 0.00021, **P = 0.0038, *P = 0.019, ****P < 0.0001, **P = 0.0028, ***P = 0.00078, ***P = 0.00024, and ****P < 0.0001 for 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h and 72h versus 0h, respectively. Dots and error bars represent mean ± standard deviation (S.D.) for three individual experiments. FIG. 10E: Acute broad-spectrum TET enzyme inhibition resulted in a caRNA m5C hypermethylation response earlier than a gDNA 5mC hypermethylation response. Abundance of DNA 5mC in genomic DNA (top) or RNA m5C chromatin-associated RNA (caRNA) (bottom) were measured in WT mESCs at indicated time points (0 hour, 4 hour, 8 hour, 12 hour, 16 hour, 20 hour, 24 hour, 36 hour, 48 hour, and 72 hour) post application of a small-molecule inhibitor of all TET enzymes (TETi-C35) in the medium at the final concentration of 5 μM. The medium was refreshed every eight hours. P values were determined using unpaired Student’s t test with Welch’s correction. *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. Top: *P = 0.0406, **P = 0.0088, and **P = 0.0014 for 36h, 48h, and 72h versus 0h, respectively. Bottom: *P = 0.0196, ***P = 0.0004, **P = 0.0016, ***P = 0.0002, ***P = 0.0003, ***P = 0.0002, ***P = 0.0004, and **P = 0.0027 for 8h, 12h, 16h, 20h, 24h, 36h, 48h and 72h versus 0h, respectively.

[0112] FIGs. 11A-11B, TET2 depletion associated increased chromatin accessibility in mESCs was caused by the oxidative activity of TET2 on RNA, rather than DNA. FIG. 11A: Shows the correlation between DNA methylation differences in Tet2 KO versus WT mESCs and changes in downstream gene transcription. The carRNAs were categorized intodifferent groups, including eRNA, paRNA, and repeat RNAs. Within each group of carRNAs, they were further divided into 50 bins based on the ranked DNA methylation differences upon Tet2 KO in mESCs. FIG. 11B: Bar graphs illustrating the overlapping ratios (Jaccard index) of the genomic binding sites of PSPC1, TET2, and CXXC5 with DNA hypermethylated regions (detected in the Tet2 KO mESCs).

[0113] FIGs.12A-12C, Decreased H2AK119ub and H3K27me3 were observed at the IAP loci following TET2 inhibition. FIG.12A: Bar graphs illustrating the overlapping ratios (Jaccard index) of regions marked by different histone modifications with DNA hypermethylated regions (detected in the Tet2 KO mESCs). FIG.12B: Volcano plot depicting the log2FC of H2AK119ub in Tet2 KO versus WT mESCs at the repeat family loci, with corresponding statistical significance. P values for the comparison of expression levels between two groups were obtained using the Wald test by DESeq2. FIG.12C: H2AK119ub at the IAP loci showed faster response to TET inhibition than H3K27me3. H2AK119ub and H3K27me3 chromatin bindings at IAP (left), MERVL (middle), and LINE (right) loci were measured through the CUT&Tag procedure followed by qPCR with loci-specific primers. P values were determined using unpaired Student’s t test with Welch’s correction by comparing values at the indicated time points with values at “0 hour”, individually. *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. Left: for H2AK119ub, ****P < 0.0001, ****P < 0.0001, ***P = 0.00014, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, and ****P < 0.0001 for 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h and 72h versus 0h, respectively; for H3K27me3, **P = 0.0044, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, and ****P < 0.0001 for 12h, 16h, 20h, 24h, 36h, 48h and 72h versus 0h, respectively. Middle: for H2AK119ub, *P = 0.020, ***P = 0.00015, ***P = 0.00056, ****P < 0.0001, ***P = 0.00067, ***P = 0.00069, ***P = 0.00045, **P = 0.0012, and **P = 0.0011 for 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h and 72h versus 0h, respectively; for H3K27me3, **P = 0.0056, *P = 0.010, ***P = 0.0003, **P = 0.007, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, and ****P < 0.0001 for 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h and 72h versus 0h, respectively. Bar plots represent mean ± standard deviation (S.D.) for three individual experiments.

[0114] FIGs. 13A-13E, MBD5 and MBD6 preferentially bound m5C-modified RNA. FIG. 13A: The MBD domains of human MBD5 and MBD6 were found to have distinct characteristics within the MBD family proteins. Sequence alignment of human MBD family proteins MBD domains (MBD1 (SEQ ID NO: 620), MBD2 (SEQ ID NO: 622), MBD3 (SEQID NO: 619), MBD4 (SEQ ID NO: 621), MBD5 (SEQ ID NO: 617), MBD6 (SEQ ID NO: 616), and MeCP2 (SEQ ID NO: 618)) showed differences in the MBD region. Conservation scores were calculated by Jalview and conserved residues among analyzed proteins were colored. FIG. 13B: Validation of Flag-MBD5 and Flag-MBD6 overexpressed mESCs. Averaged transcript expression was indicated on top of each column. P values were determined using unpaired Student’s t test with Welch’s correction by comparing values in different cell lines with WT mESCs, individually. For Mbd5,0.0001, and ***P =0.00011 for Flag- Mbd5 and Flag-Mbd6 versus WT, respectively; for Mbd6, ****P < 0.0001 for Flag-Mbd6 versus WT. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG. 13C: MBD5 and MBD6 bound significantly more RNA relative to DNA in mESCs. Nucleic acids crosslinked to MBD5 or MBD6 by UV (254 nm) were end-labeled with biotin and imaged with ECL. Samples were digested by RNase A / T1 or DNase I. The image shown is representative of three independent experiments. Western blot was quantified with ImageJ, Analyze gel module. P values were determined using unpaired Student’s t test with Welch’s correction by comparing values at the indicated time points with values of the control with no RNase or DNase treatment, individually. For Flag-Mbd5, ***P = 0.00088 and ***P = 0.00077 for RNase only and RNase + DNase, respectively; for Flag-Mbd6, **P = 0.0017 and **P = 0.0015 for RNase only and RNase + DNase, respectively. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG.13D: MBD5 enriched m5C- modified RNA in mESCs. rRNA-depleted (non-Ribo) RNAs crosslinked (CLIP) to Flag- tagged MBD5 with ultraviolet light (254 nm) were purified and digested to single nucleosides prior to mass spectrometry analysis. Abundance of m5C in RNA was obtained by normalizing the m5C level by C level in the sample. P values were determined using unpaired Student’s t test with Welch’s correction. ***P = 0.0008 for comparisons between CLIP and input samples. Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG. 13E: EMSA showing the binding preferences of the purified MBD domain of MBD6 (SEQ ID NO: 3) fused to the maltose binding protein (MBP) with C, m5C, or hm5C-containing double- stranded oligonucleotide probes. The MBP-MBD6(MBD) protein was incubated with 100 nM fluorescently labeled single-stranded or double-stranded probes. Free unbound probes were separated with RNA-protein complexes by electrophoresis. Individual dissociation coefficients (KD) were determined as described in the exemplary methods. Sequences of the oligonucleotide probes were designed according to the motif identified by CLIP in mESCs. The purity of the single-stranded probe (ss probe) and double-stranded probe (ds probe) was validated byelectrophoresis. The fluorescently labeled probes were visualized by in-gel fluorescence with FAM.

[0115] FIGs. 14A-14E, MBD6 regulated the level of H2AK119ub and the half-life of m5C-modified RNAs. FIG. 14A: Venn diagram illustrating the overlap (3558 shared sites, 45%) between the RNA binding sites of MBD5 (401 MBD5 sites, 5%) and MBD6 (3917 MBD6 sites, 50%) (CLIP-seq, this study). FIG. 14B: Depicts qPCR results showing repeat RNAs expression levels upon Mbd5 or Mbd6 knockdowns. Transcript expression levels were obtained by normalizing target RNA levels to Gapdh. P values were determined using unpaired Student’s t test with Welch’s correction. For quantification of IAP levels, *P = 0.045 and *P = 0.010 for siMbd5 and siMbd6 versus siNC; for quantification of MERVL levels, **P = 0.0098 and ***P = 0.0008 for siMbd5 and siMbd6 versus siNC; for quantification of MusD levels, *P = 0.028 for siMbd6 versus siNC. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG. 14C: Is a representative Western blot showing that MBD6 significantly regulated H2AK119ub levels in mESCs. The relative of H2AK119ub was obtained by normalizing the intensity of H2AK119ub blot to the intensity of GAPDH blot. Western blots were quantified by ImageJ analyze gel module. P values were determined using unpaired Student’s t test with Welch’s correction. *P = 0.028 for siMbd6 versus siNC. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG. 14D: MBD6 stabilized m5C-modified chromatin-associated RNAs. rRNA-depleted chromatin-associated RNAs were purified and digested to single nucleosides prior to mass spectrometry analysis. The abundance of m5C in RNA was obtained by normalizing m5C level to C in the sample. P values were determined using unpaired Student’s t test with Welch’s correction. *P = 0.020 for comparisons between siMbd6 versus siNC. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG. 14E: RT- qPCR analysis of the IAP RNA stability upon Tet2 KO or Mbd6 knockdown in mESCs. P values were determined using unpaired Student’s t test with Welch’s correction by comparing WT versus Tet2 KO or WT mESCs transfected with negative control siRNA (siNC) and siRNA targeting Mbd6 (siMbd6). Tet2 knockout and WT, *P = 0.015 and *P = 0.014 for 3 hour and 6 hour; siMbd6 and siNC, **P = 0.0082 for 3 hour. NS denotes not significant (P > 0.05). Dot plots represent mean ± standard deviation (S.D.) for three independent experiments.

[0116] FIGs. 15A-15D, Mbd6 knockdown suppressed TET2-loss-mediated abnormal HSPC proliferation in mouse LK cells. FIG.15A: Shows average ATAC-seq profiles at the gene level in WT and Tet2- / -mLK cells. FIG.15B: Are qPCR results validating efficiency ofshRNA-enabled Mbd6 knockdown in mLK cells. Transcript expression levels were obtained by normalizing target RNA levels to that of Actb. P values were determined using unpaired Student’s t test with Welch’s correction. ****P < 0.0001 and ****P < 0.0001 for shMbd6 versus shNC in WT and Tet2- / -, respectively. Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG. 15C: Are representative photographs showing colony formation during replating assays of mLK cells in WT or Tet2- / -mLK cells upon Mbd6 knockdown or control shRNA administration (shNC). FIG. 15D: Show representative flow cytometry results characterizing HSPC frequency in suspension cultures in the presence of indicated cytokines (see Methods) (top, c-Kit and Lineage as markers) and differentiation (bottom, CD11b as marker) in WT or Tet2- / -mLK cells upon Mbd6 knockdown or control shRNA administration (shNC).

[0117] FIGs.16A-16D, Targeted m5C oxidation on IAP RNA significantly suppressed the TET2-loss-mediated abnormal HSPC proliferation and differentiation of mLK cells. FIG. 16A: Flow cytometry analyses of dCas13d-TET2CD fusion protein and guide RNA transfection efficiency in mLK cells. EGFP was used to visualize guide RNA expression. mCherry was used to visualize dCas13d-TET2CD protein. Comparable transfection efficiencies were obtained for all conditions. Representative flow cytometry analyses were shown here as density plots and 10,000 gated single cells were used for each analysis. FIG. 16B: Replating assay of WT and Tet2- / -mLK cells transfected with guide RNA targeting the IAP RNA using either the RfxdCas13d fusion protein with TET2 catalytic domain (TET2CD) or the inactivated mutant (TET2HxDCD). Tet2- / -mLK cells were isolated from total BM cells of 6–8 weeks old mice prior to electroporation. Colony counts were scored every 7 days. Representative images of the 2ndreplating were shown. Numbers of colonies after each replating were quantified. Statistical analyses were conducted by comparing number of colonies between Tet2- / -TET2CD versus Tet2- / -TET2HxDCD at different replating, respectively. P values were determined using unpaired Student’s t test with Welch’s correction. ****P < 0.0001 for 2ndreplating, ****P < 0.0001 for 3rdreplating, and ****P < 0.0001 for 4threplating. Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG.16C: Flow cytometry analyses of HSPC frequency of WT and Tet2- / -mLK cells transfected with guide RNA targeting the IAP RNA using the RfxdCas13d fusion protein with TET2CD or TET2HxDCD. Lin-c-Kit+mouse HSPCs (mLK cells) were isolated from total BM cells of 6–8 weeks old mice prior to transfection by electroporation. HSPC frequency was quantified by fractions of Lin-c-Kit+cells in the pool. Representative flow cytometry analyses were shown here as density plots and 30,000 gated single cells were used for each analysis. Pvalues were determined using unpaired Student’s t test with Welch’s correction. **P = 0.0019 for Tet2- / -TET2HxDCD versus WT TET2HxDCD and *P = 0.021 for Tet2- / -TET2HxDCD versus Tet2- / -TET2CD, respectively. Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG.16D: Flow cytometry analyses of differentiation of WT and Tet2- / -mLK cells transfected with guide RNA targeting the IAP RNA using the RfxdCas13d fusion protein with TET2CD or TET2HxDCD. Lin-c-Kit+mouse HSPCs were isolated from total BM cells of 6–8 weeks old mice prior to transfection by electroporation. Differentiation towards monocyte / granulocytes and granulocytes was quantified by fractions of CD11b+cells in the pool. Representative flow cytometry analyses were shown here as density plots and 30,000 gated single cells were used for each analysis. P values were determined using unpaired Student’s t test with Welch’s correction. ****P < 0.0001 for Tet2- / -TET2HxDCD versus WT TET2HxDCD and *P = 0.027 for Tet2- / -TET2HxDCD versus Tet2- / -TET2CD, respectively. Bar plots represent mean ± standard deviation (S.D.) for three individual experiments.

[0118] FIGs. 17A-17D, Targeted ASO blockade of IAP RNA suppressed the TET2- loss-mediated abnormal HSPC proliferation and differentiation in mLK cells. FIG.17A: Flow cytometry analyses of ASO transfection efficiency in mLK cells. ASOs labeled by APC fluorophores (SEQ ID NOs: 88-90) were visualized by the corresponding laser channel. Comparable transfection efficiencies were obtained for different ASOs. Representative flow cytometry analyses were shown here as density plots and 10,000 gated single cells were used for each analysis. FIG. 17B: Replating assay of WT and Tet2- / -mLK cells transfected with control (NC ASO; SEQ ID NO: 90), MERVL-targeting (MERVL ASO; SEQ ID NO: 88) or IAP-targeting (IAP ASO; SEQ ID NO: 89) steric antisense oligos. Tet2- / -mLK were isolated from total BM cells of 6–8 weeks old mice prior to ASO transfection by electroporation. Colony counts were scored every 7 days. Numbers of colonies after each replating were quantified. Statistical analyses were conducting by comparing number of colonies between Tet2- / -NC ASO versus Tet2- / -MERVL ASO or Tet2- / -IAP ASO, respectively. P values were determined using unpaired Student’s t test with Welch’s correction. **P = 0.0055 for 1streplating, **P = 0.0048 and ***P = 0.00048 for 2ndreplating, ***P = 0.00015 and ****P < 0.0001 for 3rdreplating, ****P < 0.0001 and ****P < 0.0001 for 4threplating. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG.17C: Flow cytometry analyses of the HSPCs frequency of WT and Tet2- / -mLK cells transfected with control (NC ASO), MERVL-targeting (MERVL ASO) or IAP- targeting (IAP ASO) antisense oligos. mLK cells were isolated from total BM cells of 6–8 weeks old mice prior to ASO transfection by electroporation. The frequency of HSPCs wasquantified by fractions of Lin-c-Kit+cells in the pool. Representative flow cytometry analyses were shown here as density plots and 30,000 gated single cells were used for each analysis. P values were determined using unpaired Student’s t test with Welch’s correction. **P = 0.0057 for Tet2- / -NC ASO versus WT NC ASO. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG.17D: Flow cytometry analyses of differentiation of WT and Tet2- / -mLK cells transfected with control (NC ASO), MERVL-targeting (MERVL ASO) or IAP-targeting (IAP ASO) antisense oligos. mLK cells were isolated from total BM cells of 6–8 weeks old mice prior to ASO transfection by electroporation. Differentiation towards monocyte / macrophages and granulocytes was quantified by fractions of CD11b+cells in the pool. Representative flow cytometry analyses were shown here as density plots and 30,000 gated single cells were used for each analysis. P values were determined using unpaired Student’s t test with Welch’s correction. *P = 0.019 for Tet2- / -NC ASO versus WT NC ASO. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three individual experiments.

[0119] FIGs. 18A-18C, IAP RNA blockade suppressed gene expression profiles caused by TET2 deficiency in mLK cells. FIG.18A: Venn diagram displaying the overlap among differentially expressed genes upon Tet2 KO or the treatment with ASO targeting IAP RNA in mLK cells (total RNA-seq). Barplot depicting the odds ratio of upDEGs following Tet2 KO, in comparison to both upDEGs and downDEGs after treatment with ASO targeting IAP RNA in mLK cells. Two-tailed P values were calculated using the Fisher’s Exact Test. FIG.18B: KEGG pathway enrichment analysis of genes that were up-regulated by Tet2 KO and down-regulated by the treatment with ASO targeting IAP RNA in mLK cells. One-sided P values were calculated using Fisher's Exact test, and the size of the circle represents the level of significance, with larger circles indicating greater significance and smaller circles indicating lower significance. FIG. 18C: The correlation of gene expression log2FC between Tet2 KO versus WT and ASO targeting IAP versus control in mLK cells. The genes were associated with various signaling pathways, including MAPK signaling pathways (top) and C-type lectin receptor signaling pathways (bottom).

[0120] FIGs. 19A-19E, MBD6 knockdown potently inhibited TET2-deficient leukemia cells. FIG. 19A: Proliferation curves showing that MBD6 knockdown attenuated leukemia cell proliferation. Knockdown of MBD6 by two individual siRNAs (siMBD6-1 and siMBD6-2) in human leukemia cells (TF-1 and OCI-AML3) were performed. Cell proliferations were monitored with MTS assay at different time points post viral transduction (24 hour, 48 hour, 72 hour and 96 hour). MTS signals at different time points were normalizedto those at 24 hour to yield relative MTS signals. Non-targeting siRNA (siNC) was used as a control. P values were determined using unpaired Student’s t test with Welch’s correction by comparing MTS signals at 96 hours of siMBD6-1 or siMBD6-2 versus siNC groups, respectively. ***P = 0.0002 and ****P < 0.0001 for TF-1. NS denotes not significant (P > 0.05). Line plots represent mean ± standard deviation (S.D.) for six independent experiments. FIG.19B: (Left) Western blot analysis of TET2 levels in WT and TET2 KO K-562 and THP- 1 cells. ACTB was used as a loading control. Representative image of three individual experiments was shown. (Right) qPCR results validating efficiency of shRNA-enabled MBD6 knockdown in K-562 or THP-1 cells. Transcript expression levels were obtained by normalizing target RNA levels to that of GAPDH. P values were determined using unpaired Student’s t test with Welch’s correction. ***P = 0.0092 and ****P < 0.0001 for shMBD6 versus shNC in WT and TET2 KO K-562 cells, respectively; ***P = 0.0018 and ***P = 0.0001 for shMBD6 versus shNC in WT and TET2 KO THP-1 cells, respectively. Bar plots represent mean ± standard deviation (S.D.) for three individual experiments. FIG. 19C: Western blots assaying the global H2AK119ub level or cleaved Poly[ADP-ribose] Polymerase 1 (PARP1) upon MBD6 knockdown. Statistical tests were performed using paired Student’s t test with Welch’s correction by comparing WT versus TET2 KO groups in shNC or shMBD6 samples. For cleaved PARP1 levels, *P = 0.049 for WT versus TET2 KO of shMBD6 samples. For H2AK119ub levels, *P = 0.048 and *P = 0.036 for WT versus TET2 KO of shNC and shMBD6 samples, respectively. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG.19D: RT-qPCR validation of NSUN2 knockdown in K-562 and THP-1 cells. Relative transcript abundance was quantified by normalizing to that of GAPDH. P values were determined using unpaired Student’s t test with Welch’s correction by comparing siNSUN2 with siNC. *P = 0.010 for K-562 and *P = 0.011 for THP-1. Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG.19E: Proliferation curves showing that NSUN2 knockdown attenuated TET2 knockout mediated cell growth in K-562 and THP-1 cells. Cell proliferation was monitored with MTS assay at different time points post dilution (24 hour, 48 hour, 72 hour and 96 hour). MTS signals at different time points were normalized to those at 24 hour to yield relative MTS signals. P values were determined using unpaired Student’s t test with Welch’s correction by comparing MTS signals at 96 hours of WT versus TET2 KO groups. ***P = 0.0003 for K-562 cells; **P = 0.0026 for THP-1 cells. NS denotes not significant (P > 0.05). Line plots represent mean ± standard deviation (S.D.) for four independent experiments.

[0121] FIGs.20A-20C, MBD6 knockdown attenuated leukemia progression in mouse PDX models regardless of leukemia cell TET2 mutational status. FIG. 20A: NSG mice were intravenously injected with K-562 (FIG. 20B) or THP-1 (FIG. 20C) cells, and the animal’s overall survival was monitored and analyzed using the Kaplan-Meier estimator (WT shNC, n = 5; WT shMBD6, n = 5; TET2 KO shNC, n = 5; TET2 KO shMBD6, n = 5; see FIG. 5C). After transplantation, PB was harvested from submandibular vein, and BM was isolated from the tibias and femurs for human CD33+(for K-562) or CD33+ / CD45+(for THP-1) chimerism detection and quantifications. FIG. 20B: Fractions of CD33+cells in BM or PB isolated from NSG mice xenotransplanted with tumor cells (K-562) were quantified by flow cytometry analyses. P values were determined using unpaired Student’s t test with Welch’s correction. *P = 0.017 and *P = 0.047 for TET2 KO shNC versus TET2 KO shMBD6 in BM and PB, respectively. NS denotes not significant (P > 0.05). Bar plots represent mean ± standard deviation (S.D.) for three independent experiments. FIG. 20C: Fractions of CD33+ / CD45+cells in BM or PB isolated from NSG mice xenotransplanted with tumor cells (THP-1) were quantified by flow cytometry analyses. P values were determined using unpaired Student’s t test with Welch’s correction. *P = 0.017 and *P = 0.021 for WT shNC versus WT shMBD6 and TET2 KO shNC versus TET2 KO shMBD6 in BM; **P = 0.0043 and *P = 0.045 for WT shNC versus WT shMBD6 and TET2- / -shNC versus TET2- / -shMBD6 in PB, respectively. Bar plots represent mean ± standard deviation (S.D.) for three individual experiments.

[0122] FIGs.21A- 21F, Chromatin-associated LTR RNAs were upregulated in TET2- deficiency leukemia cells. FIG.21A: Purities of different subcellular fractions of K-562 cells were validated with western blot. GAPDH was used as cytosolic (Cyt) protein marker, SNRP70 was used as nuclear soluble (Nuc) protein marker, and H3 was used as chromatin (Chr) marker. Equal aliquots of each isolated fractions were loaded onto each lane to enable direct comparison. FIG. 21B: Bar plots showing the changes of caRNA abundance (left) and total RNA abundance (right) relative to spike-in ERCC RNAs in WT, TET2 KO and TET2 KO & MBD6 KD K-562 cells (n = 3). FIG. 21C: Volcano plots depicting the log2FC of carRNA abundance, comparing TET2 KO with WT K-562 cells, and comparing MBD6 KD with control in TET2 KO K-562. The statistical significance of the changes were also determined and indicated on the y-axis. carRNAs were categorized into eRNAs, paRNAs, and repeat RNAs. P values for the comparison of expression levels between two groups were obtained using the Wald test by DESeq2. FIG.21D: Shows the negative correlation of repeat RNAs abundance log2FC when comparing TET2 KO with WT K-562 cells and when comparing MBD6 KD withcontrol in TET2 KO K-562 cells. FIG. 21E: Shows a scatter plot demonstrating the positive correlation between repeat RNAs abundance log2FC and their target gene expression log2FC when comparing TET2 KO versus WT in K-562 cells. FIG.21F: Boxplots displays the log2FC of carRNA abundance between TET2 KO versus WT, and between MBD6 KD versus control in TET2 KO K-562 cells. The carRNAs, including eRNA, paRNAs and repeat RNAs, were categorized into two groups based on whether or not they were m5C methylated. P values were calculated by a nonparametric two-tailed Wilcoxon-Mann-Whitney test.

[0123] FIGs. 22A-22E, MBD6 enhanced the expression of TET2-regulated LTRs through H2AK119ub deubiquitylation. FIG.22A: Venn diagram illustrating the overlap of H2AK119ub peaks in control and MBD6 KD K-562 cells (4245 unique siNC peaks; 42,041 overlapping peaks; and 30,462 unique siMBD6 peaks), as well as in WT and TET2 KO K-562 cells (28,466 unique WT peaks; 36,776 overlapping peaks; and 7,214 unique TET2 KO peaks). FIG. 22B: Displays H2AK119ub modification levels at the gene level in control and MBD6 KD K-562 cells, as well as in WT and TET2 KO K-562 cells. FIG.22C: Displays cumulative curves of the log2FC of H2AK119ub between TET2 KO versus WT, and between MBD6 KD versus control K-562 cells at different genomic loci including enhancers (bottom) and promoters (top). P values were calculated by a nonparametric two-tailed Wilcoxon-Mann- Whitney test. FIG. 22D: Shows the correlation between log2FC of repeat RNAs abundance (TET2 KO vs. WT) and log2FC of H2AK119ub at the corresponding genomic loci (TET2 KO vs. WT, top; MBD6 KD vs. control, bottom) in K-562 cells. FIG.22E: Shows the correlation between the number of m5C methylated repeat RNAs and their respective m5C methylation levels in WT K-562 cells. Among LTR repeat RNAs, HERVH-int ranked top in terms of methylation enrichment, while LTR12C had the highest levels of methylation.

[0124] FIGs. 23A-23C, MBD6 deficiency resulted in downregulation of leukemia- related genes that were generally upregulated in TET2 depletion models. FIG.23A: Venn diagram illustrating the overlap of differentially expressed genes upon TET2 KO in control K- 562 cells, and upon MBD6 KD in TET2 KO K-562 cells. Barplot depicting the odds ratio of upDEGs following TET2 KO, in comparison to both upDEGs and downDEGs upon MBD6 KD in TET2 KO K-562 cells. Two-tailed P values were calculated using the Fisher’s Exact Test. FIG.23B: KEGG pathway enrichment analysis was performed on genes that were upregulated in TET2 KO in control K-562 cells, and downregulated in MBD6 KD in TET2 KO K-562 cells. One-sided P values were calculated using Fisher's Exact test, and the size of the circle represents the level of significance, with larger circles indicating greater significance and smaller circles indicating lower significance. FIG. 23C: Shows the correlations of geneexpression log2FC between TET2 KO versus WT, and MBD6 KD versus control in TET2 KO K-562 cells. The genes were found to be associated with various signaling pathways, including Th1 and Th2 cell differentiation, NF-kappa B signaling pathway, and C-type lectin receptor signaling pathways.

[0125] FIGs.24A-24B, TET2 inhibition and MBD6 inhibition acted synergistically to reduce cancer cell proliferative capacity. FIG.24A: Heatmap showing the proliferation of various cancer cell lines with shRNA-based knockdown of TET2 and / or MBD6. Expression plasmids for shRNAs were delivered into cells via lentiviral transduction with hygromycin B or puromycin resistance, respectively. Cell proliferation was measured by MTS assay (Promega) at 96-hours after transduction. FIG. 24B: Heatmap showing the proliferation of various leukemic cells with shRNA-based knockdown of TET2 and / or MBD6. The results showed that knockdown of TET2 and MBD6 exerted synergistic proliferation inhibition effects, particularly in leukemic cells and glioma cells.

[0126] FIG.25, TET2 knockout reduces H3K27me3 modifications at sites co-occupied with H2AK119ub. In human leukemia cells, H3K27me3 did not show a global change upon TET2 knockout (left panel). H3K27me3 peaks were then categorized into two subgroups according to whether or not they were overlapped with H2AK119ub (middle and right panels respectively). The results showed a dramatic reduction in H3K27me3 modification level upon TET2 knockout when these peaks were co-occupied with H2AK119ub (right panel). Similar changes were not observed at genomic regions exclusively marked with H3K27me3 modifications.

[0127] FIG. 26, Exemplary model of TET2, NSUN2, MBD6, and PR-DUB interactions with H2A. DETAILED DESCRIPTION I. Terminology

[0128] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0129] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0130] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combinationof B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or. It is specifically contemplated that A, B, or C may be specifically excluded from an aspect.

[0131] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0132] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0133] The term sequence as used herein in reference to a polynucleotide refers to the nucleotide sequence such as “A” for adenosine, “G” for guanine, “C” for cytosine, “T” for thymine, “U” for uracil, “I” for inosine, and “N” for “A” / “C” / “U” / “T” / “G” / “I”.

[0134] As used herein, the terms “individual," “subject,” and “patient” are used interchangeably and can refer to a human or non-human.

[0135] As used herein, a “protein” “peptide” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild- type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or “engineered protein” or “engineered polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as catalytic activity, RNA-binding activity, etc.

[0136] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or producedby solid phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an enzymatic domain, such as a deaminase domain, or a fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0137] In certain aspects the size of a protein or polypeptide (wild-type or modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues or nucleic acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting, localization, linking, etc.).

[0138] In certain aspects, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding an enzyme, or a fragment, derivative, or variant thereof, polynucleotides sufficient for targeting a complex to specific loci, polynucleotides sufficient to mediate caRNA methylation (e.g., m5C) level modifications, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, ancillary components of CRISPR / Cas systems, functional oligonucleotides, donor constructs, rescue constructs, and complementary sequences of the foregoing described herein. Nucleic acids encoding fusion proteins that include the proteins / polypeptides described herein are also contemplated. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids, etc.).

[0139] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (e.g., nucleic acids typically 200 residues or less, or 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0140] In certain respects, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization), or a functional RNA species, such as but not limited to, CRISPR / Cas system ancillary components, linking elements, targeting RNAs, etc. As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, mutants, and functional RNA species. A nucleic acid encoding all or part of a polypeptide and / or functional RNA species may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide and / or functional RNA species. It also is contemplated that a particular polypeptide and / or functional RNA species may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein and / or RNA species.

[0141] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods known in the art and / or described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide and / or functional RNA species that has at least 90%, 95%, or above, identity to an amino acid sequence and / or RNA sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0142] Nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, enhancers, destabilization sites, restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0143] The term “near or at”, particularly when used in the context of chromosomal distance, as used herein generally refers to a distance of less than, 4.5-5.5 kb (e.g., less than 5.5 kb, less than 5 kb, less than 4kb, less than 4.5 kb, less than 3 kb, less than 2 kb, or less than 1 kb, or any range derivable therein) between two locations.

[0144] A "diseased cell" as used herein generally refers to a cell associated with a disease or disorder comprising one or more genetic mutations relative to a healthy cell in the same individual. The one or more genetic mutations (including but not limited to, large or small truncations, point mutations, indels, complex fusions, etc.,) result in an abnormal phenotype relative to cells that do not comprise the one or more mutations (e.g. "healthy cells"). Abnormal phenotypes may include reduced or increased fitness, such as phenotypes associated with cancerous cells. In certain aspects, a diseased cell comprises one or more mutations in one or more oncogenes and / or tumor suppressor genes. In some aspects, a diseased cell may comprise one or more mutations in one or more genes encoding ten-eleven translocation (tet) methylcytosine dioxygenase 2 (TET2), ASXL transcriptional regulator 1 (ASXL1), ASXL Transcriptional Regulator 2 (ASXL2), ASXL Transcription Regulator 3 (ASXL3), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), tumor protein p53 (p53), DNA (cytosine-5-)-methyltransferase 3A (DNMT3A), Janus kinase 2 (JAK2), Protein Phosphatase Mn2+ / Mg2+-Dependent 1D (PPM1D), Spliceosome Factor 3b1 (SF3B1), Serine and ArginineRich Splicing Factor 2 (SRSF2), a component of a canonical and / or non-canonical Polycomb Repressive Complex (PRC) (e.g., E3 Ubiquitin Ligase RING1A / B, Polycomb Group Ring Finger 1 (PCGF1), Polycomb Group Ring Finger 2 (PCGF2), Polycomb Group Ring Finger 3 (PCGF3), Polycomb Group Ring Finger 4 (PCGF4), Polycomb Group Ring Finger 5 (PCGF5), and / or Polycomb Group Ring Finger 6 (PCGF6)), Polycomb Repressive-Deubiquitinase (PR- DUB) complex associated components O-linked N-acetylglucosamine Transferase (OGT), Lysine Demethylase 1B (KDM1B), Forkhead Box K1 (FOXK1), Forkhead Box K2 (FOXK2), BRCA1 Associated Protein 1 (BAP1), and / or Host Cell Factor C1 (HCFC1).

[0145] The term “proteolysis targeting chimera” or “PROTAC” as used herein refers to a chimeric molecule comprising two protein binding molecules: one capable of engaging an E3 ubiquitin ligase and another that binds to a target protein meant for degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein via the proteosome. In some aspects, a PROTAC disclosed herein may target an MBD5 protein, an MBD6 protein, a TET2 protein, an NSUN1 protein, an an NSUN2 protein, or any combination thereof. In some aspects, a PROTAC disclosed herein may target an MBD6 protein.

[0146] It is specifically contemplated that any limitation discussed with respect to one aspect of the invention may apply to any other aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an aspect set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0147] A variety of aspects are discussed throughout this application. Any aspect discussed with respect to one aspect applies to other aspects as well and vice versa. Each aspect described herein is understood to be aspects that are applicable to all aspects. It is contemplated that any aspect discussed herein can be implemented with respect to any method or composition, and vice versa. Furthermore, compositions and kits can be used to achieve methods disclosed herein.

[0148] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of” any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.II. Overview

[0149] Chromatin-associated regulatory RNAs can offer a platform for dynamic chromatin regulation. Recent studies have shown that the N6-methyladenosine (m6A) modification on carRNAs can contribute to critical roles in global and local chromatin state in mESCs and during mouse early development, with the methyltransferase METTL3 serving as a writer (e.g., installer), with binding proteins such as YTHDC1 serving as readers, and with enzymes such as FTO serving as an eraser to reversibly & dynamically guide transcriptional regulation. As described herein, the inventors have discovered that other RNA modifications may be present on caRNAs, such as carRNAs, and that these modifications can also dynamically and reversibly affect chromatin regulation.

[0150] Ten-eleven translocation enzyme 2 (TET2) is known to mediate DNA 5mC oxidation and has been established as a tumor suppressor, particularly in blood malignancies. However, TET2 mutations are known to result in global DNA hypomethylation, instead of hypermethylation. This apparent inconsistency, where loss of a DNA methylation marker oxidizer (TET2, a DNA demethylase) results in RNA hypermethylation, is a puzzle that was heretofor not explained. As described herein, the inventors have discovered that TET2 mediates RNA 5-methylcytosine (m5C) methylation, particularly on caRNAs, such as carRNAs (and in particular, in the LTR family RNAs), to regulate chromatin state and transcription. Further, as described herein, the inventors have discovered that methyl-CpG binding domain protein 6 (MBD6) preferentially recognizes RNA m5C over DNA 5mC, and particularly in repeat RNA and / or regulatory RNA sequences. MBD6 can then recruit BRCA1 associated protein-1 (BAP1) associated complexes to mediate H2AK119ub deubiquitylation for increased gene activation / open chromatin state. Relatedly, m5C oxidation by TET2 can act on these caRNAs to antagonize gene activation through the m5C-MBD6-BAP1 deubiquitylation axis. Loss of TET2 can lead to caRNA m5C hypermethylation, more open chromatin, and / or widespread DNA hypomethylation that can result in activation of genes critical to disease states (e.g., critical to leukemogenesis), helping to explain accelerated malignancies associated with TET2 inactivation (e.g., accelerated myeloid malignancy, see FIG.5K).

[0151] The results provided herein also reveal a potential bimodal function for TET2. Wherein TET2 can bind CXXC4 / 5 to mediate DNA 5mC oxidation at enhancers; but, when recruited by PSPC1, TET2 can mediate RNA m5C oxidation (such as but not limited to repeat RNA, caRNA, and / or carRNA). This RNA m5C oxidation activity by TET2 can dictate global chromatin regulation in cells, such as but not limited to mESCs, HPSCs, and leukemia cells. As described herein, the inventors have discovered a new NSUN2-TET2-MBD6-BAP1 axis inchromatin and transcription regulation that functions through caRNA m5C (e.g., repeat carRNA m5C). Practically, the results provided herein reveal targets for therapeutic intervention in disease states associated with mutants in TET2 and / or TET2 pathways (e.g., that function upstream and / or downstream of TET2 on similar and / or the same substrates, for example but not limited to, NSUN1, NSUN2, MBD5, MBD6, ASXL1, ASXL2, ASXL3, IDH1, IDH2, etc.).

[0152] 5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in chromatin regulation has hitherto remained elusive. Herein, the inventors have elucidated an epigenetic regulatory pathway that can comprise, but is not limited to, chromatin associated RNA (caRNA), Ten-eleven translocation enzyme 2 (TET2), NOP2 / Sun RNA methyltransferase 2 (NSUN2), methyl-CpG binding domain protein 6 (MBD6), and the Polycomb repressive complexes (e.g., comprising PRC1, PRC2, and / or PR- DUB), which components can interact to influence chromatin state through histone modifications (see e.g., FIG. 26), and as such, influence the transcriptome and proteome of cells. As provided herein, the inventors show that this epigenetic regulatory pathway can be perturbed in certain diseases (for example but not limited to, cancers, in particular, leukemias), and show how modifications and / or manipulations of this epigenetic regulatory pathway can treat and / or mitigate certain diseases and symptoms / phenotypes associated therewith.

[0153] As shown herein, in some aspects, TET2 mediates chromatin-associated retrotransposon RNA m5C oxidation to suppress deubiquitylation of histone H2AK119ub and gene expression in at least mESCs, HSCs, and leukemic cells.

[0154] Mutation of TET2 has been described as driving myeloid malignancy initiation and progression. TET2 deficiency has also been described as leading to a globally opened chromatin state (e.g., euchromatin) and aberrant activation of genes, contributing to aberrant cell self-renewal (e.g., aberrant hematopoietic stem cell (HSC) self-renewal). However, the open chromatin consistently observed in TET2-deficient mouse embryonic stem cells, leukemic cells, and hematopoietic progenitor and stem cells (HPSCs) is inconsistent with TET2’s described role of DNA 5mC oxidation. As described herein, the inventors have shown that chromatin-associated RNA (caRNA, e.g., chromatin-associated retrotransposon RNA) 5- methylcytosine (m5C) can be recognized by the methyl-CpG-binding domain protein MBD6, which can guide deubiquitylation of nearby histone H2AK119ub to promote an open chromatin state. TET2 can oxidize m5C and antagonize this caRNA m5C associated MBD6-dependent H2AK119ub deubiquitylation. Thereby, TET2 depletion can lead to globally decreased H2AK119ub, more open chromatin, and increased / aberrant transcription in cells, such as stemcells. As such, in certain aspects, methods of treatment of diseases associated with diseased cells with TET2 mutations, such as inactivating mutations, can comprise inhibition of MBD6. In certain cases, TET2 mutant diseases (e.g., TET2 mutant human leukemias) can become dependent on this gene activation pathway. In certain cases where TET2 is not mutated in human leukemias, inhibition of TET2 and MBD6 can result in a synergistically lethal effect upon diseased cells (e.g., cancer cells). In certain aspects, inhibiting MBD6 protein activity (including silencing expression of MBD6) can selectively inhibit proliferation of TET2 mutant cells. As shown herein, in some aspects, inhibiting MBD6 protein activity (including silencing expression of MBD6) can selectively inhibit proliferation of TET2 mutant leukemic cells in vitro and in vivo. Together, findings provided herein at least reveal novel chromatin regulation pathways that comprise TET2 interaction with caRNA m5C marks, and oxidation thereof (and lack thereof in mutant TET2 pathway conditions). Furthermore, as provided herein, the identity of the downstream reader protein influencing chromatin regulation, MBD5 and / or MBD6 has been shown. In some aspects, MBD5 and / or MBD6 are targeted for inhibition and therapeutic intervention against mutant TET2 and / or mutant TET2 pathway associated malignancies.

[0155] In certain aspects, technologies provided herein can comprise a catalytic domain fused to a targeting element (e.g., dCas13d-TET2(CD); dCas13d-MBD6(MBD domain); dCas13d-NSUN2, etc.). In certain aspects, such fusion proteins can be utilized to provide site- directed modifications to RNA, such as but not limited to, caRNA.

[0156] In some aspects, technologies provided herein (e.g., compositions, methods etc. function broadly in mammalian cells, such as human cells. In some aspects, technologies provided herein can facilitate greater than or equal to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, RNA m5C installation at user-specified or non-specified RNA loci, in a population of RNA molecules. In some aspects, technologies provided herein can facilitate greater than or equal to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, RNA m5C removal at user- specified or non-specified RNA loci in a population of RNA molecules.

[0157] In some aspects, technologies provided herein can facilitate site specific RNA m5C modification (e.g., installation, reading, or removal) activity while maintaining low off-target RNA m5C modification activity. In some aspects, technologies provided herein can facilitate site specific RNA m5C modification with low levels of sequence motif bias.

[0158] In some aspects, the current disclosure provides technologies including at least polynucleotides, proteins, polypeptides, and / or vectors, and further provides methods and / or compositions comprising any one or more of the aforementioned components. In certain aspects, polynucleotides may encode sequences comprising RNA m5C modifying catalytic domains (including isolated domains, polypeptides, and / or proteins), engineered small nucleolar RNA (snoRNA), CRISPR / Cas proteins / polypeptides, and / or ancillary RNA components (e.g., CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), single guide RNA (sgRNA), etc.), antisense oligonucleotides (ASO), etc. In certain aspects, one or more of the polynucleotides and / or polypeptides can be engineered.

[0159] In certain aspects, polynucleotides, proteins, polypeptides, and / or peptide sequences for wild type or mutant versions of various genes, such as site-specific target genes, have been previously disclosed, and may be found in the recognized computerized databases. In certain aspects, polynucleotides, proteins, polypeptides, and / or peptide sequences for wild type versions of various effector proteins and / or RNA molecules, have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

[0160] It is contemplated that in compositions of the disclosure, there can be between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0161] In certain aspects, provided herein are methods, compositions, polynucleotides, polypeptides, and / or vectors comprising engineered RNA m5C modifying (e.g., installing, reading, or erasing) catalytic domains (e.g., including complete enzymes and / or components of enzymes), engineered Cas proteins, and / or guide oligonucleotides

[0162] The oligonucleotides, polypeptides, polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to at least, exactly, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750,775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1266, 1400, 1600, 1800, or 2000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOs: 1- 625. In specific aspects, the nucleic acid encoding the peptide or polypeptide is codon optimized for expression in a mammal. In certain aspects, the peptide or polypeptide is not naturally occurring and / or is in a combination of peptides or polypeptides.

[0163] The polypeptides of the disclosure may include at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, or 422 substitutions (or any range derivable therein). In some aspects, the substitution is with an alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0164] In some aspects, the polypeptide comprises one or more substitutions at one or more amino acid positions in any one of the proteins or polynucleotides encoding the same identified in SEQ ID NOs: 1-625, wherein each substitution is independently chosen from an amino acidselected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine; and wherein the polypeptide or polynucleotide encoding the same is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) sequence identity to one of SEQ ID NOs: 1-625.

[0165] In some aspects, the protein or polypeptide, or polynucleotide encoding the same may comprise amino acids or nucleotides 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530,531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139,1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649,1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, or 2060 of SEQ ID NOs: 1-625, or any other amino acid or nucleotide noted therein, and have or have at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) sequence identity to one of SEQ ID NOs: 1-625.

[0166] In some aspects, the protein, polypeptide, or nucleic acid may comprise, comprise at least, or comprise at most 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1266, 1400, 1600, 1800, or 2000, or more (or any derivable range therein) contiguous amino acids or nucleic acids of SEQ ID NOs: 1-625.

[0167] In some aspects, the polypeptide, protein, or nucleic acid may comprise at least, at most, or exactly 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1266, 1400, 1600, 1800, or 2000, or more (or any derivable range therein) contiguous amino acids or nucleic acids of SEQ ID NOs: 1-625 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to one of SEQ ID NOs: 1-625.

[0168] In some aspects there is a nucleic acid molecule or polypeptide starting at position 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, or 422, or any other nucleotide or amino acid of any of SEQ ID NOs: 1-625 and comprising at least, at most, or exactly 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1266, 1400, 1600, 1800, or 2000, or greater than 2000 (or any derivable range therein) contiguous amino acids or nucleic acids of any of SEQ ID NOs: 1-625.III. TET2 pathway, mutations and diseases associated therewith

[0169] Aspects of the present disclosure are directed to compositions and methods for treatment of diseases associated with abnormal m5C RNA, such as abnormal m5C RNA feature levels described herein.

[0170] Somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies. Genetic studies of patients with myeloid malignancies have identified recurrent somatic alterations in the majority of patients with myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs), and acute myeloid leukemia (AML). In some aspects, provided herein are methods of treating myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs), and / or acute myeloid leukemia (AML). Several studies have identified recurrent mutations of known and putative epigenetic modifiers in patients with myeloid malignancies. These include somatic mutations in chromatin-modifying enzymes and in DNA methyltransferases. In addition, biologic studies of recurrent chromosomal translocations, including MLL fusions, have shown that leukemogenic fusion proteins alter epigenetic regulation in hematopoietic cells, resulting in changes in chromatin state at specific loci. In certain aspects, provided herein are methods of treating MLL. Taken together, these data suggest that somatic alterations in genes that regulate the epigenetic state of hematopoietic cells are a common pathogenetic event in leukemogenesis. In certain aspects, provided herein are methods of treating diseases associated with somatic alterations in genes that regulate the epigenetic state of hematopoietic cells, such as but not limited to hematopoietic stem cells.

[0171] Somatic deletions and loss-of-function mutations in the ten-eleven translocation 2 (TET2) gene were identified in 10%–20% of patients with MDS and MPN; subsequent studies identified recurrent TET2 mutations in patients with chronic myelomonocytic leukemia (CMML) and AML, and demonstrated that TET2 mutations were associated with adverse outcome in intermediate-risk AML. Within the TET family of proteins, TET1, TET2, and TET3 have been shown to modify DNA by hydroxylating 5-methylcytosine (5mC), and the TET2 mutant proteins observed in these patients with myeloid malignancies have been shown to be deficient in this enzymatic function. Mutations that impact 5-hydroxymethylation represent a mechanism of transformation in myeloid malignancies (see e.g., Kelly Moran- Crusio et al., Tet2 Loss Leads to Increased Hematopoietic Stem Cell Self-Renewal and Myeloid Transformation. Cancer Cell 2011; which is incorporated herein by reference in its entirety for the purposes described herein).

[0172] Additionally, inactivating mutations in Tet methylcytosine dioxygenase 2 (TET2) have been detected in peripheral blood cells of a remarkable 5%-10% of adults greater than 65 years of age. The TET2 inactivating mutations have been reported to impart a hematopoietic stem cell advantage and resultant clonal hematopoiesis of indeterminate potential (CHIP) with skewed myelomonocytic differentiation. CHIP has been reported to be associated with an overall increased risk of transformation to a hematological malignancy, especially myeloproliferative and myelodysplastic neoplasms (MPN, MDS) and acute myeloid leukemia (AML), of approximately 0.5% to 1% per year. However, it is becoming increasingly possible to identify individuals at greatest risk, based on CHIP mutational characteristics. CHIP, and particularly TET2-mutant CHIP, has also been reported to be a significant risk factor for cardiovascular diseases, related in part to hyper-inflammatory progeny macrophages that carry TET2 inactivating mutations. Therefore, somatic TET2 mutations, and mutations in TET2 pathways, can contribute to myeloid expansion and innate immune dysregulation with age and can contribute to prevalent diseases in the developed world (e.g., cancer and / or cardiovascular disease).

[0173] MBD5 is a member of the methyl-CpG-binding domain (MBD) family. The MBD domain consists of approximately 70 residues (e.g., ~65 to ~75 residues) and is the minimal region required for a methyl-CpG-binding protein binding specifically to methylated DNA. In addition to the MBD domain, MBD5 protein contains a PWWP domain (Pro-Trp-Trp-Pro motif), which consists of 100-150 amino acids and is found in numerous proteins that are involved in cell division, growth and differentiation. Mutations in this gene have been reported to result in an autosomal dominant type of cognitive disability. The MBD5 protein has been reported to interact with the polycomb repressive complex PR-DUB which catalyzes the deubiquitination of a lysine residue of histone 2A. Haploinsufficiency of the MBD5 encoding gene has been reported to be associated with a syndrome involving microcephaly, intellectual disabilities, severe speech impairment, and seizures. In some aspects, MBD5 functions as an m5C RNA reader protein and / or reader complex component (e.g., as a guide / reader for the PR- DUB complex). Exemplary human wildtype MBD5 protein isoforms and encoding genes are provided as SEQ ID NOs: 5-8. Additional exemplary human wildtype MBD5 protein isoforms and encoding genes, and information related thereto, can be found in the NCBI database under Gene ID: 55777. In some aspects, methods disclosed herein comprise, consist essentially of, or consist of inhibition of MBD5. In some aspects, methods disclosed herein comprise administering one or more inhibitors of MBD5. The one or more inhibitors of MBD6 may, in some aspects, comprise a polynucleotide at least partially complementary to a gene encodingMBD5 (e.g., a short hairpin RNA and / or small interfering RNA). In some aspects, the one or more inhibitors of MBD5 comprise a proteolysis targeting chimera (PROTAC) targeting MBD5.

[0174] MBD6 is a member of the methyl-CpG-binding domain (MBD) family. The human MBD6 gene and encoded protein are relatively poorly characterized relative to other members of the MBD family. MBD6 has been reported to enable chromatin binding activity, and has been found to be located in the chromocenter, the fibrillar center, and the nucleoplasm. Mutations in MBD6 have been been reported to be implicated in autism spectrum disorder. In some aspects, MBD6 functions as an m5C RNA reader protein and / or reader complex component (e.g., as a guide / reader for the PR-DUB complex). Exemplary human wildtype MBD6 protein isoforms or polypeptides derived therefrom, and encoding gene are provided as SEQ ID NOs: 1-4. Additional exemplary human wildtype MBD6 protein isoforms and encoding genes, and information related thereto, can be found in the NCBI database under Gene ID: 114785. In some aspects, methods disclosed herein comprise, consist essentially of, or consist of inhibition of MBD6. In some aspects, methods disclosed herein comprise administering one or more inhibitors of MBD6. The one or more inhibitors of MBD6 may, in some aspects, comprise a polynucleotide at least partially complementary to a gene encoding MBD6 (e.g., a short hairpin RNA and / or small interfering RNA). In some aspects, the one or more inhibitors of MBD6 comprise a proteolysis targeting chimera (PROTAC) targeting MBD6.

[0175] NSUN1 (NOP2 nucleolar protein) has been reported to enable RNA binding activity, and has been reported to be involved in positive regulation of cell population proliferation; regulation of signal transduction by p53 class mediator; and ribosomal large subunit assembly. NSUN1 has been reported to localize to the nucleolus. In some aspects, NSUN1 functions as an m5C RNA writer protein and / or writer complex component. Exemplary human wildtype NSUN1 protein isoforms and encoding genes are provided as SEQ ID NOs: 9-10. Additional exemplary human wildtype NSUN1 protein isoforms and encoding genes can be found in the NCBI database under Gene ID: 4839. In some aspects, methods disclosed herein comprise, consist essentially of, or consist of inhibition of NSUN1. In some aspects, methods disclosed herein comprise administering one or more inhibitors of NSUN1. The one or more inhibitors of NSUN1 may, in some aspects, comprise a polynucleotide at least partially complementary to a gene encoding NSUN1 (e.g., a short hairpin RNA and / or small interfering RNA). In some aspects, the one or more inhibitors of NSUN1 comprise a proteolysis targeting chimera (PROTAC) targeting NSUN1.

[0176] NSUN2 (NOP2 / Sun RNA methyltransferase 2) is a methyltransferase that has been reported to catalyze the methylation of cytosine to 5-methylcytosine (m5C) in tRNAs, such as at position 34 of intron-containing tRNA(Leu)(CAA) precursors. The m5C modification in tRNA(Leu)(CAA) is necessary to stabilize the anticodon-codon pairing and correctly translate target mRNA. Alternatively spliced transcript variants encoding different isoforms have been noted for this gene. In some aspects, NSUN2 functions as an m5C RNA writer protein and / or writer complex component. Exemplary human wildtype NSUN2 protein isoforms and encoding genes are provided as SEQ ID NOs: 11-14. Additional exemplary human wildtype NSUN1 protein isoforms and encoding genes, and information related thereto, can be found in the NCBI database under Gene ID: 54888. In some aspects, methods disclosed herein comprise, consist essentially of, or consist of inhibition of NSUN2. In some aspects, methods disclosed herein comprise administering one or more inhibitors of NSUN2. The one or more inhibitors of NSUN2 may, in some aspects, comprise a polynucleotide at least partially complementary to a gene encoding NSUN2 (e.g., a short hairpin RNA and / or small interfering RNA). In some aspects, the one or more inhibitors of NSUN2 comprise a proteolysis targeting chimera (PROTAC) targeting NSUN2.

[0177] TET2 is a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. The TET2 protein has been reported to be involved in myelopoiesis, and defects in this gene have been reported to be associated with several myeloproliferative disorders. Two variants encoding different isoforms have been found for this gene. In some aspects, TET2 functions as an m5C RNA eraser protein and / or eraser complex component. Exemplary human wildtype TET2 protein isoforms, and polypeptides derived therefrom, and encoding genes are provided as SEQ ID NOs: 15-20. Additional exemplary human wildtype TET2 protein isoforms and encoding genes, and information related thereto, can be found in the NCBI database under Gene ID: 54790. In some aspects, methods disclosed herein comprise, consist essentially of, or consist of inhibition of TET2. In some aspects, methods disclosed herein comprise administering one or more inhibitors of TET2. The one or more inhibitors of TET2 may, in some aspects, comprise a polynucleotide at least partially complementary to a gene encoding TET2 (e.g., a short hairpin RNA and / or small interfering RNA). In some aspects, the one or more inhibitors of TET2 comprise a proteolysis targeting chimera (PROTAC) targeting TET2.

[0178] In some aspects, provided herein are technologies for site-specific and / or non-site- specific RNA m5C modification activity (e.g., m5C installation, reading / binding, and / or erasing). In some aspects, technologies provided herein can be utilized to increase levels ofm5C installation at one or more RNAs (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein), such as but not limited to RNAs identified in Table 1. In some aspects, technologies provided herein can be utilized to decrease levels of m5C installation at one or more RNAs (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein), such as but not limited to RNAs identified in Table 1. In some aspects, technologies provided herein can be utilized to increase levels of m5C recognition (e.g., reading) at one or more RNAs (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein), such as but not limited to RNAs identified in Table 1. In some aspects, technologies provided herein can be utilized to decrease levels of m5C recognition (e.g., reading) at one or more RNAs (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein), such as but not limited to RNAs identified in Table 1. In some aspects, technologies provided herein can be utilized to increase levels of m5C erasing (e.g., oxidation) at one or more RNAs (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein), such as but not limited to RNAs identified in Table 1. In some aspects, technologies provided herein can be utilized to decrease levels of m5C erasing (e.g., oxidation) at one or more RNAs (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein), such as but not limited to RNAs identified in Table 1.

[0179] In certain aspects, the size of a protein or polypeptide (wild-type or modified; including m5C writers, readers, and / or erasors, and / or other proteins / polypeptides described herein) may comprise, but are not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, or 4000, or more amino acid residues or nucleic acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also or alternatively, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).

[0180] In some aspects, a protein comprises, consists essentially of, or consists of an amino acid sequence or is encoded by a polynucleotide sequence, with about, exactly, or at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity to any one of SEQ ID NOs: 1-32.

[0181] In some aspects, an inhibitor of one or more proteins (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) described herein comprises, consists essentially of, or consists of a polynucleotide sequence with about, exactly, or at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity to a contiguous stretch of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides of any one of SEQ ID NOs: 1-32. SEQ ID NO: 1 – Wild type human MBD6 amino acid sequence MNGGNESSGADRAGGPVATSVPIGWQRCVREGAVLYISPSGTELSSLEQTRSYLLSDGTCKC GLECPLNVPKVFNFDPLAPVTPGGAGVGPASEEDMTKLCNHRRKAVAMATLYRSMETTCSHS SPGEGASPQMFHTVSPGPPSARPPCRVPPTTPLNGGPGSLPPEPPSVSQAFPTLAGPGGLFP PRLADPVPSGGSSSPRFLPRGNAPSPAPPPPPAISLNAPSYNWGAALRSSLVPSDLGSPPAP HASSSPPSDPPLFHCSDALTPPPLPPSNNLPAHPGPASQPPVSSATMHLPLVLGPLGGAPTV EGPGAPPFLASSLLSAAAKAQHPPLPPPSTLQGRRPRAQAPSASHSSSLRPSQRRPRRPPTV FRLLEGRGPQTPRRSRPRAPAPVPQPFSLPEPSQPILPSVLSLLGLPTPGPSHSDGSFNLLG SDAHLPPPPTLSSGSPPQPRHPIQPSLPGTTSGSLSSVPGAPAPPAASKAPVVPSPVLQSPS EGLGMGAGPACPLPPLAGGEAFPFPSPEQGLALSGAGFPGMLGALPLPLSLGQPPPSPLLNH SLFGVLTGGGGQPPPEPLLPPPGGPGPPLAPGEPEGPSLLVASLLPPPPSDLLPPPSAPPSN LLASFLPLLALGPTAGDGEGSAEGAGGPSGEPFSGLGDLSPLLFPPLSAPPTLIALNSALLA ATLDPPSGTPPQPCVLSAPQPGPPTSSVTTATTDPGASSLGKAPSNSGRPPQLLSPLLGASLLGDLSSLTSSPGALPSLLQPPGPLLSGQLGLQLLPGGGAPPPLSEASSPLACLLQSLQIPPE QPEAPCLPPESPASALEPEPARPPLSALAPPHGSPDPPVPELLTGRGSGKRGRRGGGGLRGI NGEARPARGRKPGSRREPGRLALKWGTRGGFNGQMERSPRRTHHWQHNGELAEGGAEPKDPP PPGPHSEDLKVPPGVVRKSRRGRRRKYNPTRNSNSSRQDITLEPSPTARAAVPLPPRARPGR PAKNKRRKLAP (SEQ ID NO: 1) SEQ ID NO: 2 – Wild type human MBD6 polynucleotide coding sequence ATGAATGGGGGCAATGAGAGCAGTGGAGCAGACAGAGCTGGGGGCCCTGTGGCCACATCTGT CCCCATCGGCTGGCAGCGCTGTGTGCGAGAGGGTGCTGTGCTCTACATCAGTCCAAGTGGCA CAGAGCTGTCTTCCTTGGAGCAAACCCGGAGCTACCTCCTCAGCGATGGGACCTGCAAGTGC GGTCTGGAGTGTCCACTTAATGTCCCCAAGGTTTTCAACTTTGACCCTTTGGCCCCGGTGAC CCCGGGTGGGGCTGGGGTGGGGCCAGCATCAGAGGAGGACATGACCAAGCTGTGCAACCACC GGCGGAAAGCTGTTGCTATGGCAACTCTGTACCGCAGCATGGAGACCACCTGCTCACACTCT TCTCCTGGAGAGGGAGCGAGCCCCCAAATGTTCCACACTGTGTCCCCAGGGCCCCCCTCTGC CCGCCCTCCCTGTCGAGTTCCTCCTACAACTCCACTTAATGGGGGTCCTGGCTCCCTTCCCC CAGAACCACCCTCAGTTTCCCAGGCCTTTCCCACTCTAGCAGGCCCTGGGGGGCTTTTCCCC CCAAGGCTTGCTGACCCAGTCCCTTCTGGGGGCAGTAGCAGCCCCCGTTTCCTCCCAAGGGG CAATGCCCCCTCTCCAGCCCCACCTCCTCCACCTGCTATCAGCCTCAATGCTCCCTCATACA ACTGGGGAGCTGCCCTCAGATCCAGCCTGGTGCCCTCTGACCTGGGCTCTCCTCCGGCCCCT CATGCCTCCTCCTCACCACCTTCAGACCCTCCTCTCTTCCACTGTAGTGATGCCTTAACACC CCCTCCCCTGCCCCCGAGCAATAATCTCCCCGCCCACCCTGGTCCTGCCTCTCAGCCACCAG TGTCTTCAGCCACTATGCACCTGCCCCTGGTCCTGGGGCCCCTGGGAGGGGCCCCCACGGTG GAGGGGCCTGGGGCACCCCCCTTCCTTGCTAGCAGCCTACTCTCTGCAGCGGCCAAGGCACA GCATCCCCCACTACCCCCTCCCAGCACTTTACAGGGCCGAAGGCCCCGTGCCCAGGCACCCT CAGCTTCCCACTCCTCATCACTTCGTCCCTCTCAGCGTCGTCCCCGCAGACCCCCTACTGTA TTTCGATTGCTAGAAGGGAGAGGCCCTCAAACCCCTAGACGGAGCCGTCCTCGGGCCCCTGC TCCTGTCCCCCAACCCTTTTCTCTCCCGGAGCCATCCCAACCAATTCTCCCTTCTGTGCTGT CCCTGCTGGGACTCCCCACCCCTGGCCCTTCCCACTCTGATGGAAGCTTTAACCTTTTGGGG TCAGATGCACACCTTCCTCCTCCCCCAACCCTCTCCTCAGGGAGCCCTCCCCAGCCCAGGCA CCCCATCCAGCCCTCCCTGCCTGGGACCACCAGTGGCAGCCTCAGCAGTGTGCCAGGTGCCC CTGCCCCACCAGCTGCCTCCAAAGCCCCAGTAGTCCCCAGCCCTGTGCTTCAAAGCCCATCC GAAGGACTGGGGATGGGGGCAGGCCCGGCCTGCCCTCTGCCTCCCCTGGCTGGTGGAGAGGC TTTCCCTTTCCCCAGCCCTGAGCAGGGCCTGGCACTGAGTGGAGCTGGCTTCCCTGGGATGC TTGGGGCCTTGCCTCTCCCTCTGAGTCTGGGGCAGCCTCCACCTTCTCCATTGCTCAACCAC AGTTTATTTGGTGTGCTGACTGGGGGAGGAGGACAACCTCCCCCTGAGCCCCTGCTACCCCC ACCAGGAGGACCTGGTCCTCCCCTAGCCCCAGGAGAGCCTGAAGGGCCTTCGCTTTTGGTGG CTTCCTTGCTTCCTCCACCACCCTCAGACCTTCTTCCACCTCCTTCAGCACCTCCCAGCAAC CTCCTTGCCTCTTTCCTGCCCCTGTTGGCTCTGGGCCCCACAGCTGGGGATGGGGAGGGATC TGCAGAGGGAGCCGGGGGTCCAAGTGGGGAGCCATTTTCAGGCTTGGGAGACCTGTCCCCCC TACTTTTCCCCCCACTTTCAGCCCCCCCTACCCTCATAGCTTTAAATTCTGCGCTGCTGGCT GCCACCCTGGATCCCCCCTCGGGGACACCCCCCCAGCCCTGTGTCCTGAGTGCCCCCCAACC TGGACCACCTACCTCCAGTGTCACCACGGCAACTACTGACCCGGGGGCCTCCTCTCTGGGCA AGGCCCCCTCCAACTCAGGGAGACCCCCCCAACTCCTTAGCCCTCTGCTGGGTGCCAGCCTG CTGGGTGACCTGTCTTCACTGACCAGCAGCCCTGGAGCCCTCCCCAGCCTGTTGCAGCCTCC TGGCCCTCTTCTCTCTGGCCAGTTGGGGCTGCAGCTCCTCCCTGGGGGGGGAGCTCCTCCAC CCCTCTCAGAGGCTTCTAGTCCCCTAGCCTGCCTGCTACAGAGTCTCCAGATCCCTCCAGAG CAGCCAGAAGCCCCCTGTCTACCCCCCGAGAGCCCTGCCTCAGCCCTCGAACCAGAGCCTGC CAGGCCTCCCCTCAGTGCCTTAGCCCCACCCCATGGTTCTCCCGACCCCCCAGTCCCTGAGC TGCTCACTGGGAGGGGGTCAGGGAAACGGGGCCGGAGGGGAGGAGGGGGACTTAGGGGCATT AATGGTGAGGCCAGGCCAGCCCGGGGCCGAAAGCCTGGCAGCCGGCGGGAGCCTGGCCGACT GGCCCTCAAATGGGGGACACGTGGTGGCTTCAATGGACAAATGGAAAGGTCCCCAAGAAGAACCCACCATTGGCAGCATAATGGGGAGCTGGCTGAAGGGGGTGCTGAGCCCAAGGATCCACCC CCTCCCGGGCCCCATTCTGAGGACCTTAAGGTGCCCCCGGGAGTAGTCAGAAAGTCTCGTCG TGGCCGTAGGAGAAAATACAACCCTACCCGGAACAGCAATAGCTCCCGCCAGGACATTACCT TGGAACCCAGCCCTACAGCCCGAGCAGCTGTCCCTCTGCCTCCCCGGGCCCGCCCTGGCCGT CCTGCCAAAAACAAGAGGAGGAAACTGGCCCCATAG (SEQ ID NO: 2) SEQ ID NO: 3 – Wild type human MBD6 MBD catalytic domain amino acid sequence DRAGGPVATSVPIGWQRCVREGAVLYISPSGTELSSLEQTRSYLLSDGTCKCGLECPLNVPK VFNFDPLAP (SEQ ID NO: 3) SEQ ID NO: 4 – Wild type human MBD6 MBD catalytic domain polynucleotide coding sequence GCAGACAGAGCTGGGGGCCCTGTGGCCACATCTGTCCCCATCGGCTGGCAGCGCTGTGTGCG AGAGGGTGCTGTGCTCTACATCAGTCCAAGTGGCACAGAGCTGTCTTCCTTGGAGCAAACCC GGAGCTACCTCCTCAGCGATGGGACCTGCAAGTGCGGTCTGGAGTGTCCACTTAATGTCCCC AAGGTTTTCAACTTTGACCCTTTGGCCCCG (SEQ ID NO: 4) SEQ ID NO: 5 – Wild type human MBD5 isoform 1 amino acid sequence MNGGKECDGGDKEGGLPAIQVPVGWQRRVDQNGVLYVSPSGSLLSCLEQVKTYLLTDGTCKC GLECPLILPKVFNFDPGAAVKQRTAEDVKADEDVTKLCIHKRKIIAVATLHKSMEAPHPSLV LTSPGGGTNATPVVPSRAATPRSVRNKSHEGITNSVMPECKNPFKLMIGSSNAMGRLYVQEL PGSQQQELHPVYPRQRLGSSEHGQKSPFRGSHGGLPSPASSGSQIYGDGSISPRTDPLGSPD VFTRSNPGFHGAPNSSPIHLNRTPLSPPSVMLHGSPVQSSCAMAGRTNIPLSPTLTTKSPVM KKPMCNFSTNMEIPRAMFHHKPPQGPPPPPPPSCALQKKPLTSEKDPLGILDPIPSKPVNQN PVIINPTSFHSNVHSQVPMMNVSMPPAVVPLPSNLPLPTVKPGHMNHGSHVQRVQHSASTSL SPSPVTSPVHMMGTGIGRIEASPQRSRSSSTSSDHGNFMMPPVGPQATSSGIKVPPRSPRST IGSPRPSMPSSPSTKSDGHHQYKDIPNPLIAGISNVLNTPSSAAFPTASAGSSSVKSQPGLL GMPLNQILNQHNAASFPASSLLSAAAKAQLANQNKLAGNNSSSSSNSGAVAGSGNTEGHSTL NTMFPPTANMLLPTGEGQSGRAALRDKLMSQQKDALRKRKQPPTTVLSLLRQSQMDSSAVPK PGPDLLRKQGQGSFPISSMSQLLQSMSCQSSHLSSNSTPGCGASNTALPCSANQLHFTDPSM NSSVLQNIPLRGEAVHCHNANTNFVHSNSPVPNHHLAGLINQIQASGNCGMLSQSGMALGNS LHPNPPQSRISTSSTPVIPNSIVSSYNQTSSEAGGSGPSSSIAIAGTNHPAITKTTSVLQDG VIVTTAAGNPLQSQLPIGSDFPFVGQEHALHFPSNSTSNNHLPHPLNPSLLSSLPISLPVNQ QHLLNQNLLNILQPSAGEGKSEINLHPLGFLNPNVNAALAFLSSDMDGQVLQPVHFQLLAAL LQNQAQAAAMLPLPSFNLTISDLLQQQNTPLPSLTQMTAPPDHLPSNQSDNSRAETLLTSPL GNPLPSFAGSDTTFNPLFLPAVNGASGLMTLNPQLLGGVLNSASANTANHPEVSIATSSQAT TTTTTTSSAVAALTVSTLGGTAVVSMAETLLNISNNAGNTPGPAKLNSNSVVPQLLNPLLGT GLLGDMSSINNTLSNHQLTHLQSLLNNNQMFPPNQQQQQLLQGYQNLQAFQGQSTIPCPANN NPMACLFQNFQVRMQEDAALLNKRISTQPGLTALPENPNTTLPPFQDTPCELQPRIDPSLGQ QVKDGLVVGGPGDASVDAIYKAVVDAASKGMQVVITTAVNSTTQISPIPALSAMSAFTASIG DPLNLSSAVSAVIHGRNMGGVDHDGRLRNSRGARLPKNLDHGKNVNEGDGFEYFKSASCHTS KKQWDGEQSPRGERNRWKYEEFLDHPGHIHSSPCHERPNNVSTLPFLPGEQHPILLPPRNCP GDKILEENFRYNNYKRTMMSFKERLENTVERCAHINGNRPRQSRGFGELLSTAKQDLVLEEQ SPSSSNSLENSLVKDYIHYNGDFNAKSVNGCVPSPSDAKSISSEDDLRNPDSPSSNELIHYR PRTFNVGDLVWGQIKGLTSWPGKLVREDDVHNSCQQSPEEGKVEPEKLKTLTEGLEAYSRVR KRNRKSGKLNNHLEAAIHEAMSELDKMSGTVHQIPQGDRQMRPPKPKRRKISR (SEQ ID NO: 5)SEQ ID NO: 6 – Wild type human MBD5 isoform 1 polynucleotide coding sequence ATGAATGGAGGCAAAGAGTGTGACGGAGGGGACAAGGAAGGAGGTCTTCCAGCTATACAAGT TCCTGTGGGTTGGCAGCGTCGTGTGGATCAAAATGGAGTGCTTTATGTCAGTCCCAGTGGGT CTTTGTTATCTTGCTTGGAGCAGGTTAAAACATACCTGCTTACTGATGGAACATGCAAGTGT GGCTTGGAATGTCCTCTTATTCTTCCCAAGGTATTTAATTTTGATCCTGGAGCTGCTGTGAA ACAGAGAACCGCAGAAGATGTTAAGGCAGATGAAGATGTCACAAAGCTATGCATACATAAAA GAAAAATTATTGCAGTGGCCACACTTCATAAAAGCATGGAAGCCCCACATCCTTCTCTGGTG CTCACCAGTCCCGGAGGAGGAACAAATGCAACTCCAGTAGTACCTTCTCGGGCAGCAACTCC AAGATCAGTAAGAAATAAGTCTCATGAAGGAATTACAAATTCTGTAATGCCTGAATGTAAGA ATCCTTTCAAGTTAATGATTGGATCATCAAATGCCATGGGAAGGCTATATGTACAAGAACTG CCTGGAAGCCAACAACAAGAACTCCACCCTGTCTACCCCCGACAGAGATTGGGCAGCAGTGA ACATGGACAGAAATCTCCATTCCGTGGCAGCCATGGAGGCCTGCCCAGCCCAGCGTCATCAG GTTCCCAGATATATGGAGATGGTTCAATCTCTCCAAGGACTGACCCACTTGGAAGTCCTGAT GTTTTCACAAGAAGTAATCCTGGTTTTCATGGAGCTCCCAATTCTAGTCCTATTCACCTGAA TAGGACTCCTCTTTCTCCACCTTCAGTAATGCTACATGGTTCTCCTGTACAGTCATCCTGTG CAATGGCTGGAAGGACTAATATACCTCTTTCCCCAACCTTGACTACAAAGAGTCCAGTAATG AAAAAACCAATGTGTAATTTTTCAACTAATATGGAAATACCACGAGCAATGTTCCACCACAA ACCACCCCAAGGCCCACCTCCCCCTCCTCCACCTTCTTGTGCTCTTCAGAAAAAGCCATTAA CATCTGAGAAAGATCCACTTGGCATTCTTGACCCTATTCCTAGTAAACCAGTGAATCAGAAC CCTGTTATCATTAATCCAACCAGTTTCCATTCAAATGTCCACTCTCAGGTACCTATGATGAA TGTAAGCATGCCTCCTGCTGTTGTTCCTTTGCCAAGTAATCTCCCATTGCCAACTGTAAAAC CTGGTCACATGAATCATGGGAGTCATGTACAAAGAGTTCAGCATTCAGCTTCAACCTCCCTG TCCCCTTCTCCAGTGACATCCCCCGTGCACATGATGGGGACTGGAATTGGAAGGATTGAGGC ATCGCCCCAAAGATCACGCTCATCTTCCACATCATCAGATCATGGAAATTTCATGATGCCAC CTGTAGGACCCCAGGCCACTTCTAGTGGTATTAAGGTTCCACCCAGGTCACCAAGGTCAACA ATAGGGTCCCCAAGGCCATCAATGCCATCAAGCCCTTCTACCAAGTCCGATGGACATCATCA GTACAAGGATATCCCTAACCCATTAATTGCTGGAATAAGTAATGTACTAAATACCCCAAGCA GTGCAGCTTTTCCTACTGCATCTGCCGGAAGTAGTTCTGTAAAGAGTCAGCCTGGTTTGCTG GGAATGCCTTTAAATCAGATCTTGAACCAGCACAATGCTGCCTCCTTTCCAGCAAGTAGTTT ACTCTCAGCAGCAGCCAAAGCACAGCTAGCAAATCAAAACAAACTTGCTGGTAACAACAGTA GCAGCAGTAGCAATTCTGGAGCTGTTGCCGGCAGTGGCAACACTGAAGGACATAGCACTTTA AACACCATGTTCCCTCCTACTGCCAACATGCTTCTCCCAACAGGTGAAGGGCAAAGTGGTCG AGCAGCACTAAGAGATAAGCTGATGTCTCAGCAAAAAGACGCATTGCGGAAAAGAAAACAAC CACCTACGACAGTGTTGAGTTTGCTCAGACAGTCTCAAATGGATAGTTCTGCAGTTCCTAAA CCTGGACCTGACTTGCTAAGGAAGCAGGGTCAGGGTTCATTTCCCATCAGTTCAATGTCTCA GTTACTACAGTCTATGAGTTGTCAAAGCTCTCACTTGAGTAGCAATAGTACCCCGGGTTGTG GGGCCTCAAATACTGCTTTGCCTTGCTCTGCTAACCAGCTGCATTTTACAGATCCCAGTATG AACTCTAGTGTTCTTCAGAACATACCTTTAAGAGGGGAAGCCGTGCACTGCCACAATGCAAA CACTAACTTTGTTCACAGTAACAGTCCAGTCCCCAACCACCATCTTGCAGGTTTAATAAATC AGATTCAGGCTAGCGGGAACTGTGGGATGCTCAGTCAGTCGGGCATGGCTTTAGGAAATTCC TTACATCCCAATCCACCTCAGTCAAGAATTTCAACGTCCTCCACTCCAGTGATACCAAACAG CATTGTTAGCAGCTATAATCAAACAAGTTCTGAAGCAGGCGGTTCAGGACCATCATCCTCCA TAGCCATAGCGGGCACCAACCACCCTGCCATCACAAAGACAACATCTGTTCTTCAAGATGGC GTCATAGTCACCACTGCAGCTGGAAACCCACTGCAGAGTCAGCTACCCATTGGGAGTGATTT TCCTTTTGTTGGCCAGGAGCACGCACTTCATTTTCCATCCAACAGCACTTCAAACAACCATC TTCCACACCCCTTGAACCCCAGCCTCCTCAGTTCTCTACCTATCTCTTTGCCAGTGAATCAA CAGCATCTCCTAAACCAGAATCTATTAAATATCCTCCAGCCTTCAGCAGGAGAAGGCAAGTC TGAGATCAACCTCCACCCTTTAGGTTTTCTCAACCCGAATGTAAACGCTGCTTTAGCTTTTC TCTCCAGTGACATGGATGGGCAGGTATTGCAGCCTGTTCACTTTCAGCTCTTAGCAGCCTTG CTTCAGAACCAAGCCCAAGCAGCTGCCATGCTTCCCCTGCCATCTTTCAATCTGACCATCTC AGATCTTTTGCAACAGCAAAATACCCCTTTACCCTCATTAACACAGATGACAGCCCCACCAGACCATTTGCCAAGCAATCAGTCAGACAACAGCCGAGCTGAGACCCTTTTAACCAGCCCCCTG GGGAACCCTTTACCAAGCTTTGCAGGCAGTGACACTACTTTTAACCCCCTGTTCCTCCCAGC TGTCAATGGGGCCTCAGGATTAATGACCTTGAATCCCCAGCTGTTGGGAGGTGTCCTGAACT CGGCATCGGCCAACACCGCTAATCATCCAGAGGTTTCCATAGCAACCTCCTCCCAGGCAACC ACTACCACAACCACTACATCATCAGCAGTGGCAGCACTGACTGTCTCAACACTTGGTGGGAC AGCAGTGGTGTCAATGGCAGAAACATTGCTGAATATATCTAATAATGCTGGGAATACACCTG GTCCAGCTAAACTCAACAGTAACTCTGTGGTGCCACAGCTACTTAACCCTCTACTGGGGACA GGTCTACTTGGTGATATGTCATCAATAAACAATACTTTGAGTAACCATCAACTGACTCATCT ACAGTCGCTGTTAAACAACAATCAGATGTTTCCTCCAAATCAGCAACAGCAGCAACTTCTCC AGGGGTACCAGAATCTCCAGGCGTTCCAAGGACAGTCCACAATTCCTTGCCCAGCTAACAAT AACCCCATGGCTTGTCTGTTTCAGAACTTTCAGGTGAGAATGCAGGAAGATGCAGCTCTCCT AAACAAAAGAATAAGCACTCAGCCTGGGCTCACAGCACTTCCTGAGAATCCAAACACTACAC TTCCACCTTTTCAAGATACACCTTGTGAGTTGCAACCGAGGATTGACCCATCTCTTGGTCAA CAGGTGAAGGATGGCCTCGTTGTGGGTGGCCCAGGTGATGCTTCCGTAGATGCCATTTACAA AGCAGTTGTCGATGCAGCCAGCAAAGGAATGCAGGTTGTCATCACCACTGCAGTCAACAGTA CAACTCAGATCAGCCCCATTCCAGCTCTGAGTGCCATGAGTGCCTTCACTGCCTCAATTGGT GACCCATTAAATCTCTCCAGTGCTGTCAGTGCGGTCATTCATGGACGGAACATGGGAGGTGT TGATCATGATGGTAGGCTGAGGAATTCAAGAGGGGCTCGGCTGCCCAAGAATCTAGACCATG GGAAAAATGTGAACGAAGGAGATGGGTTTGAATATTTCAAGTCAGCAAGTTGCCACACATCC AAAAAACAGTGGGACGGGGAGCAAAGCCCCAGAGGGGAGCGAAACAGGTGGAAGTACGAGGA ATTTTTAGATCATCCAGGCCATATCCACAGTAGTCCTTGTCATGAAAGGCCCAACAATGTCT CTACACTGCCATTTCTGCCTGGGGAACAGCACCCAATACTGTTACCACCAAGAAACTGTCCA GGGGATAAAATTCTAGAGGAAAATTTCAGGTATAATAACTACAAAAGAACTATGATGAGTTT TAAGGAGAGACTAGAGAACACTGTGGAAAGATGTGCACACATAAATGGGAATAGACCTCGAC AGAGTCGGGGATTTGGAGAGCTGCTAAGCACTGCAAAGCAAGACCTGGTCCTAGAGGAGCAG TCTCCAAGTTCCTCAAATAGTTTGGAAAATTCTCTGGTCAAAGACTACATCCATTACAATGG AGACTTTAATGCCAAAAGCGTTAATGGGTGTGTGCCTAGCCCTTCAGATGCTAAAAGCATTA GTAGTGAAGATGACCTAAGGAACCCAGACTCCCCCTCTTCAAATGAATTGATACATTATAGA CCAAGGACGTTCAATGTTGGCGACTTGGTCTGGGGCCAAATCAAAGGACTGACTTCCTGGCC TGGAAAATTAGTAAGAGAAGACGACGTTCACAATTCATGTCAACAAAGCCCCGAGGAAGGGA AGGTGGAGCCCGAGAAGTTGAAGACACTAACAGAAGGTTTGGAAGCCTACAGCCGTGTCCGG AAAAGGAACAGAAAAAGTGGAAAGCTAAATAACCATTTAGAAGCTGCTATTCATGAGGCCAT GAGTGAACTGGACAAAATGTCTGGGACTGTACACCAAATCCCACAGGGTGACAGACAAATGA GACCCCCCAAACCCAAGAGGAGGAAGATCTCCAGATAA (SEQ ID NO: 6) SEQ ID NO: 7 – Wild type human MBD5 isoform 2 amino acid sequence MNGGKECDGGDKEGGLPAIQVPVGWQRRVDQNGVLYVSPSGSLLSCLEQVKTYLLTDGTCKC GLECPLILPKVFNFDPGAAVKQRTAEDVKADEDVTKLCIHKRKIIAVATLHKSMEAPHPSLV LTSPGGGTNATPVVPSRAATPRSVRNKSHEGITNSVMPECKNPFKLMIGSSNAMGRLYVQEL PGSQQQELHPVYPRQRLGSSEHGQKSPFRGSHGGLPSPASSGSQIYGDGSISPRTDPLGSPD VFTRSNPGFHGAPNSSPIHLNRTPLSPPSVMLHGSPVQSSCAMAGRTNIPLSPTLTTKSPVM KKPMCNFSTNMEIPRAMFHHKPPQGPPPPPPPSCALQKKPLTSEKDPLGILDPIPSKPVNQN PVIINPTSFHSNVHSQVPMMNVSMPPAVVPLPSNLPLPTVKPGHMNHGSHVQRVQHSASTSL SPSPVTSPVHMMGTGIGRIEASPQRSRSSSTSSDHGNFMMPPVGPQATSSGIKVPPRSPRST IGSPRPSMPSSPSTKSDGHHQYKDIPNPLIAGISNVLNTPSSAAFPTASAGSSSVKSQPGLL GMPLNQILNQHNAASFPASSLLSAAAKAQLANQNKLAGNNSSSSSNSGAVAGSGNTEGHSTL NTMFPPTANMLLPTGEGQSGRAALRDKLMSQQKDALRKRKQPPTTVLSLLRQSQMDSSAVPK PGPDLLRKQGQGSFPISSMSQLLQSMSCQSSHLSSNSTPGCGASNTALPCSANQLHFTDPSM NSSVLQNIPLRGEAVHCHNANTNFVHSNSPVPNHHLAGLINQIQASGNCGMLSQSGMALGNS LHPNPPQSRISTSSTPVIPNSIVSSYNQTSSEAGGSGPSSSIAIAGTNHPAITKTTSVLQDG VIVTTAAGNPLQSQLPIGSDFPFVGQEHALHFPSNSTSNNHLPHPLNPSLLSSLPISLPVNQQHLLNQNLLNILQPSAGEGDMSSINNTLSNHQLTHLQSLLNNNQMFPPNQQQQQLLQGYQNL QAFQGQSTIPCPANNNPMACLFQNFQVRMQEDAALLNKRISTQPGLTALPENPNTTLPPFQD TPCELQPRIDPSLGQQVKDGLVVGGPGDASVDAIYKAVVDAASKGMQVVITTAVNSTTQISP IPALSAMSAFTASIGDPLNLSSAVSAVIHGRNMGGVDHDGRLRNSRGARLPKNLDHGKNVNE GDGFEYFKSASCHTSKKQWDGEQSPRGERNRWKYEEFLDHPGHIHSSPCHERPNNVSTLPFL PGEQHPILLPPRNCPGDKILEENFRYNNYKRTMMSFKERLENTVERCAHINGNRPRQSRGFG ELLSTAKQDLVLEEQSPSSSNSLENSLVKDYIHYNGDFNAKSVNGCVPSPSDAKSISSEDDL RNPDSPSSNELIHYRPRTFNVGDLVWGQIKGLTSWPGKLVREDDVHNSCQQSPEEGKVEPEK LKTLTEGLEAYSRVRKRNRKSGKLNNHLEAAIHEAMSELDKMSGTVHQIPQGDRQMRPPKPK RRKISR (SEQ ID NO: 7) SEQ ID NO: 8 – Wild type human MBD5 isoform 2 polynucleotide coding sequence ATGAATGGAGGCAAAGAGTGTGACGGAGGGGACAAGGAAGGAGGTCTTCCAGCTATACAAGT TCCTGTGGGTTGGCAGCGTCGTGTGGATCAAAATGGAGTGCTTTATGTCAGTCCCAGTGGGT CTTTGTTATCTTGCTTGGAGCAGGTTAAAACATACCTGCTTACTGATGGAACATGCAAGTGT GGCTTGGAATGTCCTCTTATTCTTCCCAAGGTATTTAATTTTGATCCTGGAGCTGCTGTGAA ACAGAGAACCGCAGAAGATGTTAAGGCAGATGAAGATGTCACAAAGCTATGCATACATAAAA GAAAAATTATTGCAGTGGCCACACTTCATAAAAGCATGGAAGCCCCACATCCTTCTCTGGTG CTCACCAGTCCCGGAGGAGGAACAAATGCAACTCCAGTAGTACCTTCTCGGGCAGCAACTCC AAGATCAGTAAGAAATAAGTCTCATGAAGGAATTACAAATTCTGTAATGCCTGAATGTAAGA ATCCTTTCAAGTTAATGATTGGATCATCAAATGCCATGGGAAGGCTATATGTACAAGAACTG CCTGGAAGCCAACAACAAGAACTCCACCCTGTCTACCCCCGACAGAGATTGGGCAGCAGTGA ACATGGACAGAAATCTCCATTCCGTGGCAGCCATGGAGGCCTGCCCAGCCCAGCGTCATCAG GTTCCCAGATATATGGAGATGGTTCAATCTCTCCAAGGACTGACCCACTTGGAAGTCCTGAT GTTTTCACAAGAAGTAATCCTGGTTTTCATGGAGCTCCCAATTCTAGTCCTATTCACCTGAA TAGGACTCCTCTTTCTCCACCTTCAGTAATGCTACATGGTTCTCCTGTACAGTCATCCTGTG CAATGGCTGGAAGGACTAATATACCTCTTTCCCCAACCTTGACTACAAAGAGTCCAGTAATG AAAAAACCAATGTGTAATTTTTCAACTAATATGGAAATACCACGAGCAATGTTCCACCACAA ACCACCCCAAGGCCCACCTCCCCCTCCTCCACCTTCTTGTGCTCTTCAGAAAAAGCCATTAA CATCTGAGAAAGATCCACTTGGCATTCTTGACCCTATTCCTAGTAAACCAGTGAATCAGAAC CCTGTTATCATTAATCCAACCAGTTTCCATTCAAATGTCCACTCTCAGGTACCTATGATGAA TGTAAGCATGCCTCCTGCTGTTGTTCCTTTGCCAAGTAATCTCCCATTGCCAACTGTAAAAC CTGGTCACATGAATCATGGGAGTCATGTACAAAGAGTTCAGCATTCAGCTTCAACCTCCCTG TCCCCTTCTCCAGTGACATCCCCCGTGCACATGATGGGGACTGGAATTGGAAGGATTGAGGC ATCGCCCCAAAGATCACGCTCATCTTCCACATCATCAGATCATGGAAATTTCATGATGCCAC CTGTAGGACCCCAGGCCACTTCTAGTGGTATTAAGGTTCCACCCAGGTCACCAAGGTCAACA ATAGGGTCCCCAAGGCCATCAATGCCATCAAGCCCTTCTACCAAGTCCGATGGACATCATCA GTACAAGGATATCCCTAACCCATTAATTGCTGGAATAAGTAATGTACTAAATACCCCAAGCA GTGCAGCTTTTCCTACTGCATCTGCCGGAAGTAGTTCTGTAAAGAGTCAGCCTGGTTTGCTG GGAATGCCTTTAAATCAGATCTTGAACCAGCACAATGCTGCCTCCTTTCCAGCAAGTAGTTT ACTCTCAGCAGCAGCCAAAGCACAGCTAGCAAATCAAAACAAACTTGCTGGTAACAACAGTA GCAGCAGTAGCAATTCTGGAGCTGTTGCCGGCAGTGGCAACACTGAAGGACATAGCACTTTA AACACCATGTTCCCTCCTACTGCCAACATGCTTCTCCCAACAGGTGAAGGGCAAAGTGGTCG AGCAGCACTAAGAGATAAGCTGATGTCTCAGCAAAAAGACGCATTGCGGAAAAGAAAACAAC CACCTACGACAGTGTTGAGTTTGCTCAGACAGTCTCAAATGGATAGTTCTGCAGTTCCTAAA CCTGGACCTGACTTGCTAAGGAAGCAGGGTCAGGGTTCATTTCCCATCAGTTCAATGTCTCA GTTACTACAGTCTATGAGTTGTCAAAGCTCTCACTTGAGTAGCAATAGTACCCCGGGTTGTG GGGCCTCAAATACTGCTTTGCCTTGCTCTGCTAACCAGCTGCATTTTACAGATCCCAGTATG AACTCTAGTGTTCTTCAGAACATACCTTTAAGAGGGGAAGCCGTGCACTGCCACAATGCAAA CACTAACTTTGTTCACAGTAACAGTCCAGTCCCCAACCACCATCTTGCAGGTTTAATAAATC AGATTCAGGCTAGCGGGAACTGTGGGATGCTCAGTCAGTCGGGCATGGCTTTAGGAAATTCCTTACATCCCAATCCACCTCAGTCAAGAATTTCAACGTCCTCCACTCCAGTGATACCAAACAG CATTGTTAGCAGCTATAATCAAACAAGTTCTGAAGCAGGCGGTTCAGGACCATCATCCTCCA TAGCCATAGCGGGCACCAACCACCCTGCCATCACAAAGACAACATCTGTTCTTCAAGATGGC GTCATAGTCACCACTGCAGCTGGAAACCCACTGCAGAGTCAGCTACCCATTGGGAGTGATTT TCCTTTTGTTGGCCAGGAGCACGCACTTCATTTTCCATCCAACAGCACTTCAAACAACCATC TTCCACACCCCTTGAACCCCAGCCTCCTCAGTTCTCTACCTATCTCTTTGCCAGTGAATCAA CAGCATCTCCTAAACCAGAATCTATTAAATATCCTCCAGCCTTCAGCAGGAGAAGGTGATAT GTCATCAATAAACAATACTTTGAGTAACCATCAACTGACTCATCTACAGTCGCTGTTAAACA ACAATCAGATGTTTCCTCCAAATCAGCAACAGCAGCAACTTCTCCAGGGGTACCAGAATCTC CAGGCGTTCCAAGGACAGTCCACAATTCCTTGCCCAGCTAACAATAACCCCATGGCTTGTCT GTTTCAGAACTTTCAGGTGAGAATGCAGGAAGATGCAGCTCTCCTAAACAAAAGAATAAGCA CTCAGCCTGGGCTCACAGCACTTCCTGAGAATCCAAACACTACACTTCCACCTTTTCAAGAT ACACCTTGTGAGTTGCAACCGAGGATTGACCCATCTCTTGGTCAACAGGTGAAGGATGGCCT CGTTGTGGGTGGCCCAGGTGATGCTTCCGTAGATGCCATTTACAAAGCAGTTGTCGATGCAG CCAGCAAAGGAATGCAGGTTGTCATCACCACTGCAGTCAACAGTACAACTCAGATCAGCCCC ATTCCAGCTCTGAGTGCCATGAGTGCCTTCACTGCCTCAATTGGTGACCCATTAAATCTCTC CAGTGCTGTCAGTGCGGTCATTCATGGACGGAACATGGGAGGTGTTGATCATGATGGTAGGC TGAGGAATTCAAGAGGGGCTCGGCTGCCCAAGAATCTAGACCATGGGAAAAATGTGAACGAA GGAGATGGGTTTGAATATTTCAAGTCAGCAAGTTGCCACACATCCAAAAAACAGTGGGACGG GGAGCAAAGCCCCAGAGGGGAGCGAAACAGGTGGAAGTACGAGGAATTTTTAGATCATCCAG GCCATATCCACAGTAGTCCTTGTCATGAAAGGCCCAACAATGTCTCTACACTGCCATTTCTG CCTGGGGAACAGCACCCAATACTGTTACCACCAAGAAACTGTCCAGGGGATAAAATTCTAGA GGAAAATTTCAGGTATAATAACTACAAAAGAACTATGATGAGTTTTAAGGAGAGACTAGAGA ACACTGTGGAAAGATGTGCACACATAAATGGGAATAGACCTCGACAGAGTCGGGGATTTGGA GAGCTGCTAAGCACTGCAAAGCAAGACCTGGTCCTAGAGGAGCAGTCTCCAAGTTCCTCAAA TAGTTTGGAAAATTCTCTGGTCAAAGACTACATCCATTACAATGGAGACTTTAATGCCAAAA GCGTTAATGGGTGTGTGCCTAGCCCTTCAGATGCTAAAAGCATTAGTAGTGAAGATGACCTA AGGAACCCAGACTCCCCCTCTTCAAATGAATTGATACATTATAGACCAAGGACGTTCAATGT TGGCGACTTGGTCTGGGGCCAAATCAAAGGACTGACTTCCTGGCCTGGAAAATTAGTAAGAG AAGACGACGTTCACAATTCATGTCAACAAAGCCCCGAGGAAGGGAAGGTGGAGCCCGAGAAG TTGAAGACACTAACAGAAGGTTTGGAAGCCTACAGCCGTGTCCGGAAAAGGAACAGAAAAAG TGGAAAGCTAAATAACCATTTAGAAGCTGCTATTCATGAGGCCATGAGTGAACTGGACAAAA TGTCTGGGACTGTACACCAAATCCCACAGGGTGACAGACAAATGAGACCCCCCAAACCCAAG AGGAGGAAGATCTCCAGATAA (SEQ ID NO: 8) SEQ ID NO: 9 – Wild type human NSUN1 isoform 1 amino acid sequence MGRKLDPTKEKRGPGRKARKQKGAETELVRFLPAVSDENSKRLSSRARKRAAKRRLGSVEAP KTNKSPEAKPLPGKLPKGAVQTAGKKGPQSLFNAPRGKKRPAPGSDEEEEEEDSEEDGMVNH GDLWGSEDDADTVDDYGADSNSEDEEEGEALLPIERAARKQKAREAAAGIQWSEEETEDEEE EKEVTPESGPPKVEEADGGLQINVDEEPFVLPPAGEMEQDAQAPDLQRVHKRIQDIVGILRD FGAQREEGRSRSEYLNRLKKDLAIYYSYGDFLLGKLMDLFPLSELVEFLEANEVPRPVTLRT NTLKTRRRDLAQALINRGVNLDPLGKWSKTGLVVYDSSVPIGATPEYLAGHYMLQGASSMLP VMALAPQEHERILDMCCAPGGKTSYMAQLMKNTGVILANDANAERLKSVVGNLHRLGVTNTI ISHYDGRQFPKVVGGFDRVLLDAPCSGTGVISKDPAVKTNKDEKDILRCAHLQKELLLSAID SVNATSKTGGYLVYCTCSITVEENEWVVDYALKKRNVRLVPTGLDFGQEGFTRFRERRFHPS LRSTRRFYPHTHNMDGFFIAKFKKFSNSIPQSQTGNSETATPTNVDLPQVIPKSENSSQPAK KAKGAAKTKQQLQKQQHPKKASFQKLNGISKGADSELSTVPSVTKTQASSSFQDSSQPAGKA EGIREPKVTGKLKQRSPKLQSSKKVAFLRQNAPPKGTDTQTPAVLSPSKTQATLKPKDHHQP LGRAKGVEKQQLPEQPFEKAAFQKQNDTPKGPQPPTVSPIRSSRPPPAKRKKSQSRGNSQLL LS (SEQ ID NO: 9)SEQ ID NO: 10 – Wild type human NSUN1 isoform 1 polynucleotide coding sequence ATGGGGCGCAAGTTGGACCCTACGAAGGAGAAGCGGGGGCCAGGCCGAAAGGCCCGGAAGCA GAAGGGTGCCGAGACAGAACTCGTCAGATTCTTGCCTGCAGTAAGTGACGAAAATTCCAAGA GGCTGTCTAGTCGTGCTCGAAAGAGGGCAGCCAAGAGGAGATTGGGCTCTGTTGAAGCCCCT AAGACAAATAAGTCTCCTGAGGCCAAACCATTGCCTGGAAAGCTACCAAAAGGAGCTGTCCA GACAGCTGGTAAGAAGGGACCCCAGTCCCTATTTAATGCTCCTCGAGGCAAGAAGCGCCCAG CACCTGGCAGTGATGAGGAAGAGGAGGAGGAAGACTCTGAAGAAGATGGTATGGTGAACCAC GGGGACCTCTGGGGCTCCGAGGACGATGCTGATACGGTAGATGACTATGGAGCTGACTCCAA CTCTGAGGATGAGGAGGAAGGTGAAGCGTTGCTGCCCATTGAAAGAGCTGCTCGGAAGCAGA AGGCCCGGGAAGCTGCTGCTGGGATCCAGTGGAGTGAAGAGGAGACCGAGGACGAGGAGGAA GAGAAAGAAGTGACCCCTGAGTCAGGCCCCCCAAAGGTGGAAGAGGCAGATGGGGGCCTGCA GATCAATGTGGATGAGGAACCATTTGTGCTGCCCCCTGCTGGGGAGATGGAGCAGGATGCCC AGGCTCCAGACCTGCAACGAGTTCACAAGCGGATCCAGGATATTGTGGGAATTCTGCGTGAT TTTGGGGCTCAGCGGGAGGAAGGGCGGTCTCGTTCTGAATACCTGAACCGGCTCAAGAAGGA TCTGGCCATTTACTACTCCTATGGAGACTTCCTGCTTGGCAAGCTCATGGACCTCTTCCCTC TGTCTGAGCTGGTGGAGTTCTTAGAAGCTAATGAGGTGCCTCGGCCCGTCACCCTCCGGACC AATACCTTGAAAACCCGACGCCGAGACCTTGCACAGGCTCTAATCAATCGTGGGGTTAACCT GGATCCCCTGGGCAAGTGGTCAAAGACTGGACTAGTGGTGTATGATTCTTCTGTGCCCATTG GTGCTACCCCCGAGTACCTGGCTGGGCACTACATGCTGCAGGGAGCCTCCAGCATGTTGCCC GTCATGGCCTTGGCACCCCAGGAACATGAGCGGATCCTGGACATGTGTTGTGCCCCTGGAGG AAAGACCAGCTACATGGCCCAGCTGATGAAGAACACGGGTGTGATCCTTGCCAATGACGCCA ATGCTGAGCGGCTCAAGAGTGTTGTGGGCAACTTGCATCGGCTGGGAGTCACCAACACCATT ATCAGCCACTATGATGGGCGCCAGTTCCCCAAGGTGGTGGGGGGCTTTGACCGAGTACTGCT GGATGCTCCCTGCAGTGGCACTGGGGTCATCTCCAAGGATCCAGCCGTGAAGACTAACAAGG ATGAGAAGGACATCCTGCGCTGTGCTCACCTCCAGAAGGAGTTGCTCCTGAGTGCTATTGAC TCTGTCAATGCGACCTCCAAGACAGGAGGCTACCTGGTTTACTGCACCTGTTCTATCACAGT AGAAGAGAATGAGTGGGTGGTAGACTATGCTCTGAAAAAGAGGAATGTGCGACTGGTGCCCA CGGGCCTAGACTTTGGCCAGGAAGGTTTTACCCGCTTTCGAGAAAGGCGCTTCCACCCCAGT CTGCGTTCTACCCGACGCTTCTACCCTCATACCCACAATATGGATGGGTTCTTCATTGCCAA GTTCAAGAAATTTTCCAATTCTATCCCTCAGTCCCAGACAGGAAATTCTGAAACAGCCACAC CTACAAATGTAGACTTGCCTCAGGTCATCCCCAAGTCTGAGAACAGCAGCCAGCCAGCCAAG AAAGCCAAGGGGGCTGCAAAGACAAAGCAGCAGCTGCAGAAACAGCAACATCCCAAGAAGGC CTCCTTCCAGAAGCTGAATGGCATCTCCAAAGGGGCAGACTCAGAATTGTCCACTGTACCTT CTGTCACAAAGACCCAAGCTTCCTCCAGCTTCCAGGATAGCAGTCAGCCAGCTGGAAAAGCC GAAGGGATCAGGGAGCCAAAGGTGACTGGGAAGCTAAAGCAACGATCACCTAAATTACAGTC CTCCAAGAAAGTTGCTTTCCTCAGGCAGAATGCCCCTCCCAAGGGCACAGACACACAAACAC CGGCTGTGTTATCCCCATCCAAGACTCAGGCCACCCTGAAACCTAAGGACCATCATCAGCCC CTTGGAAGGGCCAAGGGGGTTGAGAAGCAGCAGTTGCCAGAGCAGCCTTTTGAGAAAGCTGC CTTCCAGAAACAGAATGATACCCCCAAGGGGCCTCAGCCTCCCACTGTGTCTCCCATCCGTT CCAGCCGCCCCCCACCAGCAAAGAGGAAGAAATCTCAGTCCAGGGGCAACAGCCAGCTGCTG CTATCTTAG (SEQ ID NO: 10) SEQ ID NO: 11 – Wild type human NSUN2 isoform 2 amino acid sequence MGRRSRGRRLQQQQRPEDAEDGAEGGGKRGEAGWEGGYPEIVKENKLFEHYYQELKIVPEGE WGQFMDALREPLPATLRITGYKSHAKEILHCLKNKYFKELEDLEVDGQKVEVPQPLSWYPEE LAWHTNLSRKILRKSPHLEKFHQFLVSETESGNISRQEAVSMIPPLLLNVRPHHKILDMCAA PGSKTTQLIEMLHADMNVPFPEGFVIANDVDNKRCYLLVHQAKRLSSPCIMVVNHDASSIPR LQIDVDGRKEILFYDRILCDVPCSGDGTMRKNIDVWKKWTTLNSLQLHGLQLRIATRGAEQL AEGGRMVYSTCSLNPIEDEAVIASLLEKSEGALELADVSNELPGLKWMPGITQWKVMTKDGQ WFTDWDAVPHSRHTQIRPTMFPPKDPEKLQAMHLERCLRILPHHQNTGGFFVAVLVKKSSMP WNKRQPKLQGKSAETRESTQLSPADLTEGKPTDPSKLESPSFTGTGDTEIAHATEDLENNGSKKDGVCGPPPSKKMKLFGFKEDPFVFIPEDDPLFPPIEKFYALDPSFPRMNLLTRTTEGKKR QLYMVSKELRNVLLNNSEKMKVINTGIKVWCRNNSGEEFDCAFRLAQEGIYTLYPFINSRII TVSMEDVKILLTQENPFFRKLSSETYSQAKDLAKGSIVLKYEPDSANPDALQCPIVLCGWRG KASIRTFVPKNERLHYLRMMGLEVLGEKKKEGVILTNESAASTGQPDNDVTEGQRAGEPNSP DAEEANSPDVTAGCDPAGVHPPR (SEQ ID NO: 11) SEQ ID NO: 12 - Wild type human NSUN2 isoform 1 polynucleotide coding sequence ATGGGGCGGCGGTCGCGGGGTCGGCGGCTCCAGCAACAGCAGCGGCCGGAGGACGCGGAGGA TGGCGCCGAGGGTGGTGGAAAGCGCGGCGAGGCGGGCTGGGAAGGAGGCTACCCCGAGATCG TCAAGGAGAACAAGCTGTTCGAGCACTACTACCAGGAGCTCAAGATCGTGCCCGAGGGCGAG TGGGGCCAGTTCATGGACGCTCTCAGGGAGCCGCTCCCGGCCACTTTAAGAATTACTGGTTA CAAAAGCCACGCAAAAGAGATTCTCCATTGCTTAAAGAACAAATATTTTAAGGAATTGGAGG ACCTGGAGGTGGACGGTCAGAAAGTTGAAGTTCCACAGCCACTGAGTTGGTATCCTGAAGAA CTTGCCTGGCACACAAATTTAAGTCGAAAAATCTTGAGAAAATCGCCACACTTGGAAAAGTT TCATCAGTTTCTAGTTAGTGAAACAGAATCTGGAAATATTAGTCGTCAAGAAGCTGTTAGCA TGATCCCACCACTGCTCCTCAACGTGCGGCCTCATCATAAGATCTTAGATATGTGTGCAGCA CCTGGCTCAAAGACCACACAGTTAATTGAAATGCTACATGCCGACATGAATGTCCCCTTTCC AGAGGGATTTGTTATTGCGAATGATGTGGACAACAAGCGCTGCTACCTGCTCGTCCATCAAG CCAAGAGGCTGAGCAGCCCCTGCATCATGGTGGTCAACCATGATGCCTCCAGCATACCCAGG CTCCAGATAGATGTGGACGGCAGGAAAGAGATCCTCTTCTATGATCGAATTTTATGTGATGT CCCTTGCAGTGGAGACGGCACTATGAGAAAAAACATTGATGTTTGGAAAAAGTGGACCACCT TAAATAGCTTGCAGCTACATGGCTTACAGCTGCGGATTGCAACACGCGGGGCTGAACAGCTG GCTGAAGGTGGAAGGATGGTGTATTCCACGTGTTCACTAAACCCTATTGAGGATGAAGCAGT CATAGCATCTTTACTGGAAAAAAGTGAAGGTGCTTTGGAGCTTGCTGATGTGTCTAATGAAC TGCCAGGGCTGAAGTGGATGCCTGGAATCACACAGTGGAAGGTAATGACGAAAGATGGGCAG TGGTTTACAGACTGGGACGCTGTTCCTCACAGCAGACACACCCAGATCCGACCTACCATGTT CCCTCCGAAGGACCCAGAAAAGCTGCAGGCCATGCACCTGGAGCGATGCCTTAGGATATTAC CCCATCATCAGAATACTGGAGGGTTTTTTGTGGCAGTATTGGTGAAAAAATCTTCAATGCCG TGGAATAAACGTCAGCCAAAGCTTCAGGGTAAATCTGCAGAGACCAGAGAAAGCACACAGCT GAGCCCTGCAGATCTCACAGAAGGGAAACCCACAGATCCCTCTAAGCTGGAAAGTCCGTCAT TCACAGGAACTGGTGACACAGAAATAGCTCATGCAACTGAGGATTTAGAGAATAATGGCAGT AAGAAAGATGGCGTGTGTGGTCCTCCTCCATCAAAGAAAATGAAGTTATTTGGATTTAAAGA AGATCCATTTGTATTTATTCCTGAAGATGACCCATTATTTCCACCTATTGAGAAATTTTATG CTTTGGATCCTTCATTCCCAAGGATGAATTTGTTAACTCGGACTACAGAAGGGAAGAAAAGG CAGCTCTACATGGTTTCTAAGGAGTTGCGGAATGTGCTGCTGAATAACAGTGAGAAGATGAA GGTTATTAACACGGGGATCAAAGTCTGGTGTAGAAATAACAGCGGTGAAGAGTTTGACTGTG CTTTCCGGCTGGCACAGGAGGGAATATATACATTGTATCCATTTATTAACTCAAGAATTATT ACTGTATCAATGGAAGATGTTAAGATACTGTTGACCCAGGAAAATCCCTTTTTTAGAAAACT CAGCAGTGAGACCTACAGTCAAGCAAAGGACCTGGCAAAGGGAAGCATCGTGCTGAAGTATG AACCAGATTCTGCGAATCCAGACGCTCTGCAGTGTCCCATCGTCTTATGCGGATGGCGGGGA AAGGCCTCCATTCGAACTTTTGTGCCCAAGAATGAACGGCTTCATTATCTCAGGATGATGGG GCTGGAGGTATTGGGAGAAAAGAAGAAGGAAGGGGTTATCCTCACAAATGAGAGTGCAGCCA GCACCGGACAGCCAGACAATGACGTGACTGAGGGACAGAGAGCAGGAGAGCCCAACAGCCCA GATGCAGAAGAGGCCAACAGTCCAGACGTGACAGCAGGCTGTGACCCGGCGGGGGTCCATCC ACCCCGGTGA (SEQ ID NO: 12) SEQ ID NO: 13 – Wild type human NSUN2 isoform 2 amino acid sequence MGRRSRGRRLQQQQRPEDAEDGAEGGGKRGEAGWEGGYPEIVKENKLFEHYYQELKIVPEGE WGQFMDALREPLPATLRITGYKRYPEELAWHTNLSRKILRKSPHLEKFHQFLVSETESGNIS RQEAVSMIPPLLLNVRPHHKILDMCAAPGSKTTQLIEMLHADMNVPFPEGFVIANDVDNKRC YLLVHQAKRLSSPCIMVVNHDASSIPRLQIDVDGRKEILFYDRILCDVPCSGDGTMRKNIDVWKKWTTLNSLQLHGLQLRIATRGAEQLAEGGRMVYSTCSLNPIEDEAVIASLLEKSEGALEL ADVSNELPGLKWMPGITQWKVMTKDGQWFTDWDAVPHSRHTQIRPTMFPPKDPEKLQAMHLE RCLRILPHHQNTGGFFVAVLVKKSSMPWNKRQPKLQGKSAETRESTQLSPADLTEGKPTDPS KLESPSFTGTGDTEIAHATEDLENNGSKKDGVCGPPPSKKMKLFGFKEDPFVFIPEDDPLFP PIEKFYALDPSFPRMNLLTRTTEGKKRQLYMVSKELRNVLLNNSEKMKVINTGIKVWCRNNS GEEFDCAFRLAQEGIYTLYPFINSRIITVSMEDVKILLTQENPFFRKLSSETYSQAKDLAKG SIVLKYEPDSANPDALQCPIVLCGWRGKASIRTFVPKNERLHYLRMMGLEVLGEKKKEGVIL TNESAASTGQPDNDVTEGQRAGEPNSPDAEEANSPDVTAGCDPAGVHPPR (SEQ ID NO: 13) SEQ ID NO: 14 - Wild type human NSUN2 isoform 2 polynucleotide coding sequence ATGGGGCGGCGGTCGCGGGGTCGGCGGCTCCAGCAACAGCAGCGGCCGGAGGACGCGGAGGA TGGCGCCGAGGGTGGTGGAAAGCGCGGCGAGGCGGGCTGGGAAGGAGGCTACCCCGAGATCG TCAAGGAGAACAAGCTGTTCGAGCACTACTACCAGGAGCTCAAGATCGTGCCCGAGGGCGAG TGGGGCCAGTTCATGGACGCTCTCAGGGAGCCGCTCCCGGCCACTTTAAGAATTACTGGTTA CAAAAGGTATCCTGAAGAACTTGCCTGGCACACAAATTTAAGTCGAAAAATCTTGAGAAAAT CGCCACACTTGGAAAAGTTTCATCAGTTTCTAGTTAGTGAAACAGAATCTGGAAATATTAGT CGTCAAGAAGCTGTTAGCATGATCCCACCACTGCTCCTCAACGTGCGGCCTCATCATAAGAT CTTAGATATGTGTGCAGCACCTGGCTCAAAGACCACACAGTTAATTGAAATGCTACATGCCG ACATGAATGTCCCCTTTCCAGAGGGATTTGTTATTGCGAATGATGTGGACAACAAGCGCTGC TACCTGCTCGTCCATCAAGCCAAGAGGCTGAGCAGCCCCTGCATCATGGTGGTCAACCATGA TGCCTCCAGCATACCCAGGCTCCAGATAGATGTGGACGGCAGGAAAGAGATCCTCTTCTATG ATCGAATTTTATGTGATGTCCCTTGCAGTGGAGACGGCACTATGAGAAAAAACATTGATGTT TGGAAAAAGTGGACCACCTTAAATAGCTTGCAGCTACATGGCTTACAGCTGCGGATTGCAAC ACGCGGGGCTGAACAGCTGGCTGAAGGTGGAAGGATGGTGTATTCCACGTGTTCACTAAACC CTATTGAGGATGAAGCAGTCATAGCATCTTTACTGGAAAAAAGTGAAGGTGCTTTGGAGCTT GCTGATGTGTCTAATGAACTGCCAGGGCTGAAGTGGATGCCTGGAATCACACAGTGGAAGGT AATGACGAAAGATGGGCAGTGGTTTACAGACTGGGACGCTGTTCCTCACAGCAGACACACCC AGATCCGACCTACCATGTTCCCTCCGAAGGACCCAGAAAAGCTGCAGGCCATGCACCTGGAG CGATGCCTTAGGATATTACCCCATCATCAGAATACTGGAGGGTTTTTTGTGGCAGTATTGGT GAAAAAATCTTCAATGCCGTGGAATAAACGTCAGCCAAAGCTTCAGGGTAAATCTGCAGAGA CCAGAGAAAGCACACAGCTGAGCCCTGCAGATCTCACAGAAGGGAAACCCACAGATCCCTCT AAGCTGGAAAGTCCGTCATTCACAGGAACTGGTGACACAGAAATAGCTCATGCAACTGAGGA TTTAGAGAATAATGGCAGTAAGAAAGATGGCGTGTGTGGTCCTCCTCCATCAAAGAAAATGA AGTTATTTGGATTTAAAGAAGATCCATTTGTATTTATTCCTGAAGATGACCCATTATTTCCA CCTATTGAGAAATTTTATGCTTTGGATCCTTCATTCCCAAGGATGAATTTGTTAACTCGGAC TACAGAAGGGAAGAAAAGGCAGCTCTACATGGTTTCTAAGGAGTTGCGGAATGTGCTGCTGA ATAACAGTGAGAAGATGAAGGTTATTAACACGGGGATCAAAGTCTGGTGTAGAAATAACAGC GGTGAAGAGTTTGACTGTGCTTTCCGGCTGGCACAGGAGGGAATATATACATTGTATCCATT TATTAACTCAAGAATTATTACTGTATCAATGGAAGATGTTAAGATACTGTTGACCCAGGAAA ATCCCTTTTTTAGAAAACTCAGCAGTGAGACCTACAGTCAAGCAAAGGACCTGGCAAAGGGA AGCATCGTGCTGAAGTATGAACCAGATTCTGCGAATCCAGACGCTCTGCAGTGTCCCATCGT CTTATGCGGATGGCGGGGAAAGGCCTCCATTCGAACTTTTGTGCCCAAGAATGAACGGCTTC ATTATCTCAGGATGATGGGGCTGGAGGTATTGGGAGAAAAGAAGAAGGAAGGGGTTATCCTC ACAAATGAGAGTGCAGCCAGCACCGGACAGCCAGACAATGACGTGACTGAGGGACAGAGAGC AGGAGAGCCCAACAGCCCAGATGCAGAAGAGGCCAACAGTCCAGACGTGACAGCAGGCTGTG ACCCGGCGGGGGTCCATCCACCCCGGTGA (SEQ ID NO: 14) SEQ ID NO: 15 – Wild type human TET2 isoform A amino acid sequence MEQDRTNHVEGNRLSPFLIPSPPICQTEPLATKLQNGSPLPERAHPEVNGDTKWHSFKSYYG IPCMKGSQNSRVSPDFTQESRGYSKCLQNGGIKRTVSEPSLSGLLQIKKLKQDQKANGERRNFGVSQERNPGESSQPNVSDLSDKKESVSSVAQENAVKDFTSFSTHNCSGPENPELQILNEQE GKSANYHDKNIVLLKNKAVLMPNGATVSASSVEHTHGELLEKTLSQYYPDCVSIAVQKTTSH INAINSQATNELSCEITHPSHTSGQINSAQTSNSELPPKPAAVVSEACDADDADNASKLAAM LNTCSFQKPEQLQQQKSVFEICPSPAENNIQGTTKLASGEEFCSGSSSNLQAPGGSSERYLK QNEMNGAYFKQSSVFTKDSFSATTTPPPPSQLLLSPPPPLPQVPQLPSEGKSTLNGGVLEEH HHYPNQSNTTLLREVKIEGKPEAPPSQSPNPSTHVCSPSPMLSERPQNNCVNRNDIQTAGTM TVPLCSEKTRPMSEHLKHNPPIFGSSGELQDNCQQLMRNKEQEILKGRDKEQTRDLVPPTQH YLKPGWIELKAPRFHQAESHLKRNEASLPSILQYQPNLSNQMTSKQYTGNSNMPGGLPRQAY TQKTTQLEHKSQMYQVEMNQGQSQGTVDQHLQFQKPSHQVHFSKTDHLPKAHVQSLCGTRFH FQQRADSQTEKLMSPVLKQHLNQQASETEPFSNSHLLQHKPHKQAAQTQPSQSSHLPQNQQQ QQKLQIKNKEEILQTFPHPQSNNDQQREGSFFGQTKVEECFHGENQYSKSSEFETHNVQMGL EEVQNINRRNSPYSQTMKSSACKIQVSCSNNTHLVSENKEQTTHPELFAGNKTQNLHHMQYF PNNVIPKQDLLHRCFQEQEQKSQQASVLQGYKNRNQDMSGQQAAQLAQQRYLIHNHANVFPV PDQGGSHTQTPPQKDTQKHAALRWHLLQKQEQQQTQQPQTESCHSQMHRPIKVEPGCKPHAC MHTAPPENKTWKKVTKQENPPASCDNVQQKSIIETMEQHLKQFHAKSLFDHKALTLKSQKQV KVEMSGPVTVLTRQTTAAELDSHTPALEQQTTSSEKTPTKRTAASVLNNFIESPSKLLDTPI KNLLDTPVKTQYDFPSCRCVEQIIEKDEGPFYTHLGAGPNVAAIREIMEERFGQKGKAIRIE RVIYTGKEGKSSQGCPIAKWVVRRSSSEEKLLCLVRERAGHTCEAAVIVILILVWEGIPLSL ADKLYSELTETLRKYGTLTNRRCALNEERTCACQGLDPETCGASFSFGCSWSMYYNGCKFAR SKIPRKFKLLGDDPKEEEKLESHLQNLSTLMAPTYKKLAPDAYNNQIEYEHRAPECRLGLKE GRPFSGVTACLDFCAHAHRDLHNMQNGSTLVCTLTREDNREFGGKPEDEQLHVLPLYKVSDV DEFGSVEAQEEKKRSGAIQVLSSFRRKVRMLAEPVKTCRQRKLEAKKAAAEKLSSLENSSNK NEKEKSAPSRTKQTENASQAKQLAELLRLSGPVMQQSQQPQPLQKQPPQPQQQQRPQQQQPH HPQTESVNSYSASGSTNPYMRRPNPVSPYPNSSHTSDIYGSTSPMNFYSTSSQAAGSYLNSS NPMNPYPGLLNQNTQYPSYQCNGNLSVDNCSPYLGSYSPQSQPMDLYRYPSQDPLSKLSLPP IHTLYQPRFGNSQSFTSKYLGYGNQNMQGDGFSSCTIRPNVHHVGKLPPYPTHEMDGHFMGA TSRLPPNLSNPNMDYKNGEHHSPSHIIHNYSAAPGMFNSSLHALHLQNKENDMLSHTANGLS KMLPALNHDRTACVQGGLHKLSDANGQEKQPLALVQGVASGAEDNDEVWSDSEQSFLDPDIG GVAVAPTHGSILIECAKRELHATTPLKNPNRNHPTRISLVFYQHKSMNEPKHGLALWEAKMA EKAREKEEECEKYGPDYVPQKSHGKKVKREPAEPHETSEPTYLRFIKSLAERTMSVTTDSTV TTSPYAFTRVTGPYNRYI (SEQ ID NO: 15) SEQ ID NO: 16 – Wild type human TET2 isoform A polynucleotide coding sequence ATGGAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATC ACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGCTCCAGAATGGAAGCCCACTGCCTG AGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATGGA ATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAG AGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCT CTGGGCTCCTTCAGATCAAGAAATTGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAAC TTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAATGTCTCCGATTTGAG TGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTT TTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAG GGGAAAAGTGCTAATTACCATGACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCTAAT GCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCCTGGAAA AAACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCAC ATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCA TACCTCAGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTG CAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATG CTAAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGAT ATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAAT TCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTC TGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCAC AGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACAC CACCACTACCCCAACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACC TGAGGCACCACCTTCCCAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGC TTTCTGAAAGGCCTCAGAATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATG ACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCAGAACACCTCAAGCATAACCCACC AATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGC AAGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCAC TATCTGAAACCAGGATGGATTGAATTGAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCT AAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCAACCCAATCTCTCCAATCAAA TGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTAC ACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATGAATCAAGG GCAGTCCCAAGGTACAGTGGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACT TCTCCAAAACAGACCATTTACCAAAAGCTCATGTGCAGTCACTGTGTGGCACTAGATTTCAT TTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAGTGTTGAAACAGCACTT GAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTGCAACATAAGCCTC ATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAG CAGCAAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAG CAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTTTC ATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTG GAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGC ATGCAAAATACAGGTTTCTTGTTCAAACAATACACACCTAGTTTCAGAGAATAAAGAACAGA CTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGCAATATTTT CCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGAGCAGAA GTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATATGTCTGGTCAAC AAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTG CCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGC TCTAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGT CTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGT ATGCACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACC TGCAAGCTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGC AGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTA AAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAACTTGA TAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAA CAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATA AAAAATTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGA GCAAATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGG CAGCTATTAGAGAAATCATGGAAGAAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAA AGAGTCATCTATACTGGTAAAGAAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGT GGTTCGCAGAAGCAGCAGTGAAGAGAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACA CCTGTGAGGCTGCAGTGATTGTGATTCTCATCCTGGTGTGGGAAGGAATCCCGCTGTCTCTG GCTGACAAACTCTACTCGGAGCTTACCGAGACGCTGAGGAAATACGGCACGCTCACCAATCG CCGGTGTGCCTTGAATGAAGAGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTG GTGCCTCCTTCTCTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTAAGTTTGCCAGA AGCAAGATCCCAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAACTGGA GTCTCATTTGCAAAACCTGTCCACTCTTATGGCACCAACATATAAGAAACTTGCACCTGATG CATATAATAATCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAA GGCCGTCCATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTT GCACAACATGCAGAATGGCAGCACATTGGTATGCACTCTCACTAGAGAAGACAATCGAGAAT TTGGAGGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACT GAGTTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGA AACTAGAAGCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAA AATGAAAAGGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGCTAA ACAGTTGGCAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGC CTCTACAGAAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAGCCACAT CACCCTCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAG ACGGCCCAATCCAGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTATGGAAGCA CCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCT AATCCCATGAACCCTTACCCTGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATG CAATGGAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTC AGCCGATGGATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCC ATCCATACACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGG TTATGGAAACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTAC ATCATGTAGGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCC ACCTCTAGATTACCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCA TTCACCTTCTCACATAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGCTCTCTTC ATGCCCTGCATCTCCAAAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCA AAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATT AAGTGATGCTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTG CAGAGGACAACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGG GGAGTGGCCGTGGCTCCAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCA TGCCACAACCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTT ACCAGCATAAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCT GAAAAAGCCCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCAGAA ATCCCATGGCAAAAAAGTGAAACGGGAGCCTGCTGAGCCACATGAAACTTCAGAGCCCACTT ACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACAGACTCCACAGTA ACTACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATATATGA (SEQ ID NO: 16) SEQ ID NO: 17 – Wild type human TET2 isoform B amino acid sequence MEQDRTNHVEGNRLSPFLIPSPPICQTEPLATKLQNGSPLPERAHPEVNGDTKWHSFKSYYG IPCMKGSQNSRVSPDFTQESRGYSKCLQNGGIKRTVSEPSLSGLLQIKKLKQDQKANGERRN FGVSQERNPGESSQPNVSDLSDKKESVSSVAQENAVKDFTSFSTHNCSGPENPELQILNEQE GKSANYHDKNIVLLKNKAVLMPNGATVSASSVEHTHGELLEKTLSQYYPDCVSIAVQKTTSH INAINSQATNELSCEITHPSHTSGQINSAQTSNSELPPKPAAVVSEACDADDADNASKLAAM LNTCSFQKPEQLQQQKSVFEICPSPAENNIQGTTKLASGEEFCSGSSSNLQAPGGSSERYLK QNEMNGAYFKQSSVFTKDSFSATTTPPPPSQLLLSPPPPLPQVPQLPSEGKSTLNGGVLEEH HHYPNQSNTTLLREVKIEGKPEAPPSQSPNPSTHVCSPSPMLSERPQNNCVNRNDIQTAGTM TVPLCSEKTRPMSEHLKHNPPIFGSSGELQDNCQQLMRNKEQEILKGRDKEQTRDLVPPTQH YLKPGWIELKAPRFHQAESHLKRNEASLPSILQYQPNLSNQMTSKQYTGNSNMPGGLPRQAY TQKTTQLEHKSQMYQVEMNQGQSQGTVDQHLQFQKPSHQVHFSKTDHLPKAHVQSLCGTRFH FQQRADSQTEKLMSPVLKQHLNQQASETEPFSNSHLLQHKPHKQAAQTQPSQSSHLPQNQQQ QQKLQIKNKEEILQTFPHPQSNNDQQREGSFFGQTKVEECFHGENQYSKSSEFETHNVQMGL EEVQNINRRNSPYSQTMKSSACKIQVSCSNNTHLVSENKEQTTHPELFAGNKTQNLHHMQYF PNNVIPKQDLLHRCFQEQEQKSQQASVLQGYKNRNQDMSGQQAAQLAQQRYLIHNHANVFPV PDQGGSHTQTPPQKDTQKHAALRWHLLQKQEQQQTQQPQTESCHSQMHRPIKVEPGCKPHAC MHTAPPENKTWKKVTKQENPPASCDNVQQKSIIETMEQHLKQFHAKSLFDHKALTLKSQKQV KVEMSGPVTVLTRQTTAAELDSHTPALEQQTTSSEKTPTKRTAASVLNNFIESPSKLLDTPI KNLLDTPVKTQYDFPSCRCVGKCQKCTETHGVYPELANLSSDMGFSFFF (SEQ ID NO: 17)SEQ ID NO: 18 – Wild type human TET2 isoform B polynucleotide coding sequence ATGGAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATC ACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGCTCCAGAATGGAAGCCCACTGCCTG AGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATGGA ATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAG AGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCT CTGGGCTCCTTCAGATCAAGAAATTGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAAC TTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAATGTCTCCGATTTGAG TGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTT TTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAG GGGAAAAGTGCTAATTACCATGACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCTAAT GCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCCTGGAAA AAACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCAC ATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCA TACCTCAGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTG CAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATG CTAAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGAT ATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAAT TCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAA CAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTC TGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCAC AGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACAC CACCACTACCCCAACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACC TGAGGCACCACCTTCCCAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGC TTTCTGAAAGGCCTCAGAATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATG ACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCAGAACACCTCAAGCATAACCCACC AATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGC AAGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCAC TATCTGAAACCAGGATGGATTGAATTGAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCT AAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCAACCCAATCTCTCCAATCAAA TGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTAC ACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATGAATCAAGG GCAGTCCCAAGGTACAGTGGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACT TCTCCAAAACAGACCATTTACCAAAAGCTCATGTGCAGTCACTGTGTGGCACTAGATTTCAT TTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAGTGTTGAAACAGCACTT GAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTGCAACATAAGCCTC ATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAG CAGCAAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAG CAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTTTC ATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTG GAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGC ATGCAAAATACAGGTTTCTTGTTCAAACAATACACACCTAGTTTCAGAGAATAAAGAACAGA CTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGCAATATTTT CCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGAGCAGAA GTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATATGTCTGGTCAAC AAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTG CCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGC TCTAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGT CTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGT ATGCACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGC AGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTA AAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAACTTGA TAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAA CAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATA AAAAATTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGG TAAGTGCCAGAAATGTACTGAGACACATGGCGTTTATCCAGAATTAGCAAATTTATCTTCAG ATATGGGATTTTCCTTCTTTTTTTAA (SEQ ID NO: 18) SEQ ID NO: 19 - Human TET2 isoform A catalytic domain amino acid sequence SVLNNFIESPSKLLDTPIKNLLDTPVKTQYDFPSCRCVEQIIEKDEGPFYTHLGAGPNVAAI REIMEERFGQKGKAIRIERVIYTGKEGKSSQGCPIAKWVVRRSSSEEKLLCLVRERAGHTCE AAVIVILILVWEGIPLSLADKLYSELTETLRKYGTLTNRRCALNEERTCACQGLDPETCGAS FSFGCSWSMYYNGCKFARSKIPRKFKLLGDDPKEEEKLESHLQNLSTLMAPTYKKLAPDAYN NQIEYEHRAPECRLGLKEGRPFSGVTACLDFCAHAHRDLHNMQNGSTLVCTLTREDNREFGG KPEDEQLHVLPLYKVSDVDEFGSVEAQEEKKRSGAIQVLSSFRRKVRMLAEPVKTCRQRKLE AKKAAAEKLSSLENSSNKNEKEKSAPSRTKQTENASQAKQLAELLRLSGPVMQQSQQPQPLQ KQPPQPQQQQRPQQQQPHHPQTESVNSYSASGSTNPYMRRPNPVSPYPNSSHTSDIYGSTSP MNFYSTSSQAAGSYLNSSNPMNPYPGLLNQNTQYPSYQCNGNLSVDNCSPYLGSYSPQSQPM DLYRYPSQDPLSKLSLPPIHTLYQPRFGNSQSFTSKYLGYGNQNMQGDGFSSCTIRPNVHHV GKLPPYPTHEMDGHFMGATSRLPPNLSNPNMDYKNGEHHSPSHIIHNYSAAPGMFNSSLHAL HLQNKENDMLSHTANGLSKMLPALNHDRTACVQGGLHKLSDANGQEKQPLALVQGVASGAED NDEVWSDSEQSFLDPDIGGVAVAPTHGSILIECAKRELHATTPLKNPNRNHPTRISLVFYQH KSMNEPKHGLALWEAKMAEKAREKEEECEKY (SEQ ID NO: 19) SEQ ID NO: 20 – Human TET2 isoform A catalytic domain polynucleotide coding sequence TCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAATTT ATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGAGCAAATTA TTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCAGCTATT AGAGAAATCATGGAAGAAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTCAT CTATACTGGTAAAGAAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGTGGTTCGCA GAAGCAGCAGTGAAGAGAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACACCTGTGAG GCTGCAGTGATTGTGATTCTCATCCTGGTGTGGGAAGGAATCCCGCTGTCTCTGGCTGACAA ACTCTACTCGGAGCTTACCGAGACGCTGAGGAAATACGGCACGCTCACCAATCGCCGGTGTG CCTTGAATGAAGAGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCC TTCTCTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTAAGTTTGCCAGAAGCAAGAT CCCAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAACTGGAGTCTCATT TGCAAAACCTGTCCACTCTTATGGCACCAACATATAAGAAACTTGCACCTGATGCATATAAT AATCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCC ATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTTGCACAACA TGCAGAATGGCAGCACATTGGTATGCACTCTCACTAGAGAAGACAATCGAGAATTTGGAGGA AAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACGTGGATGAGTT TGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACTGAGTTCTT TTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGAAACTAGAA GCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAAAATGAAAA GGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGCTAAACAGTTGG CAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGCCTCTACAG AAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAGCCACATCACCCTCA GACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAGACGGCCCA ATCCAGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCTAATCCCAT GAACCCTTACCCTGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATGCAATGGAA ACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTCAGCCGATG GATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCCATCCATAC ACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGTTATGGAA ACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTA GGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAG ATTACCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCACCTT CTCACATAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGCTCTCTTCATGCCCTG CATCTCCAAAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAAAGATGCT TCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATG CTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTGCAGAGGAC AACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGTGGC CGTGGCTCCAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGCCACAA CCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTACCAGCAT AAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGC CCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCAGAAATCCCATG GCAAAAAAGTGAAACGGGAGCCTGCTGAGCCACATGAAACTTCAGAGCCCACTTACCTGCGT TTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACAGACTCCACAGTAACTACATC TCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATATATGA (SEQ ID NO: 20) SEQ ID NO: 21 – Mouse TET2 catalytic domain amino acid sequence GSTSNGRQCAGIRPLQSQNGKCEGCNPDKDEAPYYTHLGAGPDVAAIRTLMEERYGEKGKAI RIEKVIYTGKEGKSSQGCPIAKWVYRRSSEEEKLLCLVRVRPNHTCETAVMVIAIMLWDGIP KLLASELYSELTDILGKCGICTNRRCSQNETKKKQSPPRNCCCQGENPETCGASFSFGCSWS MYYNGCKFARSKKPRKFRLHGAEPKEEERLGSHLQNLATVIAPIYKKLAPDAYNNQVEFEHQ APDCCLGLKEGRPFSGVTACLDFSAHSHRDQQNMPNGSTVVVTLNREDNREVGAKPEDEQFH VLPMYIIAPEDEFGSTEGQEKKIRMGSIEVLQSFRRRRVIRIGELPKSCKKKAEPKKAKTKK AARKHSSLENCSSRTEKGKSSSHTKLMENASHMKQMTAQPQLSGPVIRQPPTLQRHLQQGQR PQQPQPPQPQPQTTPQPQPQPQHIMPGNSQSVGSHCSGSTSVYTRQPTPHSPYPSSAHTSDI YGDTNHVNFYPTSSHASGSYLNPSNYMNPYLGLLNQNNQYAPFPYNGSVPVDNGSPFLGSYS PQAQSRDLHRYPNQDHLTNQNLPPIHTLHQQTFGDSPSKYLSYGNQNMQRDAFTTNSTLKPN VHHLATFSPYPTPKMDSHFMGAASRSPYSHPHTDYKTSEHHLPSHTVYSYTAAASGSSSSHA FHNKENDNIANGLSRVLPGFNHDRTASAQELLYSLTGSSQEKQPEVSGQDAAAVQEIEYWSD SEHNFQDPCIGGVAIAPTHGSILIECAKCEVHATTKVNDPDRNHPTRISLVLYRHKNLFLPK HCLALWEAKMAEKARKEEECGKNGSDHVSQKNHGKQEKREPTGPQEPSYLRFIQSLAENTGS VTTDSTVTTSPYAFTQVTGPYNTFV (SEQ ID NO: 21) SEQ ID NO: 22 – Mouse TET2 catalytic domain polynucleotide coding sequence ggatccactagtaacggccgccagtgtgctggaattcgccctttacaaagtcagaatggcaa atgtgaaggatgcaatccagacaaagatgaagctccttattatacccatctgggagctggtc ctgatgtggcagctattagaacactcatggaagaaaggtatggagagaagggtaaagctatt aggattgaaaaagtcatatatactggtaaagaaggcaagagctctcagggatgtcctattgc taaatgggtatatcggagatcgagtgaggaggagaaactactgtgtttggtacgagtgcgac ctaaccacacatgtgagacggcggtgatggtaattgccatcatgttgtgggacggaatccca aagctactcgcatcagaactctactcagaacttacagatatcttgggcaagtgtggcatatg caccaaccgtcgctgttctcagaatgaaacgaagaaaaagcaatcaccacccagaaactgtt gttgtcagggtgagaatccagagacctgtggtgcctccttttcttttggttgttcttggagc atgtactataatggatgtaagtttgccagaagcaagaaaccaaggaaatttaggctacatgg agctgagccaaaagaggaagagagactaggttctcatttgcaaaacctggctactgtcattgctccaatatacaagaagcttgcacccgatgcatacaataatcaggttgaatttgaacaccaa gccccagactgctgtttgggtctgaaggaaggccggccattctcaggagtcactgcatgttt ggacttctctgctcattcccacagagaccagcagaacatgccaaatggcagtacagtggtgg tcaccctcaatagagaagacaatcgagaagtcggagctaagcctgaggatgagcagttccac gtgctgcctatgtacatcatcgcccctgaggatgagtttgggagtacggaaggccaggagaa gaagatacggatggggtccattgaggttctgcagtcatttcggaggagaagggtcataagga taggagagctgcccaagagttgcaagaagaaagcggagcccaagaaagccaagaccaagaaa gcagctcgaaagcattcctctctggagaactgctccagtaggactgagaagggaaagtcttc ctcacatacaaagctgatggaaaatgcaagccatatgaaacaaatgacagcacaaccgcagc tttcgggcccggtcatccggcagccaccaacactccagaggcaccttcagcaagggcagagg ccacagcagccgcagccacctcagccgcagccgcagacgacacctcagccacagccacagcc acagcatatcatgcccggtaactctcagtctgttggttctcattgttctggatccaccagtg tctacacgagacagcctactcctcacagtccttatcccagctcagcacacacctcagatatt tatggagataccaaccatgtgaacttttaccccacttcatctcatgcctcgggttcatattt gaatccttctaattacatgaacccctaccttgggcttttgaatcagaataaccaatatgcac cttttccatacaatgggagtgtgccagtggacaatggttcccctttcttaggttcttattcc ccccaggctcagtccagggatctacatagatatccaaaccaggaccatctcaccaatcagaa cttaccacccatccacacccttcaccaacagacgtttggggacagtccctctaagtacttaa gttatggaaaccaaaatatgcagagagatgccttcactactaactccaccctaaaaccaaat gtacaccacctagcaacgttttctccttaccccacccccaagatggatagtcatttcatggg agctgcctccagatcaccatacagccacccacacactgactacaaaaccagtgagcatcatc taccctctcacacggtctacagctacacggcagcagcttcggggagcagttccagccacgcc ttccacaacaaggagaatgacaacatagccaatgggctctcaagagtgcttccagggtttaa tcatgatagaactgcttctgcccaagaactattatacagtctgactggcagcagtcaggaga agcagcctgaggtgtcaggccaggatgcagctgctgtgcaggaaattgagtattggtcagat agtgagcacaactttcaggatccttgcattggaggggtggctatagccccaactcatgggtc aattcttattgagtgtgcaaagtgtgaggttcatgccacaaccaaagtaaacgatcccgacc ggaatcaccccaccaggatctcacttgtactgtataggcataagaatttgtttctaccaaaa cattgtttggctctctgggaagccaaaatggctgaaaaggcccggaaagaggaagagtgcgg aaagaatggatcagaccacgtgtctcagaaaaatcatggcaaacaggaaaagcgtgagccca cagggccacaggaacccagttacctgcgtttcatccagtctcttgctgagaacacagggtct gtgactacggattctaccgtgactacatcaccatatgctttcactcaggtcacagggcctta caacacatttgta (SEQ ID NO: 22) SEQ ID NO: 23 – Mouse TET2 catalytic domain (dead) amino acid sequence GSTSNGRQCAGIRPLQSQNGKCEGCNPDKDEAPYYTHLGAGPDVAAIRTLMEERYGEKGKAI RIEKVIYTGKEGKSSQGCPIAKWVYRRSSEEEKLLCLVRVRPNHTCETAVMVIAIMLWDGIP KLLASELYSELTDILGKCGICTNRRCSQNETKKKQSPPRNCCCQGENPETCGASFSFGCSWS MYYNGCKFARSKKPRKFRLHGAEPKEEERLGSHLQNLATVIAPIYKKLAPDAYNNQVEFEHQ APDCCLGLKEGRPFSGVTACLDFSAHSYRAQQNMPNGSTVVVTLNREDNREVGAKPEDEQFH VLPMYIIAPEDEFGSTEGQEKKIRMGSIEVLQSFRRRRVIRIGELPKSCKKKAEPKKAKTKK AARKHSSLENCSSRTEKGKSSSHTKLMENASHMKQMTAQPQLSGPVIRQPPTLQRHLQQGQR PQQPQPPQPQPQTTPQPQPQPQHIMPGNSQSVGSHCSGSTSVYTRQPTPHSPYPSSAHTSDI YGDTNHVNFYPTSSHASGSYLNPSNYMNPYLGLLNQNNQYAPFPYNGSVPVDNGSPFLGSYS PQAQSRDLHRYPNQDHLTNQNLPPIHTLHQQTFGDSPSKYLSYGNQNMQRDAFTTNSTLKPN VHHLATFSPYPTPKMDSHFMGAASRSPYSHPHTDYKTSEHHLPSHTVYSYTAAASGSSSSHA FHNKENDNIANGLSRVLPGFNHDRTASAQELLYSLTGSSQEKQPEVSGQDAAAVQEIEYWSD SEHNFQDPCIGGVAIAPTHGSILIECAKCEVHATTKVNDPDRNHPTRISLVLYRHKNLFLPK HCLALWEAKMAEKARKEEECGKNGSDHVSQKNHGKQEKREPTGPQEPSYLRFIQSLAENTGS VTTDSTVTTSPYAFTQVTGPYNTFV (SEQ ID NO: 23)SEQ ID NO: 24 – Mouse TET2 catalytic domain (dead) polynucleotide coding sequence GGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGCCCTTTACAAAGTCAGAATGGCAA ATGTGAAGGATGCAATCCAGACAAAGATGAAGCTCCTTATTATACCCATCTGGGAGCTGGTC CTGATGTGGCAGCTATTAGAACACTCATGGAAGAAAGGTATGGAGAGAAGGGTAAAGCTATT AGGATTGAAAAAGTCATATATACTGGTAAAGAAGGCAAGAGCTCTCAGGGATGTCCTATTGC TAAATGGGTATATCGGAGATCGAGTGAGGAGGAGAAACTACTGTGTTTGGTACGAGTGCGAC CTAACCACACATGTGAGACGGCGGTGATGGTAATTGCCATCATGTTGTGGGACGGAATCCCA AAGCTACTCGCATCAGAACTCTACTCAGAACTTACAGATATCTTGGGCAAGTGTGGCATATG CACCAACCGTCGCTGTTCTCAGAATGAAACGAAGAAAAAGCAATCACCACCCAGAAACTGTT GTTGTCAGGGTGAGAATCCAGAGACCTGTGGTGCCTCCTTTTCTTTTGGTTGTTCTTGGAGC ATGTACTATAATGGATGTAAGTTTGCCAGAAGCAAGAAACCAAGGAAATTTAGGCTACATGG AGCTGAGCCAAAAGAGGAAGAGAGACTAGGTTCTCATTTGCAAAACCTGGCTACTGTCATTG CTCCAATATACAAGAAGCTTGCACCCGATGCATACAATAATCAGGTTGAATTTGAACACCAA GCCCCAGACTGCTGTTTGGGTCTGAAGGAAGGCCGGCCATTCTCAGGAGTCACTGCATGTTT GGACTTCTCTGCTCATTCCTACAGAGCCCAGCAGAACATGCCAAATGGCAGTACAGTGGTGG TCACCCTCAATAGAGAAGACAATCGAGAAGTCGGAGCTAAGCCTGAGGATGAGCAGTTCCAC GTGCTGCCTATGTACATCATCGCCCCTGAGGATGAGTTTGGGAGTACGGAAGGCCAGGAGAA GAAGATACGGATGGGGTCCATTGAGGTTCTGCAGTCATTTCGGAGGAGAAGGGTCATAAGGA TAGGAGAGCTGCCCAAGAGTTGCAAGAAGAAAGCGGAGCCCAAGAAAGCCAAGACCAAGAAA GCAGCTCGAAAGCATTCCTCTCTGGAGAACTGCTCCAGTAGGACTGAGAAGGGAAAGTCTTC CTCACATACAAAGCTGATGGAAAATGCAAGCCATATGAAACAAATGACAGCACAACCGCAGC TTTCGGGCCCGGTCATCCGGCAGCCACCAACACTCCAGAGGCACCTTCAGCAAGGGCAGAGG CCACAGCAGCCGCAGCCACCTCAGCCGCAGCCGCAGACGACACCTCAGCCACAGCCACAGCC ACAGCATATCATGCCCGGTAACTCTCAGTCTGTTGGTTCTCATTGTTCTGGATCCACCAGTG TCTACACGAGACAGCCTACTCCTCACAGTCCTTATCCCAGCTCAGCACACACCTCAGATATT TATGGAGATACCAACCATGTGAACTTTTACCCCACTTCATCTCATGCCTCGGGTTCATATTT GAATCCTTCTAATTACATGAACCCCTACCTTGGGCTTTTGAATCAGAATAACCAATATGCAC CTTTTCCATACAATGGGAGTGTGCCAGTGGACAATGGTTCCCCTTTCTTAGGTTCTTATTCC CCCCAGGCTCAGTCCAGGGATCTACATAGATATCCAAACCAGGACCATCTCACCAATCAGAA CTTACCACCCATCCACACCCTTCACCAACAGACGTTTGGGGACAGTCCCTCTAAGTACTTAA GTTATGGAAACCAAAATATGCAGAGAGATGCCTTCACTACTAACTCCACCCTAAAACCAAAT GTACACCACCTAGCAACGTTTTCTCCTTACCCCACCCCCAAGATGGATAGTCATTTCATGGG AGCTGCCTCCAGATCACCATACAGCCACCCACACACTGACTACAAAACCAGTGAGCATCATC TACCCTCTCACACGGTCTACAGCTACACGGCAGCAGCTTCGGGGAGCAGTTCCAGCCACGCC TTCCACAACAAGGAGAATGACAACATAGCCAATGGGCTCTCAAGAGTGCTTCCAGGGTTTAA TCATGATAGAACTGCTTCTGCCCAAGAACTATTATACAGTCTGACTGGCAGCAGTCAGGAGA AGCAGCCTGAGGTGTCAGGCCAGGATGCAGCTGCTGTGCAGGAAATTGAGTATTGGTCAGAT AGTGAGCACAACTTTCAGGATCCTTGCATTGGAGGGGTGGCTATAGCCCCAACTCATGGGTC AATTCTTATTGAGTGTGCAAAGTGTGAGGTTCATGCCACAACCAAAGTAAACGATCCCGACC GGAATCACCCCACCAGGATCTCACTTGTACTGTATAGGCATAAGAATTTGTTTCTACCAAAA CATTGTTTGGCTCTCTGGGAAGCCAAAATGGCTGAAAAGGCCCGGAAAGAGGAAGAGTGCGG AAAGAATGGATCAGACCACGTGTCTCAGAAAAATCATGGCAAACAGGAAAAGCGTGAGCCCA CAGGGCCACAGGAACCCAGTTACCTGCGTTTCATCCAGTCTCTTGCTGAGAACACAGGGTCT GTGACTACGGATTCTACCGTGACTACATCACCATATGCTTTCACTCAGGTCACAGGGCCTTA CAACACATTTGTATGA (SEQ ID NO: 24) A. Targeting Proteins and Polypeptides

[0182] In certain aspects, proteins capable of RNA m5C installation, reading / binding, and / or erasing, or polypeptides derived therefrom, are guided to a target RNA molecule (suchas but not limited to, an RNA molecule comprising a m5C RNA feature) by a targeting element comprising a protein, polypeptide, and / or RNA molecule. In some aspects, a targeting element comprising a protein and / or polypeptide can be guided to a target RNA element by a complementary, or at least partially complementary, RNA molecule.

[0183] In some aspects, a targeting element comprises a Cas protein. In some aspects, a targeting element comprises an engineered Cas protein with reduced and / or absent gene-editing activity relative to non-engineered Cas proteins, such proteins may be referenced as “dead” Cas (dCas) proteins. In some aspects, dCas proteins such as but not limited to dCas13d, can have less than or equal to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any range derivable therein, less potent gene-editing activity relative to non-engineered parental Cas proteins. In some aspects, a Cas protein can be, but is not limited to a wild type and / or modified variant of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, Cas13a, Cas13b, Cas13c, Cas13d, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some aspects, a Cas protein can be, or can be derived from, a type I, type II, type III, type IV, type V, and / or type VI, CRISPR systems.

[0184] In some aspects, a targeting element, such as a targeting protein, can be linked to a protein (or polypeptide derived therefrom) capable of RNA m5C installation, reading (e.g., binding), and / or erasing (e.g., oxidation). In some aspects, a targeting protein can be directly fused to a protein (or polypeptide derived therefrom) capable of RNA m5C installation, reading (e.g., binding), and / or erasing (e.g., oxidation). In some aspects, a targeting protein can be linked to a protein (or polypeptide derived therefrom) capable of RNA m5C installation, reading (e.g., binding), and / or erasing (e.g., oxidation) via a linker. In some aspects, a linker comprises a polypeptide. In some aspects, a linker polypeptide comprises or is a flexible polypeptide. In some aspects, a linker polypeptide comprises a sequence that is, is about, or is at least about 80%, 85% 90%, 95%, 98%, 99%, 100%, or any range derivable therein, identical to SEQ ID NO: 33.

[0185] In some aspects, a targeting element comprises, consists essentially of, or consists of an amino acid sequence or is encoded by a polynucleotide sequence, that is, is about, or is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity to any one of SEQ ID NOs: 25-30. SEQ ID NO: 25 - Exemplary catalytically dead Cas13d (dCas13d) amino acid sequence NIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENNENLWFHPVMSHLYNAKN GYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLK MYRDLTNAYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDK RFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRL PIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSA EKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCI DGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDT YTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKK TEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRL TVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGE NKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPAN AVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELP RQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQWNRNYRYMDMLKG EYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNRQMRNASSEEIETILDKRL SNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMP MSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKEDGSKRTADGSE F (SEQ ID NO: 25) SEQ ID NO: 26 - Exemplary catalytically dead Cas13d (dCas13d) polynucleotide sequence aacatccccgctctggtggaaaaccagaagaagtactttggcacctacagcgtgatggccat gctgaacgctcagaccgtgctggaccacatccagaaggtggccgatattgagggcgagcaga acgagaacaacgagaatctgtggtttcaccccgtgatgagccacctgtacaacgccaagaac ggctacgacaagcagcccgagaaaaccatgttcatcatcgagcggctgcagagctacttccc attcctgaagatcatggccgagaaccagagagagtacagcaacggcaagtacaagcagaacc gcgtggaagtgaacagcaacgacatcttcgaggtgctgaagcgcgccttcggcgtgctgaag atgtacagggacctgaccaacgcatacaagacctacgaggaaaagctgaacgacggctgcga gttcctgaccagcacagagcaacctctgagcggcatgatcaacaactactacacagtggccc tgcggaacatgaacgagagatacggctacaagacagaggacctggccttcatccaggacaag cggttcaagttcgtgaaggacgcctacggcaagaaaaagtcccaagtgaataccggattctt cctgagcctgcaggactacaacggcgacacacagaagaagctgcacctgagcggagtgggaa tcgccctgctgatctgcctgttcctggacaagcagtacatcaacatctttctgagcaggctg cccatcttctccagctacaatgcccagagcgaggaacggcggatcatcatcagatccttcgg catcaacagcatcaagctgcccaaggaccggatccacagcgagaagtccaacaagagcgtgg ccatggatatgctcaacgaagtgaagcggtgccccgacgagctgttcacaacactgtctgcc gagaagcagtcccggttcagaatcatcagcgacgaccacaatgaagtgctgatgaagcggag cagcgacagattcgtgcctctgctgctgcagtatatcgattacggcaagctgttcgaccaca tcaggttccacgtgaacatgggcaagctgagatacctgctgaaggccgacaagacctgcatc gacggccagaccagagtcagagtgatcgagcagcccctgaacggcttcggcagactggaaga ggccgagacaatgcggaagcaagagaacggcaccttcggcaacagcggcatccggatcagag acttcgagaacatgaagcgggacgacgccaatcctgccaactatccctacatcgtggacacc tacacacactacatcctggaaaacaacaaggtcgagatgtttatcaacgacaaagaggacag cgccccactgctgcccgtgatcgaggatgatagatacgtggtcaagacaatccccagctgcc ggatgagcaccctggaaattccagccatggccttccacatgtttctgttcggcagcaagaaa accgagaagctgatcgtggacgtgcacaaccggtacaagagactgttccaggccatgcagaa agaagaagtgaccgccgagaatatcgccagcttcggaatcgccgagagcgacctgcctcagaagatcctggatctgatcagcggcaatgcccacggcaaggatgtggacgccttcatcagactg accgtggacgacatgctgaccgacaccgagcggagaatcaagagattcaaggacgaccggaa gtccattcggagcgccgacaacaagatgggaaagagaggcttcaagcagatctccacaggca agctggccgacttcctggccaaggacatcgtgctgtttcagcccagcgtgaacgatggcgag aacaagatcaccggcctgaactaccggatcatgcagagcgccattgccgtgtacgatagcgg cgacgattacgaggccaagcagcagttcaagctgatgttcgagaaggcccggctgatcggca agggcacaacagagcctcatccatttctgtacaaggtgttcgcccgcagcatccccgccaat gccgtcgagttctacgagcgctacctgatcgagcggaagttctacctgaccggcctgtccaa cgagatcaagaaaggcaacagagtggatgtgcccttcatccggcgggaccagaacaagtgga aaacacccgccatgaagaccctgggcagaatctacagcgaggatctgcccgtggaactgccc agacagatgttcgacaatgagatcaagtcccacctgaagtccctgccacagatggaaggcat cgacttcaacaatgccaacgtgacctatctgatcgccgagtacatgaagagagtgctggacg acgacttccagaccttctaccagtggaaccgcaactaccggtacatggacatgcttaagggc gagtacgacagaaagggctccctgcagcactgcttcaccagcgtggaagagagagaaggcct ctggaaagagcgggcctccagaacagagcggtacagaaagcaggccagcaacaagatccgca gcaaccggcagatgagaaacgccagcagcgaagagatcgagacaatcctggataagcggctg agcaacagccggaacgagtaccagaaaagcgagaaagtgatccggcgctacagagtgcagga tgccctgctgtttctgctggccaaaaagaccctgaccgaactggccgatttcgacggcgaga ggttcaaactgaaagaaatcatgcccgacgccgagaagggaatcctgagcgagatcatgccc atgagcttcaccttcgagaaaggcggcaagaagtacaccatcaccagcgagggcatgaagct gaagaactacggcgacttctttgtgctggctagcgacaagaggatcggcaacctgctggaac tcgtgggcagcgacatcgtgtccaaagaggatggatccaaaagaaccgccgacggcagcgaa ttc (SEQ ID NO: 26) SEQ ID NO: 27 - Exemplary catalytically dead Cas13d (dCas13d) fused to mouse TET2 catalytic domain (TET(CD)) amino acid (shown with optional HA tag, SV40 NLS, and Flag Tag, each of which are underlined) MYPYDVPDYASPKKKRKVNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNEN NENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVE VNSNDIFEVLKRAFGVLKMYRDLTNAYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVALRN MNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIAL LICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMD MLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRF HVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFE NMKRDDANPANYPYIVDTYTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMS TLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKIL DLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLA DFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGT TEPHPFLYKVFARSIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTP AMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDF QTFYQWNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNR QMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFK LKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVG SDIVSKEDGSKRTADGSEFDYKDDDDKGSTSNGRQCAGIRPLQSQNGKCEGCNPDKDEAPYY THLGAGPDVAAIRTLMEERYGEKGKAIRIEKVIYTGKEGKSSQGCPIAKWVYRRSSEEEKLL CLVRVRPNHTCETAVMVIAIMLWDGIPKLLASELYSELTDILGKCGICTNRRCSQNETKKKQ SPPRNCCCQGENPETCGASFSFGCSWSMYYNGCKFARSKKPRKFRLHGAEPKEEERLGSHLQ NLATVIAPIYKKLAPDAYNNQVEFEHQAPDCCLGLKEGRPFSGVTACLDFSAHSHRDQQNMP NGSTVVVTLNREDNREVGAKPEDEQFHVLPMYIIAPEDEFGSTEGQEKKIRMGSIEVLQSFR RRRVIRIGELPKSCKKKAEPKKAKTKKAARKHSSLENCSSRTEKGKSSSHTKLMENASHMKQ MTAQPQLSGPVIRQPPTLQRHLQQGQRPQQPQPPQPQPQTTPQPQPQPQHIMPGNSQSVGSHCSGSTSVYTRQPTPHSPYPSSAHTSDIYGDTNHVNFYPTSSHASGSYLNPSNYMNPYLGLLN QNNQYAPFPYNGSVPVDNGSPFLGSYSPQAQSRDLHRYPNQDHLTNQNLPPIHTLHQQTFGD SPSKYLSYGNQNMQRDAFTTNSTLKPNVHHLATFSPYPTPKMDSHFMGAASRSPYSHPHTDY KTSEHHLPSHTVYSYTAAASGSSSSHAFHNKENDNIANGLSRVLPGFNHDRTASAQELLYSL TGSSQEKQPEVSGQDAAAVQEIEYWSDSEHNFQDPCIGGVAIAPTHGSILIECAKCEVHATT KVNDPDRNHPTRISLVLYRHKNLFLPKHCLALWEAKMAEKARKEEECGKNGSDHVSQKNHGK QEKREPTGPQEPSYLRFIQSLAENTGSVTTDSTVTTSPYAFTQVTGPYNTFVV (SEQ ID NO: 27) SEQ ID NO: 28 - Exemplary catalytically dead Cas13d (dCas13d) fused to mouse TET2 catalytic domain (TET(CD)) polynucleotide coding sequence (shown with optional HA tag, SV40 NLS, and Flag Tag, each of which are underlined) ATGtacccatacgatgttccagattacgcttcgccaaagaagaagcggaaagtcaacatccc cgctctggtggaaaaccagaagaagtactttggcacctacagcgtgatggccatgctgaacg ctcagaccgtgctggaccacatccagaaggtggccgatattgagggcgagcagaacgagaac aacgagaatctgtggtttcaccccgtgatgagccacctgtacaacgccaagaacggctacga caagcagcccgagaaaaccatgttcatcatcgagcggctgcagagctacttcccattcctga agatcatggccgagaaccagagagagtacagcaacggcaagtacaagcagaaccgcgtggaa gtgaacagcaacgacatcttcgaggtgctgaagcgcgccttcggcgtgctgaagatgtacag ggacctgaccaacgcatacaagacctacgaggaaaagctgaacgacggctgcgagttcctga ccagcacagagcaacctctgagcggcatgatcaacaactactacacagtggccctgcggaac atgaacgagagatacggctacaagacagaggacctggccttcatccaggacaagcggttcaa gttcgtgaaggacgcctacggcaagaaaaagtcccaagtgaataccggattcttcctgagcc tgcaggactacaacggcgacacacagaagaagctgcacctgagcggagtgggaatcgccctg ctgatctgcctgttcctggacaagcagtacatcaacatctttctgagcaggctgcccatctt ctccagctacaatgcccagagcgaggaacggcggatcatcatcagatccttcggcatcaaca gcatcaagctgcccaaggaccggatccacagcgagaagtccaacaagagcgtggccatggat atgctcaacgaagtgaagcggtgccccgacgagctgttcacaacactgtctgccgagaagca gtcccggttcagaatcatcagcgacgaccacaatgaagtgctgatgaagcggagcagcgaca gattcgtgcctctgctgctgcagtatatcgattacggcaagctgttcgaccacatcaggttc cacgtgaacatgggcaagctgagatacctgctgaaggccgacaagacctgcatcgacggcca gaccagagtcagagtgatcgagcagcccctgaacggcttcggcagactggaagaggccgaga caatgcggaagcaagagaacggcaccttcggcaacagcggcatccggatcagagacttcgag aacatgaagcgggacgacgccaatcctgccaactatccctacatcgtggacacctacacaca ctacatcctggaaaacaacaaggtcgagatgtttatcaacgacaaagaggacagcgccccac tgctgcccgtgatcgaggatgatagatacgtggtcaagacaatccccagctgccggatgagc accctggaaattccagccatggccttccacatgtttctgttcggcagcaagaaaaccgagaa gctgatcgtggacgtgcacaaccggtacaagagactgttccaggccatgcagaaagaagaag tgaccgccgagaatatcgccagcttcggaatcgccgagagcgacctgcctcagaagatcctg gatctgatcagcggcaatgcccacggcaaggatgtggacgccttcatcagactgaccgtgga cgacatgctgaccgacaccgagcggagaatcaagagattcaaggacgaccggaagtccattc ggagcgccgacaacaagatgggaaagagaggcttcaagcagatctccacaggcaagctggcc gacttcctggccaaggacatcgtgctgtttcagcccagcgtgaacgatggcgagaacaagat caccggcctgaactaccggatcatgcagagcgccattgccgtgtacgatagcggcgacgatt acgaggccaagcagcagttcaagctgatgttcgagaaggcccggctgatcggcaagggcaca acagagcctcatccatttctgtacaaggtgttcgcccgcagcatccccgccaatgccgtcga gttctacgagcgctacctgatcgagcggaagttctacctgaccggcctgtccaacgagatca agaaaggcaacagagtggatgtgcccttcatccggcgggaccagaacaagtggaaaacaccc gccatgaagaccctgggcagaatctacagcgaggatctgcccgtggaactgcccagacagat gttcgacaatgagatcaagtcccacctgaagtccctgccacagatggaaggcatcgacttca acaatgccaacgtgacctatctgatcgccgagtacatgaagagagtgctggacgacgacttccagaccttctaccagtggaaccgcaactaccggtacatggacatgcttaagggcgagtacga cagaaagggctccctgcagcactgcttcaccagcgtggaagagagagaaggcctctggaaag agcgggcctccagaacagagcggtacagaaagcaggccagcaacaagatccgcagcaaccgg cagatgagaaacgccagcagcgaagagatcgagacaatcctggataagcggctgagcaacag ccggaacgagtaccagaaaagcgagaaagtgatccggcgctacagagtgcaggatgccctgc tgtttctgctggccaaaaagaccctgaccgaactggccgatttcgacggcgagaggttcaaa ctgaaagaaatcatgcccgacgccgagaagggaatcctgagcgagatcatgcccatgagctt caccttcgagaaaggcggcaagaagtacaccatcaccagcgagggcatgaagctgaagaact acggcgacttctttgtgctggctagcgacaagaggatcggcaacctgctggaactcgtgggc agcgacatcgtgtccaaagaggatggatccaaaagaaccgccgacggcagcgaattcgacta caaagacgatgacgacaagggatccactagtaacggccgccagtgtgctggaattcgccctt tacaaagtcagaatggcaaatgtgaaggatgcaatccagacaaagatgaagctccttattat acccatctgggagctggtcctgatgtggcagctattagaacactcatggaagaaaggtatgg agagaagggtaaagctattaggattgaaaaagtcatatatactggtaaagaaggcaagagct ctcagggatgtcctattgctaaatgggtatatcggagatcgagtgaggaggagaaactactg tgtttggtacgagtgcgacctaaccacacatgtgagacggcggtgatggtaattgccatcat gttgtgggacggaatcccaaagctactcgcatcagaactctactcagaacttacagatatct tgggcaagtgtggcatatgcaccaaccgtcgctgttctcagaatgaaacgaagaaaaagcaa tcaccacccagaaactgttgttgtcagggtgagaatccagagacctgtggtgcctccttttc ttttggttgttcttggagcatgtactataatggatgtaagtttgccagaagcaagaaaccaa ggaaatttaggctacatggagctgagccaaaagaggaagagagactaggttctcatttgcaa aacctggctactgtcattgctccaatatacaagaagcttgcacccgatgcatacaataatca ggttgaatttgaacaccaagccccagactgctgtttgggtctgaaggaaggccggccattct caggagtcactgcatgtttggacttctctgctcattcccacagagaccagcagaacatgcca aatggcagtacagtggtggtcaccctcaatagagaagacaatcgagaagtcggagctaagcc tgaggatgagcagttccacgtgctgcctatgtacatcatcgcccctgaggatgagtttggga gtacggaaggccaggagaagaagatacggatggggtccattgaggttctgcagtcatttcgg aggagaagggtcataaggataggagagctgcccaagagttgcaagaagaaagcggagcccaa gaaagccaagaccaagaaagcagctcgaaagcattcctctctggagaactgctccagtagga ctgagaagggaaagtcttcctcacatacaaagctgatggaaaatgcaagccatatgaaacaa atgacagcacaaccgcagctttcgggcccggtcatccggcagccaccaacactccagaggca ccttcagcaagggcagaggccacagcagccgcagccacctcagccgcagccgcagacgacac ctcagccacagccacagccacagcatatcatgcccggtaactctcagtctgttggttctcat tgttctggatccaccagtgtctacacgagacagcctactcctcacagtccttatcccagctc agcacacacctcagatatttatggagataccaaccatgtgaacttttaccccacttcatctc atgcctcgggttcatatttgaatccttctaattacatgaacccctaccttgggcttttgaat cagaataaccaatatgcaccttttccatacaatgggagtgtgccagtggacaatggttcccc tttcttaggttcttattccccccaggctcagtccagggatctacatagatatccaaaccagg accatctcaccaatcagaacttaccacccatccacacccttcaccaacagacgtttggggac agtccctctaagtacttaagttatggaaaccaaaatatgcagagagatgccttcactactaa ctccaccctaaaaccaaatgtacaccacctagcaacgttttctccttaccccacccccaaga tggatagtcatttcatgggagctgcctccagatcaccatacagccacccacacactgactac aaaaccagtgagcatcatctaccctctcacacggtctacagctacacggcagcagcttcggg gagcagttccagccacgccttccacaacaaggagaatgacaacatagccaatgggctctcaa gagtgcttccagggtttaatcatgatagaactgcttctgcccaagaactattatacagtctg actggcagcagtcaggagaagcagcctgaggtgtcaggccaggatgcagctgctgtgcagga aattgagtattggtcagatagtgagcacaactttcaggatccttgcattggaggggtggcta tagccccaactcatgggtcaattcttattgagtgtgcaaagtgtgaggttcatgccacaacc aaagtaaacgatcccgaccggaatcaccccaccaggatctcacttgtactgtataggcataa gaatttgtttctaccaaaacattgtttggctctctgggaagccaaaatggctgaaaaggccc ggaaagaggaagagtgcggaaagaatggatcagaccacgtgtctcagaaaaatcatggcaaacaggaaaagcgtgagcccacagggccacaggaacccagttacctgcgtttcatccagtctct tgctgagaacacagggtctgtgactacggattctaccgtgactacatcaccatatgctttca ctcaggtcacagggccttacaacacatttgtagtttaa (SEQ ID NO: 28) SEQ ID NO: 29 - Exemplary catalytically dead Cas13d (dCas13d) fused to mouse TET2 catalytic domain dead (TET(HxD)) amino acid (shown with optional HA tag, SV40 NLS, and Flag Tag, each of which are underlined) MYPYDVPDYASPKKKRKVNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNEN NENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVE VNSNDIFEVLKRAFGVLKMYRDLTNAYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVALRN MNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIAL LICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMD MLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRF HVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFE NMKRDDANPANYPYIVDTYTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMS TLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKIL DLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLA DFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGT TEPHPFLYKVFARSIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTP AMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDF QTFYQWNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNR QMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFK LKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVG SDIVSKEDGSKRTADGSEFDYKDDDDKGSTSNGRQCAGIRPLQSQNGKCEGCNPDKDEAPYY THLGAGPDVAAIRTLMEERYGEKGKAIRIEKVIYTGKEGKSSQGCPIAKWVYRRSSEEEKLL CLVRVRPNHTCETAVMVIAIMLWDGIPKLLASELYSELTDILGKCGICTNRRCSQNETKKKQ SPPRNCCCQGENPETCGASFSFGCSWSMYYNGCKFARSKKPRKFRLHGAEPKEEERLGSHLQ NLATVIAPIYKKLAPDAYNNQVEFEHQAPDCCLGLKEGRPFSGVTACLDFSAHSYRAQQNMP NGSTVVVTLNREDNREVGAKPEDEQFHVLPMYIIAPEDEFGSTEGQEKKIRMGSIEVLQSFR RRRVIRIGELPKSCKKKAEPKKAKTKKAARKHSSLENCSSRTEKGKSSSHTKLMENASHMKQ MTAQPQLSGPVIRQPPTLQRHLQQGQRPQQPQPPQPQPQTTPQPQPQPQHIMPGNSQSVGSH CSGSTSVYTRQPTPHSPYPSSAHTSDIYGDTNHVNFYPTSSHASGSYLNPSNYMNPYLGLLN QNNQYAPFPYNGSVPVDNGSPFLGSYSPQAQSRDLHRYPNQDHLTNQNLPPIHTLHQQTFGD SPSKYLSYGNQNMQRDAFTTNSTLKPNVHHLATFSPYPTPKMDSHFMGAASRSPYSHPHTDY KTSEHHLPSHTVYSYTAAASGSSSSHAFHNKENDNIANGLSRVLPGFNHDRTASAQELLYSL TGSSQEKQPEVSGQDAAAVQEIEYWSDSEHNFQDPCIGGVAIAPTHGSILIECAKCEVHATT KVNDPDRNHPTRISLVLYRHKNLFLPKHCLALWEAKMAEKARKEEECGKNGSDHVSQKNHGK QEKREPTGPQEPSYLRFIQSLAENTGSVTTDSTVTTSPYAFTQVTGPYNTFV (SEQ ID NO: 29) SEQ ID NO: 30 - Exemplary catalytically dead Cas13d (dCas13d) fused to mouse TET2 catalytic domain dead (TET(HxD)) polynucleotide coding sequence (shown with optional HA tag, SV40 NLS, and Flag Tag, each of which are underlined) ATGtacccatacgatgttccagattacgcttcgccaaagaagaagcggaaagtcaacatccc cgctctggtggaaaaccagaagaagtactttggcacctacagcgtgatggccatgctgaacg ctcagaccgtgctggaccacatccagaaggtggccgatattgagggcgagcagaacgagaac aacgagaatctgtggtttcaccccgtgatgagccacctgtacaacgccaagaacggctacga caagcagcccgagaaaaccatgttcatcatcgagcggctgcagagctacttcccattcctga agatcatggccgagaaccagagagagtacagcaacggcaagtacaagcagaaccgcgtggaa gtgaacagcaacgacatcttcgaggtgctgaagcgcgccttcggcgtgctgaagatgtacag ggacctgaccaacgcatacaagacctacgaggaaaagctgaacgacggctgcgagttcctgaccagcacagagcaacctctgagcggcatgatcaacaactactacacagtggccctgcggaac atgaacgagagatacggctacaagacagaggacctggccttcatccaggacaagcggttcaa gttcgtgaaggacgcctacggcaagaaaaagtcccaagtgaataccggattcttcctgagcc tgcaggactacaacggcgacacacagaagaagctgcacctgagcggagtgggaatcgccctg ctgatctgcctgttcctggacaagcagtacatcaacatctttctgagcaggctgcccatctt ctccagctacaatgcccagagcgaggaacggcggatcatcatcagatccttcggcatcaaca gcatcaagctgcccaaggaccggatccacagcgagaagtccaacaagagcgtggccatggat atgctcaacgaagtgaagcggtgccccgacgagctgttcacaacactgtctgccgagaagca gtcccggttcagaatcatcagcgacgaccacaatgaagtgctgatgaagcggagcagcgaca gattcgtgcctctgctgctgcagtatatcgattacggcaagctgttcgaccacatcaggttc cacgtgaacatgggcaagctgagatacctgctgaaggccgacaagacctgcatcgacggcca gaccagagtcagagtgatcgagcagcccctgaacggcttcggcagactggaagaggccgaga caatgcggaagcaagagaacggcaccttcggcaacagcggcatccggatcagagacttcgag aacatgaagcgggacgacgccaatcctgccaactatccctacatcgtggacacctacacaca ctacatcctggaaaacaacaaggtcgagatgtttatcaacgacaaagaggacagcgccccac tgctgcccgtgatcgaggatgatagatacgtggtcaagacaatccccagctgccggatgagc accctggaaattccagccatggccttccacatgtttctgttcggcagcaagaaaaccgagaa gctgatcgtggacgtgcacaaccggtacaagagactgttccaggccatgcagaaagaagaag tgaccgccgagaatatcgccagcttcggaatcgccgagagcgacctgcctcagaagatcctg gatctgatcagcggcaatgcccacggcaaggatgtggacgccttcatcagactgaccgtgga cgacatgctgaccgacaccgagcggagaatcaagagattcaaggacgaccggaagtccattc ggagcgccgacaacaagatgggaaagagaggcttcaagcagatctccacaggcaagctggcc gacttcctggccaaggacatcgtgctgtttcagcccagcgtgaacgatggcgagaacaagat caccggcctgaactaccggatcatgcagagcgccattgccgtgtacgatagcggcgacgatt acgaggccaagcagcagttcaagctgatgttcgagaaggcccggctgatcggcaagggcaca acagagcctcatccatttctgtacaaggtgttcgcccgcagcatccccgccaatgccgtcga gttctacgagcgctacctgatcgagcggaagttctacctgaccggcctgtccaacgagatca agaaaggcaacagagtggatgtgcccttcatccggcgggaccagaacaagtggaaaacaccc gccatgaagaccctgggcagaatctacagcgaggatctgcccgtggaactgcccagacagat gttcgacaatgagatcaagtcccacctgaagtccctgccacagatggaaggcatcgacttca acaatgccaacgtgacctatctgatcgccgagtacatgaagagagtgctggacgacgacttc cagaccttctaccagtggaaccgcaactaccggtacatggacatgcttaagggcgagtacga cagaaagggctccctgcagcactgcttcaccagcgtggaagagagagaaggcctctggaaag agcgggcctccagaacagagcggtacagaaagcaggccagcaacaagatccgcagcaaccgg cagatgagaaacgccagcagcgaagagatcgagacaatcctggataagcggctgagcaacag ccggaacgagtaccagaaaagcgagaaagtgatccggcgctacagagtgcaggatgccctgc tgtttctgctggccaaaaagaccctgaccgaactggccgatttcgacggcgagaggttcaaa ctgaaagaaatcatgcccgacgccgagaagggaatcctgagcgagatcatgcccatgagctt caccttcgagaaaggcggcaagaagtacaccatcaccagcgagggcatgaagctgaagaact acggcgacttctttgtgctggctagcgacaagaggatcggcaacctgctggaactcgtgggc agcgacatcgtgtccaaagaggatggatccaaaagaaccgccgacggcagcgaattcgacta caaagacgatgacgacaagggatccactagtaacggccgccagtgtgctggaattcgccctt tacaaagtcagaatggcaaatgtgaaggatgcaatccagacaaagatgaagctccttattat acccatctgggagctggtcctgatgtggcagctattagaacactcatggaagaaaggtatgg agagaagggtaaagctattaggattgaaaaagtcatatatactggtaaagaaggcaagagct ctcagggatgtcctattgctaaatgggtatatcggagatcgagtgaggaggagaaactactg tgtttggtacgagtgcgacctaaccacacatgtgagacggcggtgatggtaattgccatcat gttgtgggacggaatcccaaagctactcgcatcagaactctactcagaacttacagatatct tgggcaagtgtggcatatgcaccaaccgtcgctgttctcagaatgaaacgaagaaaaagcaa tcaccacccagaaactgttgttgtcagggtgagaatccagagacctgtggtgcctccttttc ttttggttgttcttggagcatgtactataatggatgtaagtttgccagaagcaagaaaccaaggaaatttaggctacatggagctgagccaaaagaggaagagagactaggttctcatttgcaa aacctggctactgtcattgctccaatatacaagaagcttgcacccgatgcatacaataatca ggttgaatttgaacaccaagccccagactgctgtttgggtctgaaggaaggccggccattct caggagtcactgcatgtttggacttctctgctcattcctacagagcccagcagaacatgcca aatggcagtacagtggtggtcaccctcaatagagaagacaatcgagaagtcggagctaagcc tgaggatgagcagttccacgtgctgcctatgtacatcatcgcccctgaggatgagtttggga gtacggaaggccaggagaagaagatacggatggggtccattgaggttctgcagtcatttcgg aggagaagggtcataaggataggagagctgcccaagagttgcaagaagaaagcggagcccaa gaaagccaagaccaagaaagcagctcgaaagcattcctctctggagaactgctccagtagga ctgagaagggaaagtcttcctcacatacaaagctgatggaaaatgcaagccatatgaaacaa atgacagcacaaccgcagctttcgggcccggtcatccggcagccaccaacactccagaggca ccttcagcaagggcagaggccacagcagccgcagccacctcagccgcagccgcagacgacac ctcagccacagccacagccacagcatatcatgcccggtaactctcagtctgttggttctcat tgttctggatccaccagtgtctacacgagacagcctactcctcacagtccttatcccagctc agcacacacctcagatatttatggagataccaaccatgtgaacttttaccccacttcatctc atgcctcgggttcatatttgaatccttctaattacatgaacccctaccttgggcttttgaat cagaataaccaatatgcaccttttccatacaatgggagtgtgccagtggacaatggttcccc tttcttaggttcttattccccccaggctcagtccagggatctacatagatatccaaaccagg accatctcaccaatcagaacttaccacccatccacacccttcaccaacagacgtttggggac agtccctctaagtacttaagttatggaaaccaaaatatgcagagagatgccttcactactaa ctccaccctaaaaccaaatgtacaccacctagcaacgttttctccttaccccacccccaaga tggatagtcatttcatgggagctgcctccagatcaccatacagccacccacacactgactac aaaaccagtgagcatcatctaccctctcacacggtctacagctacacggcagcagcttcggg gagcagttccagccacgccttccacaacaaggagaatgacaacatagccaatgggctctcaa gagtgcttccagggtttaatcatgatagaactgcttctgcccaagaactattatacagtctg actggcagcagtcaggagaagcagcctgaggtgtcaggccaggatgcagctgctgtgcagga aattgagtattggtcagatagtgagcacaactttcaggatccttgcattggaggggtggcta tagccccaactcatgggtcaattcttattgagtgtgcaaagtgtgaggttcatgccacaacc aaagtaaacgatcccgaccggaatcaccccaccaggatctcacttgtactgtataggcataa gaatttgtttctaccaaaacattgtttggctctctgggaagccaaaatggctgaaaaggccc ggaaagaggaagagtgcggaaagaatggatcagaccacgtgtctcagaaaaatcatggcaaa caggaaaagcgtgagcccacagggccacaggaacccagttacctgcgtttcatccagtctct tgctgagaacacagggtctgtgactacggattctaccgtgactacatcaccatatgctttca ctcaggtcacagggccttacaacacatttgtatga (SEQ ID NO: 30) SEQ ID NO: 31 - Exemplary dCas13d fused to human TET2 isoform A catalytic domain amino acid NIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENNENLWFHPVMSHLYNAKN GYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLK MYRDLTNAYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDK RFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRL PIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSA EKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCI DGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDT YTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKK TEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRL TVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGE NKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPAN AVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELP RQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQWNRNYRYMDMLKG EYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNRQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMP MSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKEDGSKRTADGSE FSVLNNFIESPSKLLDTPIKNLLDTPVKTQYDFPSCRCVEQIIEKDEGPFYTHLGAGPNVAA IREIMEERFGQKGKAIRIERVIYTGKEGKSSQGCPIAKWVVRRSSSEEKLLCLVRERAGHTC EAAVIVILILVWEGIPLSLADKLYSELTETLRKYGTLTNRRCALNEERTCACQGLDPETCGA SFSFGCSWSMYYNGCKFARSKIPRKFKLLGDDPKEEEKLESHLQNLSTLMAPTYKKLAPDAY NNQIEYEHRAPECRLGLKEGRPFSGVTACLDFCAHAHRDLHNMQNGSTLVCTLTREDNREFG GKPEDEQLHVLPLYKVSDVDEFGSVEAQEEKKRSGAIQVLSSFRRKVRMLAEPVKTCRQRKL EAKKAAAEKLSSLENSSNKNEKEKSAPSRTKQTENASQAKQLAELLRLSGPVMQQSQQPQPL QKQPPQPQQQQRPQQQQPHHPQTESVNSYSASGSTNPYMRRPNPVSPYPNSSHTSDIYGSTS PMNFYSTSSQAAGSYLNSSNPMNPYPGLLNQNTQYPSYQCNGNLSVDNCSPYLGSYSPQSQP MDLYRYPSQDPLSKLSLPPIHTLYQPRFGNSQSFTSKYLGYGNQNMQGDGFSSCTIRPNVHH VGKLPPYPTHEMDGHFMGATSRLPPNLSNPNMDYKNGEHHSPSHIIHNYSAAPGMFNSSLHA LHLQNKENDMLSHTANGLSKMLPALNHDRTACVQGGLHKLSDANGQEKQPLALVQGVASGAE DNDEVWSDSEQSFLDPDIGGVAVAPTHGSILIECAKRELHATTPLKNPNRNHPTRISLVFYQ HKSMNEPKHGLALWEAKMAEKAREKEEECEKY (SEQ ID NO: 31) SEQ ID NO: 32 - Exemplary dCas13d fused to human TET2 isoform A catalytic domain polynucleotide coding sequence aacatccccgctctggtggaaaaccagaagaagtactttggcacctacagcgtgatggccat gctgaacgctcagaccgtgctggaccacatccagaaggtggccgatattgagggcgagcaga acgagaacaacgagaatctgtggtttcaccccgtgatgagccacctgtacaacgccaagaac ggctacgacaagcagcccgagaaaaccatgttcatcatcgagcggctgcagagctacttccc attcctgaagatcatggccgagaaccagagagagtacagcaacggcaagtacaagcagaacc gcgtggaagtgaacagcaacgacatcttcgaggtgctgaagcgcgccttcggcgtgctgaag atgtacagggacctgaccaacgcatacaagacctacgaggaaaagctgaacgacggctgcga gttcctgaccagcacagagcaacctctgagcggcatgatcaacaactactacacagtggccc tgcggaacatgaacgagagatacggctacaagacagaggacctggccttcatccaggacaag cggttcaagttcgtgaaggacgcctacggcaagaaaaagtcccaagtgaataccggattctt cctgagcctgcaggactacaacggcgacacacagaagaagctgcacctgagcggagtgggaa tcgccctgctgatctgcctgttcctggacaagcagtacatcaacatctttctgagcaggctg cccatcttctccagctacaatgcccagagcgaggaacggcggatcatcatcagatccttcgg catcaacagcatcaagctgcccaaggaccggatccacagcgagaagtccaacaagagcgtgg ccatggatatgctcaacgaagtgaagcggtgccccgacgagctgttcacaacactgtctgcc gagaagcagtcccggttcagaatcatcagcgacgaccacaatgaagtgctgatgaagcggag cagcgacagattcgtgcctctgctgctgcagtatatcgattacggcaagctgttcgaccaca tcaggttccacgtgaacatgggcaagctgagatacctgctgaaggccgacaagacctgcatc gacggccagaccagagtcagagtgatcgagcagcccctgaacggcttcggcagactggaaga ggccgagacaatgcggaagcaagagaacggcaccttcggcaacagcggcatccggatcagag acttcgagaacatgaagcgggacgacgccaatcctgccaactatccctacatcgtggacacc tacacacactacatcctggaaaacaacaaggtcgagatgtttatcaacgacaaagaggacag cgccccactgctgcccgtgatcgaggatgatagatacgtggtcaagacaatccccagctgcc ggatgagcaccctggaaattccagccatggccttccacatgtttctgttcggcagcaagaaa accgagaagctgatcgtggacgtgcacaaccggtacaagagactgttccaggccatgcagaa agaagaagtgaccgccgagaatatcgccagcttcggaatcgccgagagcgacctgcctcaga agatcctggatctgatcagcggcaatgcccacggcaaggatgtggacgccttcatcagactg accgtggacgacatgctgaccgacaccgagcggagaatcaagagattcaaggacgaccggaa gtccattcggagcgccgacaacaagatgggaaagagaggcttcaagcagatctccacaggca agctggccgacttcctggccaaggacatcgtgctgtttcagcccagcgtgaacgatggcgag aacaagatcaccggcctgaactaccggatcatgcagagcgccattgccgtgtacgatagcgg cgacgattacgaggccaagcagcagttcaagctgatgttcgagaaggcccggctgatcggcaagggcacaacagagcctcatccatttctgtacaaggtgttcgcccgcagcatccccgccaat gccgtcgagttctacgagcgctacctgatcgagcggaagttctacctgaccggcctgtccaa cgagatcaagaaaggcaacagagtggatgtgcccttcatccggcgggaccagaacaagtgga aaacacccgccatgaagaccctgggcagaatctacagcgaggatctgcccgtggaactgccc agacagatgttcgacaatgagatcaagtcccacctgaagtccctgccacagatggaaggcat cgacttcaacaatgccaacgtgacctatctgatcgccgagtacatgaagagagtgctggacg acgacttccagaccttctaccagtggaaccgcaactaccggtacatggacatgcttaagggc gagtacgacagaaagggctccctgcagcactgcttcaccagcgtggaagagagagaaggcct ctggaaagagcgggcctccagaacagagcggtacagaaagcaggccagcaacaagatccgca gcaaccggcagatgagaaacgccagcagcgaagagatcgagacaatcctggataagcggctg agcaacagccggaacgagtaccagaaaagcgagaaagtgatccggcgctacagagtgcagga tgccctgctgtttctgctggccaaaaagaccctgaccgaactggccgatttcgacggcgaga ggttcaaactgaaagaaatcatgcccgacgccgagaagggaatcctgagcgagatcatgccc atgagcttcaccttcgagaaaggcggcaagaagtacaccatcaccagcgagggcatgaagct gaagaactacggcgacttctttgtgctggctagcgacaagaggatcggcaacctgctggaac tcgtgggcagcgacatcgtgtccaaagaggatggatccaaaagaaccgccgacggcagcgaa ttcTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAA TTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGAGCAAA TTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCAGCT ATTAGAGAAATCATGGAAGAAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGT CATCTATACTGGTAAAGAAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGTGGTTC GCAGAAGCAGCAGTGAAGAGAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACACCTGT GAGGCTGCAGTGATTGTGATTCTCATCCTGGTGTGGGAAGGAATCCCGCTGTCTCTGGCTGA CAAACTCTACTCGGAGCTTACCGAGACGCTGAGGAAATACGGCACGCTCACCAATCGCCGGT GTGCCTTGAATGAAGAGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCC TCCTTCTCTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTAAGTTTGCCAGAAGCAA GATCCCAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAACTGGAGTCTC ATTTGCAAAACCTGTCCACTCTTATGGCACCAACATATAAGAAACTTGCACCTGATGCATAT AATAATCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCG TCCATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTTGCACA ACATGCAGAATGGCAGCACATTGGTATGCACTCTCACTAGAGAAGACAATCGAGAATTTGGA GGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACGTGGATGA GTTTGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACTGAGTT CTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGAAACTA GAAGCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAAAATGA AAAGGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGCTAAACAGT TGGCAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGCCTCTA CAGAAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAGCCACATCACCC TCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAGACGGC CCAATCCAGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTATGGAAGCACCAGC CCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCTAATCC CATGAACCCTTACCCTGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATGCAATG GAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTCAGCCG ATGGATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCCATCCA TACACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGTTATG GAAACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACATCAT GTAGGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTC TAGATTACCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCAC CTTCTCACATAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGCTCTCTTCATGCC CTGCATCTCCAAAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAAAGAT GCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATGCTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTGCAGAG GACAACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGT GGCCGTGGCTCCAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGCCA CAACCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTACCAG CATAAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAA AGCCCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCAGAAATCCC ATGGCAAAAAAGTGAAACGGGAGCCTGCTGAGCCACATGAAACTTCAGAGCCCACTTACCTG CGTTTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACAGACTCCACAGTAACTAC ATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATATATGA (SEQ ID NO: 32) IV. m5C marked RNA as disease biomarkers and / or targets for therapeutic intervention

[0186] In certain aspects, provided herein are methods of diagnosis and / or treatment comprising analysis and / or modification of RNA, such as but not limited to chromatin associated RNA (caRNA), including at least chromatin associated regulatory RNA (carRNA). In some aspects, methods of diagnosis and / or treatment comprising analysis and / or modification of RNA may be loci and / or sequence specific, or may be loci and / or sequence non-specific.

[0187] In certain aspects, provided herein are methods of identifying features, (e.g., such as biomarker features), and evaluating the presence, absence, or levels of said features in a sample. In some aspects, one or more (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein) features are used to classify (e.g., diagnose) a disease state and / or identify one or more effective treatment options for a patient with a disease associated with aberrant transcription (e.g., aberrant transcription associated with increased euchromatin). In some aspects, one or more features are used to diagnose a disease state and / or identify one or more effective treatment options for a patient with cancer.

[0188] In some aspects, one or more features (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein) are used to diagnose a disease state and / or identify one or more effective treatment options for a patient with a blood cancer. In some aspects, one or more features are used to diagnose a disease state and / or identify one or more effective treatment options for a patient with a leukemia. In some aspects, one or more features are used to diagnose a disease state and / or identify one or more effective treatment options for a patientwith a myeloid malignancy. In some aspects, one or more features are used to diagnose a disease state and / or identify one or more effective treatment options for a patient with acute myeloid leukemia. In some aspects, one or more features are used to diagnose a disease state and / or identify one or more effective treatment options for a patient with chronic myelomonocytic leukemia. In some aspects, one or more features are used to diagnose a disease state and / or identify one or more effective treatment options for a patient with a glioma. In some aspects, one or more features are used to diagnose a disease state and / or identify one or more effective treatment options for a patient with glioblastoma.

[0189] In some aspects, one or more (for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein) features are used to diagnose a disease state and / or identify one or more effective treatment options for a patient diagnosed with a pre-cancerous condition, such as but not limited to, clonal hematopoiesis of indeterminate potential (CHIP).

[0190] In some aspects, a feature comprises one or more m5C RNA modifications in one or more RNA populations. In some aspects, m5C RNA modification features are determined in caRNA, carRNA, and / or other RNA species. In some aspects, m5C RNA modification features are comprised, consist essentially of, or consist of one or more caRNA described in Table 1. In some aspects, m5C RNA modification features are comprised, consist essentially of, or consist of one or more caRNA described in Table 2.

[0191] In some aspects, m5C RNA modification features are comprised, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1266, 1400, 1500, or more, or any range derivable therein, caRNA described in Table 1.

[0192] In some aspects, m5C RNA modification features are comprised, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875,900, 925, 950, 1000, 1200, 1266, 1400, 1500, or more, or any range derivable therein, caRNA described in Table 2.

[0193] In some aspects, m5C RNA modification features are comprised, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, or more than 500 features, or any range derivable therein, that are comprised in a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% (or any range derivable therein) complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length relative of any one or more of SEQ ID NOs: 100-614.

[0194] In some aspects, m5C RNA modification features are comprised, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, or more than 500 features that are comprised in a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% (or any range derivable therein) complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length relative of any one or more of SEQ ID NOs: 104 or 107.

[0195] In certain aspects, provided herein are methods of treatment comprising analyzing m5C RNA modification levels in suspected diseased cells, determining if m5C RNA modification levels are aberrant, and if m5C RNA modification levels are aberrant, contacting the cells with one or more TET2, NSUN2, and / or MBD6 inhibitors. In certain aspects, provided herein are methods of treatment comprising analyzing m5C RNA modification levels in suspected diseased cells, determining if m5C RNA modification levels are aberrant, and if m5C RNA modification levels are aberrant, contacting the cells with one or more site-specific caRNA targeting agents, such as but not limited to, dCas13b::TET2(CD) and / or dCas13b::MBD6 fusion proteins that may be guided a polynucleotide sequence at least partially complementary to an m5C RNA feature described in Table 1 or Table 2.A. chromatin associate RNA (caRNA)

[0196] In some aspects, m5C RNA features are comprised within one or more chromatin associated RNA (caRNA) species. In ertain aspects, a caRNA may comprise caRNAs with potential regulatory functions, such as but not limited to, promoter-associated RNA (paRNA), enhancer RNA (eRNA), and / or RNA transcribed from transposable elements (repeat RNA), these caRNA species can be referred to herein as chromosome-associated regulatory RNAs (carRNAs).

[0197] In certain aspects, caRNAs of interest as m5C RNA features are described herein in Table 1, and in particular, Table 2. In certain aspects, caRNAs of particular interest as m5C RNA features are described herein in Table 1 as SEQ ID NOs: 100-614 (e.g., SEQ ID NOs: 104 and 107 in Table 2). In certain aspects, caRNAs of interest as m5C RNA features comprise, consist essentially of, or consist of HERVH-int repeats. In certain aspects, caRNAs of interest as m5C RNA features comprise, consist essentially of, or consist of HERVH-int repeats identified in Table 1 (shaded rows) on Chr1, Chr2, Chr3, Chr4, Chr8, Chr11, Chr212, and ChrX. In certain aspects, caRNAs of interest as m5C RNA features in leukemia cells comprise, consist essentially of, or consist of any one or more of HERVH-int repeats: chr1:22997913- 23003991;-;HERVH-int,LTR,ERV1;2,7713,(0) || chr1:35132025-35137611;-;HERVH- int,LTR,ERV1;1,7680,(33) || chr11:67842303-67856609;-;HERVH-int,LTR,ERV1;1,7713,(0) || chr11:71737927-71752300;+;HERVH-int,LTR,ERV1;1,7713,(0) || chr12:8279422- 8293739;-;HERVH-int,LTR,ERV1;1,7713,(0) || chr2:64252898-64257196;+;HERVH- int,LTR,ERV1;1,7713,(0) || chr2:170977859-170982983;-;HERVH-int,LTR,ERV1;1,7713,(0) || chr3:44345027-44350026;-;HERVH-int,LTR,ERV1;1,7713,(0) || chr3:130138040- 130152338;-;HERVH-int,LTR,ERV1;1,7713,(0) || chr4:71031921-71036834;+;HERVH- int,LTR,ERV1;1,7713,(0) || chr8:8169077-8183461;+;HERVH-int,LTR,ERV1;1,7713,(0) || chr8:12482202-12496589;-;HERVH-int,LTR,ERV1;1,7713,(0) || chrX:71264927- 71272291;+;HERVH-int,LTR,ERV1;1,7713,(0).

[0198] In some aspects, technologies described herein may comprise increasing one or more m5C marks at one or more loci, such as but not limited to, increasing m5C marks by 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 2.0x, 3.0x, 4.0x, 5.0x, 6.0x, 7.0x, 8.0x, 9.0x, 10.0x, 15.0x, 20.0x, 25.0x, 50.0x, 100.0x, or greater than 100x (or any range derivable therein) relative to a control level, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein, m5C marks.

[0199] In some aspects, technologies described herein may comprise decreasing one or more m5C marks at one or more loci, such as but not limited to, decreasing m5C marks by 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 2.0x, 3.0x, 4.0x, 5.0x, 6.0x, 7.0x, 8.0x, 9.0x, 10.0x, 15.0x, 20.0x, 25.0x, 50.0x, 100.0x, or greater than 100x (or any range derivable therein) relative to a control level, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein, m5C marks.

[0200] In some aspects, technologies described herein may comprise decreasing recognition (e.g., reading) of one or more m5C marks at one or more loci by one or more complexes comprising deubiquitination activity, (e.g., PR-DUB, PCR1, PCR2, etc.) such as but not limited to, decreasing recognition of m5C marks by 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 2.0x, 3.0x, 4.0x, 5.0x, 6.0x, 7.0x, 8.0x, 9.0x, 10.0x, 15.0x, 20.0x, 25.0x, 50.0x, 100.0x, or greater than 100x (or any range derivable therein) relative to a control level, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000, or more than 1000, or any range derivable therein, m5C marks.260-T-32IHCU / O W5970P.DCR A260-T-32IHCU / O W5970P.DCR AB. Targeting RNAs

[0201] In certain aspects, provided herein are targeting RNAs that may guide a protein and / or ribonucleoprotein complex to a target RNA of interest.

[0202] In certain aspects, a targeting RNA is driven by a promoter comprising a Polymerase III promoter (i.e., a promoter that can drive Pol III mediated transcription). In certain aspects, transcription of a targeting RNA is driven by one or more U6 promoters. In certain aspects, a targeting RNA is transcribed by Polymerase III. In certain aspects, a targeting RNA is driven by a promoter comprising a Polymerase II promoter (i.e., a promoter that can drive Pol II mediated transcription). In some aspects, a targeting RNA is designed to be comprised in an intronic sequence. In some aspects, a targeting RNA is created ex-vivo. In some aspects, a targeting RNA is created in-vivo. In some aspects, a targeting RNA can guide an endogenous protein and / or ribonucleoprotein complex to a target RNA of interest.

[0203] In some aspects, provided herein are CRISPR / Cas system ancillary components, such as functional targeting RNA species, for example but not limited to, CRISPR RNA, trans- activating CRISPR RNA (tracrRNA), and / or gRNA.

[0204] In some aspects, provided herein are targeting RNA species that mediate greater than or equal to about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, RNA m5C installation or erasure in a target polynucleotide population. In some aspects, targeting RNA species comprise gRNA molecules. In some aspects, a gRNA molecule is engineered to provide improved functionality relative to a non-engineered gRNA.

[0205] In some aspects, an RNA targeting element comprises a gRNA sequence, which may comprise or consist of a polynucleotide sequence, with about, exactly, or at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity to any one of SEQ ID NOs: 100-614 or to any sequence complementary thereto that isat least, exactly, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. V. Vectors and Nanoparticles A. Vectors

[0206] Among other things, the present disclosure provides that in some aspects, polypeptides and / or oligonucleotides described herein are encoded by a polynucleotide, such as a vector comprising a polynucleotide (e.g., a polynucleotide construct). Vectors comprising polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno associated viral constructs) that incorporate a polynucleotide encoding a polypeptide with m5C RNA writer, reader, and / or erasor functionality and / or targeting element described herein, or characteristic portions thereof (e.g., as utilized herein, a “characteristic portion thereof” refers to the portion of said protein required to perform the desired function, e.g., it comprises the ability to a impact m5C activity and / or levels, for example, a polypeptide with m5C RNA writer, reader, and / or erasor functionality, or the ability to inhibit the same, in a site-specific or non-site specific manner). Those of skill in the art will be capable of selecting suitable constructs, as well as cells, for making any of the polynucleotides described herein. In some aspects, a construct is a plasmid (i.e., a circular DNA molecule that can autonomously replicate inside a cell). In some aspects, a construct can be a cosmid (e.g., pWE or sCos series).

[0207] In some aspects, a construct is a viral construct. In some aspects, a viral construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus construct. In some aspects, a construct is an adeno-associated virus (AAV) construct (see, e.g., Asokan et al., Mol. Ther.20: 699-7080, 2012, which is incorporated herein by reference for the purposes described herein). In some aspects, a viral construct is an adenovirus construct. In some aspects, a viral construct may also be based on or derived from an alphavirus. Alphaviruses include but are not limited to, Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus. Generally, the genome of such viruses encode nonstructural (e.g., replicon) and structural proteins (e.g.,capsid and envelope) that can be translated in the cytoplasm of the host cell. Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop viral constructs for coding sequence delivery. Pseudotyped viruses may be formed by combining alphaviral envelope glycoproteins and retroviral capsids. Examples of alphaviral constructs can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819; constructs and methods of their making are incorporated herein by reference for the purposes described herein.

[0208] In some aspects, constructs provided herein can be of different sizes. In some aspects, a construct is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb. In some aspects, a construct is a plasmid and can have a total length in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.

[0209] In some aspects, a construct is a viral construct and can have a total number of nucleotides of up to 10 kb. In some aspects, a viral construct can have a total number of nucleotides in the range of about 4.5 kb to 5 kb, or about 4.7 kb. In some aspects, a viral construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 1 O kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about 10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or about 9 kb to about 10 kb.

[0210] In some aspects, a construct is a lentivirus construct and can have a total number of nucleotides of up to 8 kb. In some examples, a lentivirus construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb.

[0211] In some aspects, a construct is an adenovirus construct and can have a total number of nucleotides of up to 8 kb. In some aspects, an adenovirus construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0212] Any of the constructs described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and / or additional untranslated regions which may house pre- or post-transcriptional regulatory and / or control elements. In some aspects, a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of control sequences are described herein. 1. AAV particles

[0213] Among other things, the present disclosure provides AAV particles that comprise a polynucleotide construct encoding a polypeptide with m5C RNA writer, reader, and / or erasor functionality and / or targeting element, and an AAV capsid. In some aspects, AAV particles can be described as having a serotype, which is a description of the construct strain and the capsid strain. For example, in some aspects an AAV particle may be described as AAV2,wherein the particle has an AAV2 capsid and a construct that comprises characteristic AAV2 Inverted Terminal Repeats (ITRs). In some aspects, an AAV particle may be described as a pseudotype, wherein the capsid and construct are derived from different AAV strains, for example, AAV2 / 9 would refer to an AAV particle that comprises a construct utilizing the AAV2 ITRs and an AAV9 capsid. Additional examples of pseudotyped AAV vectors include, but are not limited to, AAV2 / 1, AAV2 / 2, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8 and AAV2 / 9.

[0214] In some aspects, AAV particles suitable for use according to the present disclosure may comprise or be derived from any natural or recombinant AAV serotype. In some aspects, an AAV according to the present disclosure can be selected from natural serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12; or pseudotypes, chimeras, and variants thereof.

[0215] As used herein, the term "chimera" when referring to an AAV vector, or a "chimeric AAV vector", refers to an AAV vector which comprises a capsid containing VP1, VP2 and VP3 proteins from at least two different AAV serotypes; or alternatively, which comprises VP1, VP2 and VP3 proteins, at least one of which comprises at least a portion from another AAV serotype. Examples of chimeric AAV vectors include, but are not limited to, AAV-DJ, AAV-DJ / 8, AAV2G9, AAV2i8, AAV2i8G9, AAV8G9, and AAV9i1.

[0216] In some aspects, an AAV serotype and / or pseudotype according to the present invention is selected from the group comprising or consisting of AAV1, AAV2, AAV3, AAV 4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.1 / hu.43, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu.11, AAV16.3, AAV16.8 / hu.10, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV1-7 / rh.48, AAV1- 8 / rh.49, AAV2i8, AAV2i8G9, AAV2-15 / rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3 / rh.61, AAV24.1, AAV2-4 / rh.50, AAV2- 5 / rh.51, AAV2.5T, AAV27.3, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV2G9, AAV3B, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-11 / rh.53, AAV3-3, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4-19 / rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-1b, AAV42-2, AAV42-3a, AAV42- 3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV4-8 / rh.64, AAV4-9 / rh.54, AAV52.1 / hu.20,AAV52 / hu.19, AAV5-22 / rh.58, AAV5-3 / rh.57, AAV54.1 / hu.21,AAV54.2 / hu.22,AAV54.4R / hu.27, AAV54.5 / hu.23, AAV54.7 / hu.24, AAV58.2 / hu.25, AAV6.1, AAV6.1.2, AAV6.2, AAV7m8, AAV7.2, AAV7.3 / hu.7, AAV-8b, AAV8G9, AAV- 8h, AAV9i1, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.5R1, AAVcy.5R2, AAVcy.5R3, AAVcy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.12, AAVhu.13, AAVhu.14 / 9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh8R R533A mutant, AAVrh8R A586R mutant, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAV- PHP.B, AAVPHP.A, AAV-G2B-26, AAV-G2B-13, AAV-TH1.1-32, AAVTH1.1-35, AAV- PHP.B2, AAV-PHP.B3, AAV-PHP.N / PHP.B-DGT, AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.BATP, AAV-PHP.B-ATT-T, AAV-PHP.B-DGT-T, AAV-PHP.B-GGT-T, AAV- PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAV-PHP.B-SNP(3), AAV-PHP.B- SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B-EGS, AAV-PHP.BSGN, AAV- PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.BDST, AAV-PHP.B-STP, AAV-PHP.B-PQP, AAV-PHP.BSQP, AAV-PHP.B-Q1P, AAV-PHP.B-TMP, AAV-PHP.BTTP, AAV- PHP.S / G2A12, AAV-G2A15 / G2A3, AAV-G2B4, AAV-G2B5, PHP.S, AAAV, AAV A3.3, AAV A3.4, AAV A3.5, AAV A3.7, AAV CBr-7.3, AAV CBr-7.1, AAV CBr-7.10, AAV CBr- 7.2, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr- B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd- N4, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKdB4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-M9, AAV CLv-R6, AAV CLv-1, AAV CLv1-1, AAV CLv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV CLv1-7, AAV CLv1-8, AAV CLv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLvM7, AAV CLv-M8, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-8.10, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAVLK08, AAV-LK15, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, AAV SM 10-8, AAV.VR-355, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.5R1, AAV-DJ, AAV-DJ8, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, AAV-h, AAVH-1 / hu.1,AAVH2,AAVH-5 / hu.3,AAVH6,AAVhE1.1, AAVhEr1.14, AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5, AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG- 9 / hu.39, AAVLG-9 / hu.39, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAVLK07, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK16, AAV-LK17, AAVLK18, AAV-LK19, AAVN721- 8 / rh.43, AAV-PAEC, AAVPAEC12, AAV-PAEC11, AAV-PAEC2, AAV-PAEC4,AAVPAEC6, AAV-PAEC7, AAV-PAECS, Anc80, Anc80L65, Anc81, Anc82, Anc83, Anc84, Anc94, Anc110, Anc113, Anc126, Anc127, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprine AAV, Japanese AAV10 serotype, UPENN AAV10, VOY101, and VOY201.

[0217] In some aspects, an AAV is an AAV variant that has been genetically modified, e.g., by substitution, deletion or addition of one or several amino acid residues in one or more capsid proteins. Examples of such variants include, but are not limited to, AAV2 with one or more of Y444F, Y500F, Y730F and / or S662V mutations; AAV3 with one or more of Y705F, Y731F and / or T492V mutations; and AAV6 with one or more of S663V and / or T492V mutations.

[0218] In some aspects, an AAV capsid is modified to comprise at least one surface-bound saccharide or a derivative thereof. As used herein, the term "surface-bound", when referring to the at least one saccharide, means that said at least one saccharide is bound to and exposed at the outer surface of the AAV vector. Suitable examples of saccharides include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, and derivatives thereof. 2. AAV constructs

[0219] In some aspects, the present disclosure provides polynucleotide vectors (e.g., polynucleotide constructs) that comprise a nucleotide sequence encoding a polypeptide with m5C RNA writer, reader, and / or erasor functionality and / or targeting element. In some aspects described herein, a polynucleotide vector comprising a nucleotide sequence encoding a polypeptide with m5C RNA writer, reader, and / or erasor functionality and / or targeting element, can be comprised in an AAV capsid to produce an AAV particle (e.g., an AAV particle comprises an AAV construct comprised in an AAV capsid).

[0220] In some aspects, a polynucleotide construct comprises one or more components derived from or modified from a naturally occurring AAV genomic construct. In some aspects, a sequence derived from an AAV construct is an AAV1 construct, an AAV2 construct, an AAV3 construct, an AAV4 construct, an AAV5 construct, an AAV6 construct, an AAV7 construct, an AAV8 construct, an AAV DJ / 8 construct, an AAV9 construct, an AAV2.7m8 construct, an AAV8BP2 construct, an AAV293 construct, an AAVPhp.B construct, or AAVPhp.eB construct (see e.g., Chan et al., 2017). Additional exemplary AAV constructs that can be used herein are known in the art. See, e.g., Kanaan et al., Mol. Ther. Nucleic Acids 8: 184-197, 2017; Li et al., Mol. Ther.16(7): 1252-1260, 2008; Adachi et al., Nat. Commun.5: 3075, 2014; Isgrig et al., Nat. Commun. 10(1): 427, 2019; and Gao et al., J. Virol. 78(12):6381-6388, 2004; each of which are incorporated herein by reference for the purposes described herein).

[0221] In some aspects, AAV derived sequences (e.g., which are comprised in a polynucleotide construct) typically include the cis-acting 5' and 3' ITR sequences (see, e.g., B. J. Carter, in "Handbook of Parvoviruses," ed., P. Tijsser, CRC Press, pp.155168, 1990, which is incorporated herein by reference for the purposes described herein). Typical AAV2-derived ITR sequences are approximately 145 nucleotides in length. In some aspects, at least or exactly 80% of a typical ITR sequence (e.g., at least or exactly 85%, at least or exactly 90%, at least or exactly 95%, or at least or exactly 100%, etc.) is incorporated into a construct provided herein. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York, 1989; and K. Fisher et al., J Virol.70:520532, 1996, each of which is incorporated herein by reference for the purposes described herein). In some aspects, any of the coding sequences and / or constructs described herein are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types.

[0222] In some aspects, polynucleotide constructs described in accordance with this disclosure and in a pattern known to the art (see, e.g., Asokan et al., Mal. Ther.20: 699- 7080, 2012, which is incorporated herein by reference for the purposes described herein) are typically comprised of, a coding sequence or a portion thereof, at least one and / or control sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). In some aspects, provided constructs can be packaged into a capsid to create an AAV particle. An AAV particle may be delivered to a selected target cell. In some aspects, provided constructs comprise an additional optional coding sequence that is a nucleic acid sequence (e.g., inhibitory nucleic acid sequence), heterologous to the construct sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. In some aspects, a nucleic acid coding sequence is operatively linked to and / or control components in a manner that permits coding sequence transcription, translation, and / or expression in a cell of a target tissue.

[0223] In some aspects, an unmodified AAV endogenous genome includes two open reading frames, "cap" and "rep," which are flanked by ITRs. In some aspects, recombinant AAV constructs similarly comprise one or more open reading frames flanked by ITR sequences. In some aspects, an AAV construct also comprises conventional control elements that are operably linked to the coding sequence in a manner that permits its transcription,translation and / or expression in a cell transfected with the polynucleotide construct or infected with a virus particle produced by the disclosure. In some aspects, an AAV construct optionally comprises a promoter, an enhancer, an untranslated region (e.g., a 5' UTR, 3' UTR), a Kozak sequence, an internal ribosomal entry site (IRES), splicing sites (e.g., an acceptor site, a donor site), a polyadenylation site, or any combination thereof.

[0224] In some aspects, a construct is an AAV construct. In some aspects, an AAV construct can include at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, at least 4.5 kb, or at least 4.7 kb. In some aspects, an AAV construct can include at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, at most 4 kb, at most 3.5 kb, at most 3 kb, or at most 2.5 kb. In some aspects, an AAV construct can include about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

[0225] Any of the constructs described herein can further include regulatory and / or control sequences, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and / or any combination thereof. In some aspects, a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of control sequences are described herein and others are known in the art 3. AAV capsids

[0226] In some aspects, the present disclosure provides one or more polynucleotide constructs packaged into an AAV capsid. In some aspects, an AAV capsid is from or is derived from an AAV capsid of an AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rhl0, rh39, rh43 or Ancestral serotype, or one or more hybrids thereof. In some aspects, an AAV capsid is from an AAV ancestral serotype. In some aspects, an AAV capsid is an ancestral (Anc) AAV capsid. An Anc capsid is created from a construct sequence that is constructed using evolutionary probabilities and evolutionary modeling to determine a probable ancestral sequence. Thus, an Anc capsid / construct sequence is not known to have existed in nature. As provided herein, in some aspects, any combination of AAV capsids and AAV constructs (e.g., comprising AAV ITRs) may be used in recombinant AAV particles of the present disclosure. 4. Exemplary AAV construct componentsa. Inverted Terminal Repeat Sequences (ITRs)

[0227] AAV derived sequences of a construct typically comprises the cis-acting 5' and 3' ITRs (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155168 (1990), which is incorporated herein by reference for the purposes described herein). Generally, ITRs are able to form a hairpin. The ability to form a hairpin can contribute to an ITRs ability to self-prime, allowing primase-independent synthesis of a second DNA strand. ITRs can also aid in efficient encapsidation of an AAV construct in an AAV particle.

[0228] An AAV particle of the present disclosure can comprise an AAV construct comprising a coding sequence (e.g., encoding a polypeptide with m5C RNA writer, reader, and / or erasor functionality and / or targeting element) and associated elements flanked by a 5' and a 3' AAV ITR sequences. In some aspects, an ITR is or comprises approximately 130 nucleic acids. In some aspects, an ITR is or comprises approximately 145 nucleic acids. In some aspects, an ITR is or comprises 125 to 150 nucleic acids. In some aspects, all or substantially all of a sequence encoding an ITR is used. In some aspects, an AAV ITR sequence may be obtained from any known AAV, including presently identified mammalian AAV types. In some aspects an ITR is an AAV2 ITR. In some aspects, an ITR is an AAV9 ITR.

[0229] A non-limiting example of a polynucleotide construct of the present disclosure is a "cisacting" construct comprising a coding sequence, in which said sequence and any associated regulatory elements are flanked by 5' or "left" and 3' or "right" AAV ITR sequences.5' and left designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction. For example, in some aspects, a 5' or left ITR is an ITR that is closest to a promoter (e.g., as opposed to a polyadenylation sequence) for a given construct, when a construct is depicted in a sense orientation, linearly. Concurrently, 3' and right designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction. For example, in some aspects, a 3' or right ITR is an ITR that is closest to a polyadenylation sequence and / or stop codon (e.g., as opposed to a promoter sequence) for a given construct, when a construct is depicted in a sense orientation, linearly. In general, ITRs as provided herein are depicted in 5' to 3' order in accordance with a sense strand. Accordingly, one of skill in the art will appreciate that a 5' or "left" orientation ITR can also be depicted as a 3' or "right" ITR when converting from sense to anti sense direction. Further, it is well within the ability of one of skill in the art to transform a given sense ITR sequence (e.g., a 5' / left AAV ITR) into an antisense sequence (e.g., 3' / right ITR sequence). One of ordinary skill in the art would understand how to modify a given ITR sequence for use as either a 5' / left or 3' / right ITR, or an antisense version thereof.b. Promoters

[0230] In some aspects, a construct (e.g., an AAV construct) comprises a promoter. The term "promoter" refers to a DNA sequence recognized by enzymes / proteins that can promote and / or initiate transcription of an operably linked gene. For example, a promoter typically refers to, e.g., a nucleotide sequence to which an RNA polymerase and / or any associated factor binds and from which it can initiate transcription. Thus, in some aspects, a construct (e.g., an AAV construct) comprises a promoter operably linked to one of the non-limiting example promoters described herein.

[0231] In some aspects, a promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art. In some aspects, a promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some aspects, a promoter is a RNA polymerase III promoter, including, but not limited to, a HI promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter. A promoter will generally be one that is able to promote transcription in a mammalian cell.

[0232] A variety of promoters are known in the art, which in some aspects, can be used herein. Nonlimiting examples of promoters that can be used herein in some aspects include: human EFlα, human cytomegalovirus (CMV) (US Patent No.5,168,062, which is incorporated herein by reference for the purposes described herein), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40), β-globin, β-actin, α- fetoprotein, γ-globin, β-interferon, γ-glutamyl transferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g., human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and GRP94), α-2-macroglobulin, vimentin, MHC class I gene H-2K b, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone a gene, immunoglobulin light chain, T-cell receptor, HLA DQa and DQ, interleukin-2 receptor, MHC class II, MHC class II HLA-DRa, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponin I (TN I), duchenne muscular dystrophy, human immunodeficiency virus, and Gibbon Ape Leukemia Virus (GAL V) promoters. Additional examples of promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology,Freeman and Company, New York 2007, each of which is incorporated herein by reference for the purposes described herein. In some aspects, a promoter is the CMV immediate early promoter. In some aspects, the promoter is a CAG promoter and / or a CAG / CBA promoter.

[0233] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked with a nucleic acid encoding a gene (e.g., encoding a polypeptide with m5C RNA writer, reader, and / or erasor functionality and / or targeting element), causes RNA to be transcribed from the nucleic acid in a cell under most or all physiological conditions. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41:521- 530, 1985, which is incorporated herein by reference for the purposes described herein), the SV 40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl-alpha promoter (Invitrogen).

[0234] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art. Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (see e.g., WO 98 / 10088, which is incorporated herein by reference for the purposes described herein); the ecdysone insect promoter (see e.g., No et al., Proc. Natl. Acad Sci. U.S.A 93:3346-3351, 1996, which is incorporated herein by reference for the purposes described herein), the tetracycline-repressible system (see e.g., Gossen et al., Proc. Natl. Acad Sci. U.S.A 89:5547-5551, 1992, which is incorporated herein by reference for the purposes described herein), the tetracycline-inducible system (see e.g., Gossen et al., Science 268: 1766-1769, 1995, see also Harvey et al., Curr. Opin. Chem. Biol.2:512-518, 1998, each of which is incorporated herein by reference for the purposes described herein), the RU486-inducible system (see e.g., Wang et al., Nat. Biotech. 15:239- 243, 1997, and Wang et al., Gene Ther.4:432-441, 1997, each of which is incorporated herein by reference for the purposes described herein), and the rapamycin-inducible system (see e.g., Magari et al., J Clin. Invest.100:2865-2872, 1997, which is incorporated herein by reference for the purposes described herein).

[0235] The term "tissue-specific" promoter refers to a promoter that is active only in certain specific cell types and / or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory and / or control proteins that bind to the tissue-specific promoter). In some aspects, regulatory and / or control sequences impart tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory and / or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner. In some aspects, a tissue-specific promoter is a neuron-specific promoter. In some aspects, a tissue-specific promoter is hematopoietic lineage cell-specific promoter. In some aspects, a tissue-specific promoter is an immune cell-specific promoter. c. Enhancers

[0236] In some aspects, a construct can include an enhancer sequence. The term "enhancer" as used herein refers to a nucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein and / or RNA molecule of interest (e.g., a polypeptide with m5C RNA writer, reader, and / or erasor functionality and / or targeting element), and / or increase or modify the translational efficiency of a transcript following transcription. In some aspects, enhancer sequences (generally 50-1500 bp in length) generally increase the level of transcription by providing additional binding sites for transcription-associated proteins (e.g., transcription factors), and / or stabilize or modify post-transcriptional regulatory machinery. In some aspects, an enhancer sequence is found within an intronic sequence. In some aspects, an enhancer sequence is found in a 3ʹ and / or 5ʹ UTR. In some aspects, an enhancer region is found downstream of a coding sequence comprising a transgene and proximal to a poly adenylation sequence. Unlike promoter sequences, enhancer sequences can act at much larger distance away from the transcription start site (e.g., as compared to a promoter). Non-limiting examples of enhancers include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), RSV enhancer, a CMV enhancer, and / or a SV40 enhancer. d. Flanking untranslated regions, 5ʹ UTR and 3ʹ UTR

[0237] In some aspects, any of the constructs described herein can include an untranslated region (UTR), such as a 5' UTR or a 3' UTR. UTRs of a gene are transcribed but not translated. A 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. A 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory and / or control features of a UTR can be incorporated into any of the constructs, particles, polynucleotides, compositions, kits, or methods as described herein to enhance or otherwise modulate the expression of a gene.

[0238] Natural 5' UTRs include a sequence that plays a role in translation initiation. In some aspects, a 5' UTR can comprise sequences, like Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus sequence CCR(A / G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another “G”. In some aspects, 5' UTRs also form secondary structures that are involved in elongation factor b...

Claims

CLAIMS What is claimed is:

1. A method of treating a disease in an individual, comprising the step of administering one or more inhibitors of methyl-CpG-binding domain protein 6 (MBD6) to an individual in need thereof.

2. The method of claim 1, wherein the disease comprises cancer of the blood, lung, brain, breast, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, and / or cervix.

3. The method of claim 1, wherein the disease comprises clonal hematopoiesis of indeterminate potential (CHIP).

4. The method of claim 3, wherein the disease is characterized by atherosclerosis, myocardial fibrosis, and / or heart failure.

5. The method of claim 2, wherein the cancer comprises a blood cancer.

6. The method of claim 5, wherein the blood cancer comprises a leukemia.

7. The method of claim 5, wherein the blood cancer comprises a myeloid malignancy.

8. The method of claim 7, wherein the myeloid malignancy comprises acute myeloid leukemia.

9. The method of claim 7, wherein the myeloid malignancy comprises chronic myelomonocytic leukemia.

10. The method of claim 2, wherein the cancer comprises a glioma.

11. The method of claim 10, wherein the glioma comprises glioblastoma.

12. The method of claim 1, comprising reducing proliferation of a cancer and / or pre- cancerous cell.

13. The method of claim 1, wherein the disease is associated with diseased cells comprising one or more mutations in one or more genes encoding a ten-eleven translocation (tet) methylcytosine dioxygenase 2 (TET2), ASXL transcriptional regulator 1 (ASXL1),isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), tumor protein p53 (p53), DNA (cytosine-5-)-methyltransferase 3A (DNMT3A), Janus kinase 2 (JAK2), Protein Phosphatase Mn2+ / Mg2+-Dependent 1D (PPM1D), Spliceosome Factor 3b1 (SF3B1), and / or Serine and Arginine Rich Splicing Factor 2 (SRSF2).

14. The method of claim 1, wherein the disease is associated with diseased cells with one or more mutations in one or more genes encoding components of a canonical and / or non- canonical Polycomb Repressive Complex (PRC).

15. The method of claim 14, wherein the one or more mutations in one or more genes encoding components of PRC comprises one or more loss of function mutations.

16. The method of claim 14, wherein the one or more mutations in one or more genes encoding components of PRC comprises one or more mutations in E3 Ubiquitin Ligase RING1A / B, Polycomb Group Ring Finger 1 (PCGF1), Polycomb Group Ring Finger 2 (PCGF2), Polycomb Group Ring Finger 3 (PCGF3), Polycomb Group Ring Finger 4 (PCGF4), Polycomb Group Ring Finger 5 (PCGF5), and / or Polycomb Group Ring Finger 6 (PCGF6).

17. The method of claim 1, wherein the disease is associated with diseased cells with one or more mutations in one or more genes encoding Polycomb Repressive-Deubiquitinase (PR- DUB) complex associated components O-linked N-acetylglucosamine Transferase (OGT), Lysine Demethylase 1B (KDM1B), Forkhead Box K1 (FOXK1), Forkhead Box K2 (FOXK2), BRCA1 Associated Protein 1 (BAP1), ASXL Transcriptional Regulator 1 (ASXL1), ASXL Transcriptional Regulator 2 (ASXL2), ASXL Transcription Regulator 3 (ASXL3), and / or Host Cell Factor C1 (HCFC1).

18. The method of claim 17, wherein the one or more mutations in PR-DUB complex associated components OGT, KDM1B, FOXK1, FOXK2, BAP1, ASXL1, ASXL2, ASXL3, and / or HCFC1 comprises one or more gain of function mutations.

19. The method of claim 13, wherein the disease is associated with diseased cells with one or more mutations in a TET2 encoding gene.

20. The method of claim 19, wherein the one or more mutations in a TET2 encoding gene comprises one or more loss of function mutations.

21. The method of claim 20, wherein the disease is associated with diseased cells comprising one or more mutations in one or more genes encoding ASXL1, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2.

22. The method of claim 1, wherein the individual is administered an additional therapy.

23. The method of claim 22, wherein the additional therapy is surgery, radiation, chemotherapy, hormone therapy, and / or immunotherapy.

24. The method of claim 22, wherein the additional therapy comprises administration of an inhibitor of TET2.

25. The method of claim 24, wherein the inhibitor of TET2 comprises C35 and / or TETi76.

26. The method of claim 22, wherein the additional therapy comprises administration of an inhibitor of NSUN1 and / or NSUN2.

27. The method of claim 26, wherein the additional therapy comprises administration of an inhibitor of NSUN2.

28. The method of claim 22, further comprising administration of 5-AzaC.

29. The method of claim 1, further comprising a step of diagnosing the disease in the individual.

30. The method of claim 1, wherein the disease is is associated with diseased cells characterized as comprising an open chromatin state relative to non-diseased cells of the same developmental lineage.

31. The method of claim 30, wherein administering the one or more MBD6 inhibitors results in promotion of a closed chromatin state in one or more diseased cells in the individual.

32. The method of claim 1, wherein the disease is characterized as pro-inflammatory.

33. The method of claim 1, comprising inhibition of diseased cell proliferation.

34. The method of claim 1, wherein administering the one or more MBD6 inhibitors decreases m5C levels in one or more chromatin associated RNA (caRNA) and / or decreases association of one or more caRNA with a PR-DUB complex in one or more diseased cells in the individual.

35. The method of claim 34, wherein administering the one or more MBD6 inhibitors increases oxidation of m5C in the one or more caRNA and / or inhibits installation of m5C in the one or more caRNA in one or more diseased cells in the individual.

36. The method of claim 34, wherein the one or more caRNA comprises or consists essentially of Long Terminal Repeat (LTR) RNAs.

37. The method of claim 34, wherein the one or more caRNA comprises one or more of the caRNAs described in Table 1.

38. The method of claim 34, wherein the one or more caRNA comprises one or more of the caRNAs described in Table 2.

39. The method of claim 34, wherein the one or more caRNA comprise a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 100-614.

40. The method of claim 34, wherein the one or more caRNA comprise a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs 104 or 107.

41. The method of claim 34, wherein the one or more caRNAs comprise between 20-50 caRNAs.

42. The method of claim 34, wherein the one or more caRNAs comprise a chromatin associated regulatory RNA (carRNA) and / or a chromatin associated repeat RNA sequence.

43. The method of claim 34, wherein the one or more caRNAs comprise or consists essentially of endogenous retroviral RNAs.

44. The method of claim 43, wherein the endogenous retroviral RNAs comprise or consists essentially of endogenous retrovirus-K (ERVK).

45. The method of claim 1, wherein histone ubiquitination is increased in one or more diseased cells after administration of the one or more MBD6 inhibitors.

46. The method of claim 45, wherein the histone ubiquitination comprises or consists of ubiquitination on H2A.

47. The method of claim 46, wherein the histone ubiquitination occurs at a site comprising or consisting essentially of H2AK119.

48. The method of claim 1, wherein administering the one or more MBD6 inhibitors modifies histone methylation in one or more diseased cells in the individual.

49. The method of claim 48, wherein a histone that comprises modified methylation comprises or consists essentially of H3.

50. The method of claim 49, wherein the histone comprises modified methylation on a methylation site comprising or consisting essentially of H3K27me3.

51. The method of claim 48, wherein the histone that comprises modified methylation is localized near or at a genetic locus overlapping with a histone that comprises modified histone ubiquitination.

52. The method of claim 1, wherein the one or more inhibitors of MBD6 comprise a polynucleotide at least partially complementary to a gene encoding MBD6.

53. The method of claim 52, wherein the polynucleotide comprises a short hairpin RNA and / or small interfering RNA.

54. The method of claim 52, wherein the polynucleotide comprises a sequence at least 80% complementary to at least 15, 20, 25, or more than 25 contiguous nucleotides of any one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.

55. The method of claim 52, wherein the polynucleotide comprises modified RNA phosphoramidites.

56. The method of claim 52, wherein the polynucleotide comprises RNA with one or more 2ʹ-O-Methyl (2ʹ-OMe) or 2ʹ-O-Methoxyethyl (2ʹ-MOE) modifications.

57. The method of claim 56, wherein the polynucleotide comprises RNA with every nucleotide comprising a 2ʹ-MOE modification.

58. The method of claim 52, wherein the polynucleotide comprises RNA with one or more phosphorothioate bonds.

59. The method of claim 52, wherein the polynucleotide is comprised within a lentiviral particle and / or nanoparticle.

60. The method of claim 52, wherein the inhibitor of MBD6 comprises more than one polynucleotide.

61. The method of claim 1, wherein the one or more inhibitors of MBD6 comprise a proteolysis targeting chimera.

62. A method of promoting histone ubiquitination in a cell comprising contacting the cell with one or more inhibitors of methyl-CpG-binding domain protein 6 (MBD6).

63. The method of claim 62, wherein promoting histone ubiquitination comprises or consists essentially of promoting ubiquitination on H2A.

64. The method of claim 63, wherein the histone ubiquitination site comprises or consists essentially of H2AK119.

65. The method of claim 62, wherein the cell comprises one or more mutations in one or more genes encoding TET2, ASXL1, IDH1, IDH2, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2.

66. The method of claim 62, wherein the cell comprises one or more mutations in one or more genes encoding RING1A / B, PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, PCGF6, OGT, KDM1B, FOXK1, FOXK2, BAP1, ASXL1, ASXL2, ASXL3, and / or HCFC1.

67. A method of decreasing m5C levels in chromatin associated RNA (caRNA) in a cell, comprising contacting the cell with one or more inhibitors of MBD6.

68. The method of claim 67, wherein the caRNA comprise or consists essentially of LTR RNAs.

69. The method of claim 67, wherein the caRNA comprise or consists essentially of endogenous retroviral RNAs.

70. The method of claim 69, wherein the endogenous retroviral RNAs comprise or consists essentially of endogenous retrovirus-K (ERVK).

71. A method of treating a disease in an individual, wherein the disease is characterized by cells comprising one or more mutations in TET2, ASXL1, IDH1, IDH2, p53, DNMT3A, JAK2, PPM1D, SF3B1, and / or SRSF2 encoding genes, the method comprising administering one or more inhibitors of MBD6, TET2, and / or NSUN2 to the individual in need thereof.

72. A manufactured article for use in performing the method of any one of claims 1-71.

73. A kit comprising means for performining the method according to any one of claims 1-71.

74. A fusion protein comprising a sequence at least 80%, 85%, 90%, 95%, or 100% identical to any one or more of SEQ ID NOs: 1-4, 11-14, or 15-20.

75. The fusion protein of claim 74, wherein the fusion protein comprises a sequence at least 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 19-20.

76. A composition comprising the fusion protein of claim 74.

77. A method of treating a disease associated with aberrant m5C RNA methylation in an individual, comprising administering the fusion protein and / or composition of any one of claims 74-76.

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