Compositions and methods for the targeting of apolipoprotein c3

Abstract

Provided herein are systems comprising Class 2, Type V CRISPR proteins and guide nucleic acids (gRNA) useful in the modification of a target nucleic acid of an apolipoprotein-C3 gene (APOC3). The systems are also useful for introduction into cells, for example eukaryotic cells. Also provided are methods for treating subjects having a ApoC3-related disease and methods for optimizing the encoding sequences.

Description

CROSS REFERENCE TO RELATED APPLCATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 649,154, filed on May 17, 2024, and to U.S. Provisional Patent Application No. 63 / 719,744, filed on November 13, 2024, the content of which are incorporated by reference herein in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SCRB_065_02WO_SeqList_ST26.xml; Size: 2,017,874 bytes; and Date of Creation: May 14, 2025) are herein incorporated by reference in their entirety. BACKGROUND

[0003] Cardiovascular diseases (CVDs) are the leading cause of death globally. At the beginning of the 20th century CVD represented only 10% of deaths worldwide. By 2001, CVD represented 30% of all deaths, and now, in 2024, CVD is the leading cause of death and disability worldwide. (See e.g., Gaziano T, Reddy KS, Paccaud F, et al. Cardiovascular Disease. In: Jamison DT, Breman JG, Measham AR, et al., editors. Disease Control Priorities in Developing Countries. 2nd edition. Washington (DC): The International Bank for Reconstruction and Development / The World Bank; 2006. Chapter 33. Available on the internet at ncbi.nlm.nih.gov / books / NBKl 1767 / Co-published by Oxford University Press, New York).

[0004] The occurrence of CVD can be positively affected by dietary and behavioral changes. However, genetics also play a role in CVD risk. Indeed, a family history of CVD influences future risk of CVD depending on the number and age of affected first-degree relatives, (see e.g., Kolber M.R., et al. (2014) Can Fam Physician.60:1016). Thus, even the most conscientious individuals may experience elevated risk for cardiovascular disease if they have family members with diabetes, high blood pressure or high cholesterol, which can have genetic predisposition.

[0005] Therefore, new approaches are needed to decrease the risk of CVD. As will be apparent from the detailed description that follows, the present disclosure provides for these and other needs. SUMMARY

[0006] The present disclosure provides systems comprising or encoding engineered X-editor proteins and guide ribonucleic acid variants (gRNA) that have utility in the modification of a target nucleic acid of an apolipoprotein C3 gene (APOC3) in eukaryotic cells. The systems and compositions are useful in methods for target nucleic acid modification, which methods are also provided. The present disclosure also provides vectors and LNP formulations for the delivery of the systems to cells for the modification of the APOC3 gene. The present disclosure also provides methods for treating subjects having an APOC3-related disease. The present disclosure also provides methods for optimizing the encoding sequences.

[0007] In some aspects, disclosed herein are systems for modifying an apolipoprotein C3 (APOC3) gene target nucleic acid sequence, comprising: an mRNA comprising a sequence encoding an engineered X-editor protein; and a guide ribonucleic acid (gRNA), wherein the gRNA comprises a scaffold and a linked targeting sequence complementary to the APOC3 gene target nucleic acid sequence, wherein the gRNA is capable of forming a ribonucleoprotein (RNP) with the engineered X-editor protein and binding and modifying the target nucleic acid, wherein the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the APOC3 gene. In some embodiments, the gRNA is a single-molecule gRNA (sgRNA).

[0008] In some embodiments, the targeting sequence is complementary to an APOC3 gene target nucleic acid sequence located between about -5000 base pairs (bp) to about +4100 bp from the transcription start site (TSS) of the APOC3 gene. In some embodiments, targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 3.2 kb of a TSS of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 1.0 kb upstream to 1.0 kb downstream of a TSS of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 500 bps upstream to 500 bps downstream of a TSS of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 500 bps upstream to 500 bps downstream of a TSS of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 100 bps upstream to 100 bps downstream of a TSS of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid within exon 2, within exon 3, within exon 4, or is located at the intron 1-exon 1 boundary of the APOC3 gene. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 458-713, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the fulllength sequence, or a sequence having 1, 2, 3, 4, or 5 mismatches thereto. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 458-713. In some embodiments, the targeting sequence comprises a sequence of SEQ ID NOS: 458-713 with 1, 2, 3, 4, or 5 nucleotide(s) removed from the 3’ end of the sequence. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 458-470, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the full-length sequence, or a sequence having 1, 2, 3, 4, or 5 mismatches thereto. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 458-470. In some embodiments, the targeting sequence comprises a sequence of SEQ ID NOs: 458-470 with 1, 2, 3, 4, or 5 nucleotide(s) removed from the 3’ end of the sequence. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458-464. In some embodiments, the targeting sequence consists of a sequence selected from the group consisting of SEQ ID NO: 458, 459, 460, 461, 462 and 463.

[0009] In some embodiments, the gRNA scaffold comprises a scaffold stem loop comprising the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 435), or a sequence having 1, 2, 3, 4, or 5 mismatches thereto. In some embodiments, the gRNA scaffold comprises a sequence selected from the group consisting of SEQ ID NOS: 357-434 or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the gRNA scaffold comprises a sequence selected from the group consisting of SEQ ID NOS: 357-434. In some embodiments, the gRNA scaffold consists of a sequence selected from the group consisting of SEQ ID NOS: 357-359. In some embodiments, the gRNA scaffold comprises a sequence of SEQ ID NO: 359, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity 3 to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the gRNA scaffold comprises a sequence of SEQ ID NO: 359. In some embodiments, the gRNA scaffold consists of the sequence of SEQ ID NO: 359.

[0010] In some embodiments, the gRNA is chemically modified. In some embodiments, the chemical modification results in an enhanced property compared to a gRNA that is not modified. In some embodiments, the chemical modification to the gRNA comprises an addition of a 2’0-methyl group to one or more nucleotides of the gRNA. In some embodiments, one or more nucleotides located 1, 2, 3, or 4 nucleotides from the 5’ terminal end, the 3’ terminal end, or both terminal ends of the gRNA are modified by an addition of a 2’O-methyl group. In some embodiments, the chemical modification to the gRNA comprises an addition of a 2’O-methyl group to 5 or more, 10 or more, 15 or more, 20 or more, or 25 or more nucleotides of the gRNA. In some embodiments, the chemical modification to the gRNA comprises an addition of a 2’0-methyl group 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 or 30 nucleotides of the gRNA. In some embodiments, the chemical modification to the gRNA comprises an addition of a 2’O-methyl group to 27 nucleotides of the gRNA. In some embodiments, one or more nucleotides located in the scaffold stem loop of the gRNA, the extended stem loop of the gRNA, or both are modified by an addition of a 2’O-methyl group. In some embodiments, the chemical modification to the gRNA comprises an addition of a 2’0-methyl group to at least 1, at least 2 at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nucleotides of the scaffold stem loop. In some embodiments, the chemical modification to the gRNA comprises an addition of a 2’O-methyl group to at least about 1, at least about 2 at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12 nucleotides of the scaffold extended stem loop. In some embodiments, one or more nucleotides selected from nucleotide positions 1, 2, 3, 37, 42, 43, 44, 45, 48, 49, 50, 51, 64, 65, 66, 67, 68, 69, 76, 77, 78, 79, 80, 81, 107, 108, and 109 as compared to SEQ ID NO: 274 are modified by an addition of a 2’O-methyl group. In some embodiments, nucleotide positions 1, 2, 3, 37, 42, 43, 44, 45, 48, 49, 50, 51, 64, 65, 66, 67, 68, 69, 76, 77, 78, 79, 80, 81, 107, 108, and 109, as compared to SEQ ID NO: 274. are modified by an addition of a 2’O-methyl group. In some embodiments, the chemical modification to the gRNA is a substitution of a phosphorothioate bond between two or more nucleosides of the gRNA, optionally wherein the chemical modification comprises a substitution of phosphorothioate bonds between two or more nucleosides located 1, 2, 3 or 4 nucleosides from the 5’ terminal end, the 3’ terminal end, or both terminal ends of the gRNA. In some embodiments, the chemical modification comprises a substitution of phosphorothioate bonds between two or more nucleosides located 1, 2, 3 or 4 nucleosides the from the 5’ terminal end, the 3’ terminal end, or both terminal ends of the gRNA. In some embodiments, the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1116-1128. In some embodiments, the chemically modified gRNA consists of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

[0011] In some embodiments, the enhanced property of the chemically modified gRNA is one or more of improved manufacturability, increased ability to recover full-length gRNA, a reduction in truncations during or after synthesis, increased stability, reduced off-target effects, increased nuclease resistance by cellular RNase, increased duplex bond formation, and a reduced ability to elicit an immune response as compared to a gRNA that is not chemically modified. In some embodiments, the enhanced property comprises recovery of at least about 10%, of at least about 15%, of at least about 20%, or at least about 25% greater amounts of full-length gRNA in the manufacture of the gRNA compared to a gRNA that has not been modified. In some embodiments, the enhanced property comprises an increase in stability of the chemically modified gRNA of at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% in an in vitro stability assay compared to a gRNA that has not been modified. In some embodiments, the chemically modified gRNA is capable of forming a RNP with the engineered X-editor protein and binding and modifying the APOC3 target nucleic acid with an editing rate in an in vitro assay of at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more the editing rate a gRNA that has not been modified.

[0012] In some embodiments, the chemically modified gRNA is capable of forming a RNP with the engineered X-editor protein and binding and modifying the APOC3 target nucleic acid in an in vitro assay with off-target effects equal to or lower than the off-target effects of a gRNA that has not been modified. In some embodiments, the engineered X-editor protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 22-25, or a sequence having at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the engineered X-editor protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 22-25. In some embodiments, the engineered X-editor protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 22-25. In some embodiments, the engineered X-editor protein comprises the amino acid sequence of SEQ ID NO: 23, or a sequence having at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96% , or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the engineered X-editor protein comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the engineered X-editor protein consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the engineered X-editor protein comprises one or more nuclear localization signals (NLS).

[0013] In some embodiments, the one or more NLS comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 67-136. In some embodiments, the one or more NLS is an SV40 NLS or a c-MYC NLS. In some embodiments, the one or more NLS comprises an amino acid sequence selected from SEQ ID NO: 67 or SEQ ID NO: 69.

[0014] In some aspects, disclosed herein are nucleic acids comprising: (a) a sequence that encodes the engineered X-editor of the disclosure; (b) a sequence that encodes or comprises a gRNA of the disclosure; or a sequence that encodes the engineered X-editor of (a) and the gRNA of (b). In some embodiments, the sequence encoding the engineered X-editor is codon-optimized. In some embodiments, the sequence encoding the engineered X-editor is codon-optimized for expression in a eukaryotic cell. In some embodiments, the sequence encoding the engineered X-editor and / or the gRNA is a DNA sequence. In some embodiments, the sequence encoding the engineered X-editor is a messenger RNA (mRNA). In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 47-50, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96% , or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the mRNA is chemically modified. In some embodiments, the sequence encoding the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 51-54, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 52, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the sequence encoding the mRNA comprises a sequence of SEQ ID NO: 52.

[0015] In some aspects, disclosed herein are lipid nanoparticles (LNPs) comprising: (a) a gRNA of the disclosure; (b) an mRNA of the disclosure; or (c) a gRNA of the disclosure and a mRNA of the disclosure. In some embodiments, the LNP comprises one or more components selected from the group consisting of one or more ionizable lipids, one or more helper phospholipids, one or more polyethylene glycol (PEG)-modified lipids, and cholesterol or a derivative thereof. In some embodiments, the LNP comprises an ionizable lipid, a helper phospholipid, a polyethylene glycol (PEG)-modified lipid, and cholesterol or a derivative thereof.

[0016] In some aspects, disclosed herein are vectors comprising the nucleic acid system of the disclosure. In some embodiments, the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex virus (HSV) vector, a plasmid, a minicircle, a nanoplasmid, a DNA vector, and an RNA vector.

[0017] In some aspects, disclosed herein are host cells comprising a vector of the disclosure. In some embodiments, the host cell is selected from the group consisting of a Baby Hamster Kidney fibroblast (BHK), a human embryonic kidney 293 (HEK293) cell, a human embryonic 7 kidney 293T (HEK293T) cell, aNSO cell, a SP2 / 0 cell, a YO myeloma cell, a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell, a hybridoma cell, a NIH3T3 cell, a CV-1 (simian) in Origin with SV40 genetic material (COS) cell, a HeLa cell, a Chinese hamster ovary (CHO) cell, and a yeast cell.

[0018] In some aspects, disclosed herein are pharmaceutical compositions comprising: a system of the disclosure; a nucleic acid of the disclosure; an LNP of the disclosure; or a vector of the disclosure, and one or more pharmaceutically suitable excipients. In some embodiments, the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intraarterial, intraportal vein injection, intraperitoneal, intramuscular, intraocular, subcutaneous, and oral routes.

[0019] In some aspects, disclosed herein are methods of enhancing the manufacture and recovery of full-length gRNA, comprising incorporating one or more 2’O-methyl groups at least about 10, at least about 15, or at least about 20 nucleotides distal to the 5' and 3' ends of the gRNA. In some embodiments, the method comprises incorporating at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 2’O-methyl groups into scaffold stem loop nucleotides of the gRNA. In some embodiments, the method comprises incorporating at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12 2’O-methyl groups into extended stem loop nucleotides of the gRNA. In some embodiments, the method comprises an enhanced recovery of at least about 10%, of at least about 15%, of at least about 20%, or at least about 25% greater amounts of full-length gRNA compared to a gRNA that has not been modified, or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA. In some embodiments, the 2’O-methyl groups are incorporated into a gRNA selected from the group consisting of SEQ ID NOS: 742, 743, 748, 749, 756, and 757.

[0020] In some aspects, disclosed herein are methods of modifying an APOC3 target nucleic acid sequence in a population of cells, the method comprising introducing into cells of the population: (a) a system of the disclosure; (b) a nucleic acid system of the disclosure; (c) an LNP of the disclosure; (d) a vector of the disclosure; (e) a pharmaceutical composition the disclosure; or (f) combinations of two or more of (a)-(e), wherein the APOC3 target nucleic acid sequence of the cells targeted by the gRNA is modified by the engineered X-editor protein. In some embodiments, the modifying comprises introducing a single-stranded break in the APOC3 target nucleic acid sequence of the cells of the population. In some embodiments, the modifying comprises introducing a double-stranded break in the APOC3 target nucleic acid sequence of the cells of the population. In some embodiments, the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the APOC3 target nucleic acid of the cells of the population. In some embodiments, the modifying produces off-target effects of less than about 5%, less than about 4%, less than 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than 0.1% in the cells. In some embodiments, the APOC3 target nucleic acid is modified in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% or more of the cells of the population. In some embodiments, the modifying results in a knocking down expression of the APOC3 gene in the cells of the population such that expression of apolipoprotein C3 is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in comparison to a cell where the APOC3 gene has not been modified. In some embodiments, the APOC3 gene of the cells of the population is modified such that at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the modified cells do not express a detectable level of apolipoprotein C3. In some embodiments, the cells are eukaryotic cells. In some embodiments, the eukaryotic cells are selected from the group consisting of rodent cells, mouse cells, rat cells, and non-human primate cells. In some embodiments, the eukaryotic cells are human cells. In some embodiments, the eukaryotic cells are selected from the group consisting of hepatocytes, intestine cells, kidney cells, and cells of arterial endothelial cells. In some embodiments, the modifying of the APOC3 gene target nucleic acid sequence of the population of cells occurs in vitro or ex vivo. In some embodiments, the modifying of the APOC3 gene target nucleic acid sequence of the population of cells occurs in vivo in a subject. In some embodiments, the subject is selected from the group consisting of a rodent, a mouse, a rat, and a non-human primate. In some embodiments, the subject is a human.

[0021] In some aspects, disclosed herein are methods of treating an ApoC 3-related disease in a subject in need thereof, comprising administering a therapeutically effective dose of: (a) a system of the disclosure; (b) a nucleic acid system of the disclosure; (c) an LNP of the disclosure; (d) a vector of the disclosure; (e) a pharmaceutical composition of the disclosure; or (f) a combination of two or more of (a)-(e), wherein the APOC3 target nucleic acid sequence of cells of the subject targeted by the gRNA are modified by the engineered X-editor protein. In some embodiments, the modifying comprises introducing a single-stranded break in the APOC3 gene of the cells. In some embodiments, the modifying comprises introducing a double-stranded break in the APOC3 gene of the cells. In some embodiments, the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the APOC3 gene of the cells. In some embodiments, the modifying results in a knocking down of the expression of the APOC3 gene in the modified cells of the subject such that expression of apolipoprotein C3 in the subject is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in comparison to a subject where the APOC3 gene has not been modified. In some embodiments, the modifying results in a knocking down expression of the APOC3 gene in the modified cells of the subject such that expression of apolipoprotein C3 in the subject is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in comparison to a subject where the APOC3 gene has not been modified. In some embodiments, the subject is selected from the group consisting of a rodent, a mouse, a rat, and a non-human primate. In some embodiments, the subject is a human. In some embodiments, the cells that are modified are selected from the group consisting of hepatocytes, intestine cells, kidney cells, and arterial endothelial cells. In some embodiments, the subject is treated with a therapeutically effective dose of the LNP. In some embodiments, the LNP is administered by a route of administration selected from the group consisting of intravenous, intraarterial, intraportal vein injection, intraperitoneal, intramuscular, subcutaneous, and oral routes. In some embodiments, the ApoC3-related disease is selected from the group consisting of: hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or abnormal cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia induced pancreatitis, metabolic syndrome, type II diabetes mellitus, familial chylomicronemia syndrome, and familial partial lipodystrophy or combinations thereof. In some embodiments, the method results in improvement in at least one clinically-relevant endpoint associated with ApoC3-related disease. In some embodiments, the method results in improvement in at least two clinically-relevant endpoints associated with ApoC3-related disease. In some embodiments, the clinically-relevant endpoint is selected from the group consisting of: percent change in fasting triglyceride levels from baseline, percent change from baseline in apoC-III, percent change from baseline in total cholesterol, percent change from baseline in LDL-C, percent change from baseline in HDL-C, percent change from baseline in non-HDL-C, percent change from baseline in VLDL-C, percent change from baseline in apoB, percent change from baseline in apoA-I, percent change from baseline in lipoprotein(a) and percent change from baseline in angiopoietin-like 3 (ANGPTL3).

[0022] In some aspects, disclosed herein are gRNAs comprising a sequence selected from the group consisting of SEQ ID NOS: 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0023] In some aspects, disclosed herein are gRNAs consisting of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0024] In some aspects, disclosed herein are compositions comprising a gRNA consisting of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

[0025] In some aspects, disclosed herein are compositions comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0026] In some aspects, disclosed herein are compositions comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

[0027] In some aspects, disclosed herein are compositions comprising a gRNA consisting of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

[0028] In some aspects, disclosed herein are compositions consisting of the sequence of SEQ ID NO: 1115.

[0029] In some aspects, disclosed herein are compositions comprising an mRNA encoding an engineered X-editor protein, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 33-54, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, and comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0030] In some aspects, disclosed herein are kits comprising a system of the disclosure, a nucleic acid system of the disclosure, an LNP of the disclosure, a vector of the disclosure, a composition of the disclosure, or combinations thereof, and a suitable container. In some embodiments, the kit comprises a buffer, an excipient, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[0031] In some aspects, disclosed herein are methods of reducing a level of apolipoprotein C3 in a subject, comprising administering a therapeutically effective dose of: (a) a system of the disclosure; (b) a nucleic acid system of the disclosure; (c) an LNP of the disclosure; (d) a vector of the disclosure; (e) a pharmaceutical composition of the disclosure; or (f) a combination of two or more of (a)-(e), wherein the APOC3 target nucleic acid sequence of cells of the subject targeted by the gRNA are modified by the engineered X-editor protein.

[0032] Other features, objects, and advantages will be apparent from the disclosure that follows. INCORPORATION BY REFERENCE

[0033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The contents of WO 2024 / 206620, WO 2025 / 072293, WO2020 / 247882, WO2020 / 247883, WO2021 / 113772, WO2022 / 120095, WO2022 / 125843, WO2022 / 261150, WO2022 / 261149, WO2023 / 049872, WO2023 / 235818, and WO2023 / 240162, which disclose engineered X-editor proteins and gRNA variants, and methods of delivering same, are hereby expressly incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0035] FIG. 1 is a schematic of gRNA scaffold variant 174, as described in Example 2. Structural motifs are highlighted (gRNA scaffold variant 174 sequence: SEQ ID NO: 357).

[0036] FIG. 2 is a schematic of gRNA scaffold variant 235, as described in Example 2. Highlighted structural motifs are the same as in FIG. 1. The differences between variant 174 and variant 235 lie in the extended stem motif and several single-nucleotide changes (indicated with asterisks).

[0037] FIG. 3 is a schematic of gRNA scaffold variant 316, as described in Example 2. Highlighted structural motifs are the same as in FIG. 1 (gRNA scaffold variant 316 sequence: SEQ ID NO: 359). Variant 316 maintains the shorter extended stem from variant 174 (FIG. 1) but harbors the four substitutions found in scaffold 235 (FIG. 2).

[0038] FIG. 4 is a schematic illustrating versions 1-3 (“vl-v3”) of chemical modifications made to gRNA scaffold variant 235, as described in Example 2. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified (2'-O-methylation) ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond. For the v2 profile, the addition of three 3’ uracils (3’UUU) is annotated with “U”s in the relevant circles.

[0039] FIG. 5 is a schematic illustrating versions 4-6 (“v4-v6”) of chemical modifications made to gRNA scaffold variant 235, as described in Example 2. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0040] FIG. 6 is a schematic illustrating versions 7-9 (“v7-v9”) of chemical modifications made to gRNA scaffold variant 316, as described in Example 2. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0041] FIG. 7 is a schematic illustrating versions 1-3 (“vl-v3”) of chemical modifications made to gRNA scaffold variant 316, as described in Example 2. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0042] FIG. 8 is a schematic illustrating versions 4-6 (“v4-v6”) of chemical modifications made to gRNA scaffold variant 316, as described in Example 2. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0043] FIG. 9 is a plot displaying a correlation between indel rate (depicted as edit fraction) at the PCSK9 locus as measured by NGS (x-axis) and secreted PCSK9 levels (ng / mL) detected by enzyme-linked immunosorbent assay (ELISA) (y-axis) in HepG2 cells lipofected with CasX 491 mRNA and PCSXP-targeting gRNAs containing the indicated scaffold variant and spacer combination, as described in Example 2.

[0044] FIG. 10 is a plot illustrating the quantification of percent knockout of B2M in HepG2 cells co-transfected with 100 ng of CasX 491 mRNA and with the indicated doses of end-modified (vl) or unmodified (vO) B2M-targeting gRNAs with spacer 7.37, as described in Example 2. Editing level was determined by flow cytometry as the population of cells with loss of surface presentation of the HLA complex due to successful editing at the B2M locus.

[0045] FIG. 11A is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the human B2Mlocus in HepG2 cells treated with the indicated doses of LNPs formulated with CasX 491 mRNA and the indicated B2A7-targeting gRNA, as described in Example 2.

[0046] FIG. 1 IB is a plot illustrating the quantification of percent knockout of B2M in HepG2 cells treated with the indicated doses of LNPs formulated with CasX 491 mRNA and the indicated B2A7-targeting gRNA, as described in Example 2. Editing level was determined by flow cytometry as population of cells that did not have surface presentation of the HLA complex due to successful editing at the B2Mlocus.

[0047] FIG. 12A is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the mouse ROSA26 locus in Hepal-6 cells treated with the indicated doses of LNPs formulated with CasX 676 mRNA #2 and the indicated ROSA 26-targeting gRNA with either the vl or v5 modification profile, as described in Example 2.

[0048] FIG. 12B is a plot illustrating the quantification of percent editing measured as indel rate detected by NGS at the ROSA26 locus in mice treated with LNPs formulated with CasX 676 mRNA #2 and the indicated chemically-modified RO SA 26-targeting gRNA, as described in Example 2.

[0049] FIG. 13 is a bar graph showing the results of the editing assay measured as indel rate detected by NGS as the mouse PCSK9 locus in mice treated with LNPs formulated with CasX 676 mRNA #1 and the indicated chemically-modified PGS'O-targeting gRNA, as described in Example 2. Untreated mice served as experimental control.

[0050] FIG. 14 is a bar graph showing the results of the editing assay measured as indel rate detected by next generation sequencing (NGS) at the human APOC3 locus in Lot #131 Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified APOC3-targeting gRNA, as described in Example 4.

[0051] FIG. 15 is a bar graph showing the results of the editing assay measured as indel rate detected by NGS at the human APOC3 locus in Lot #271 Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified APOC3-targeting gRNA, as described in Example 4.

[0052] FIG. 16 is a bar graph showing the results of the APOC3 mRNA level assay measured by qPCR in Lot #131 Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified APOC 3-targeting gRNA, as described in Example 4.

[0053] FIG. 17 is a bar graph showing the results of the APOC3 mRNA level assay measured by qPCR in Lot #271 Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified APOC 3-targeting gRNA, as described in Example 4.

[0054] FIG. 18 is a bar graph showing the results of the APOC3 ELISA assay in Lot #131 Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified APOCJ-targeting gRNA, as described in Example 4.

[0055] FIG. 19 is a bar graph showing the results of the APOC3 ELISA assay in Lot #271 Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified ZPOC3-targeting gRNA, as described in Example 4.

[0056] FIG. 20 is a bar graph displaying the level of off-target editing at select off-target sites in HEK293 cells expressing X-editor 515, along with gRNA spacers TG-49-021, TG-49-024, TG-49-025, and TG-49-027 targeting the APOC3 locus, which was assessed using a CSLseq assay as described in Example 5. The number of off-target CSLseq reads for the 10 most frequent off-target (“OT”) sites was normalized relative to the number of on-target CSLseq reads, such that each bar represents the relative level of editing at a unique off-target site in the genome.

[0057] FIG. 21A is a bar graph showing the results of an editing assay, measured as indel rate detected by NGS, at the on-target site (“Target site”) and the one off-target (“OT”) site nominated by CSLseq, in PHH cells. Cells were harvested six days post-transfection with a lentiviral plasmid encoding the X-editor 515 with gRNA scaffold 316 and spacer TG-49-021, as described in Example 5. Indel rate was normalized to transfection efficiency.

[0058] FIG. 2 IB is a bar graph showing the results of an editing assay, measured as indel rate detected by NGS, at the on-target site (“Target site”) and the four off-target (“OT”) sites nominated by CSLseq, in PHH cells. Cells were harvested six days post-transfection with a lentiviral plasmid encoding the X-editor 515 with gRNA scaffold 316 and spacer TG-49-024, as described in Example 5. Indel rate was normalized to transfection efficiency.

[0059] FIG. 2IC is a bar graph showing the results of an editing assay, measured as indel rate detected by NGS, at the on-target site (“Target site”) and the eight off-target (“OT”) sites nominated by CSLseq, in PHH cells. Cells were harvested six days post-transfection with a lentiviral plasmid encoding the X-editor 515 with gRNA scaffold 316 and spacer TG-49-025, as described in Example 5. Indel rate was normalized to transfection efficiency.

[0060] FIG. 22 is a schematic illustrating version 105 (“vl05”) of chemical modifications made to gRNA scaffold variant 316, as described in Example 7. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified (2'-O- methylation) ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0061] FIG. 23 is a bar graph showing the results of the editing assay measured as indel rate detected by NGS at the human APOC3 locus in Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified APOC3-targeting gRNAs, as described in Example 7.

[0062] FIG. 24 is a bar graph showing the results of the APOC3 mRNA level assay measured by qPCR in Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified APOC 3-targeting gRNAs, as described in Example 7.

[0063] FIG. 25 is a bar graph showing the results of the APOC3 ELISA assay in Primary Human Hepatocytes (PHH) treated with LNPs formulated with CasX 515 and the indicated chemically-modified APOC3-targeting gRNAs, as described in Example 7.

[0064] FIG. 26 is an image of a polyacrylamide gel comparing the migration pattern for gRNAs comprising either the vl or vl05 chemical modification profile for the gRNA scaffold variant 316 with three different APOC3 targeting sequences, as described in Example 7.

[0065] FIG. 27 is a bar graph depicting the % full-length product quantified for the polyacrylamide gel depicted in FIG. 26 for gRNAs comprising either the vl or vl05 chemical modification profile for the gRNA scaffold variant 316 with three different APOC3 targeting sequences, as described in Example 7. DETAILED DESCRIPTION Definitions

[0066] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[0067] As used herein, the singular forms “a,” “an,” and “the,” include the plural referents unless the context clearly indicates otherwise.

[0068] The term “about,” as used herein, indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value± 10%, ± 5%, or± 1%. In certain embodiments, the term “about” indicates the designated value ± one standard deviation of that value.

[0069] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also include any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 1-5 members refers to groups having 1, 2, 3, 4, or 5 members, and so forth.

[0070] The term “combinations thereof includes every possible combination of elements to which the term refers.

[0071] The term “exemplary” as used herein, refers to an example or illustration, and is not intended to imply any preference or value.

[0072] The term “reference CasX protein,” as used herein, refers to naturally-occurring or wild-type CasX proteins such as e.g., CasX proteins having amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0073] The terms “engineered X-editor,” or “engineered X-editor protein,” as used herein, refers to a highly engineered CasX protein (interchangeably referred to herein as “CasX variants”) that comprises sequence modifications that confer one or more improved characteristics relative to a wild-type CasX protein. In some embodiments, an engineered X-editor protein is a chimeric CasX comprising regions from two or more reference CasX protein. Exemplary engineered X-editor proteins include, e.g., proteins having an amino acid sequence according to SEQ ID NOS: 22-25.

[0074] The term “apolipoprotein C3,” “apolipoprotein C-III,” “apo-CHI,” “APOC3P “Apo-C3,” or “ApoC-3,” as used herein, refers to a protein having at least about 80% amino acid sequence identity, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100% identity to SEQ ID NO: 342, see also UniProtKB-P02656. In humans, APOC3 is encoded by the APOC3 gene (SEQ ID NO: 343 see also GenBank Accession NG 008949). APOC3 is known to be a component of triglyceride-rich very low density lipoproteins (VLDL) and high density lipoproteins (HDL) in plasma (see e.g., Yao Z., and Wang Y., (2012) Curr. Opin. Lipidol. 23:206-212) and plays a multifaceted role in triglyceride homeostasis (see e.g., Chan, D.C. et al (2008) Int. J. Clin. Pract. 62:799-809).

[0075] As used herein, “specificity,” refers to the degree to which a CRISPR / Cas system ribonucleoprotein complex edits off-target sequences that are similar, but not identical to the target nucleic acid sequence.

[0076] The term “off-target effects” as used herein refers to unintended cleavage and mutations at untargeted genomic sites showing a similar but not an identical sequence compared to the target site. In some embodiments the off-target effects are determined in an in vitro cell-free assay. In some embodiments the off-target effects are determined in a cell-based assay.

[0077] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass single-stranded DNA; doublestranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multistranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0078] “Hybridizable” or “complementary” are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and / or G / U base pairs, “anneal,” or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still hybridize to the target nucleic acid. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a 'bulge', ‘bubble’ and the like). Thus, the skilled artisan will appreciate that while individual bases within a sequence may not be complementary to another sequence, the sequence as a whole is still considered to be complementary.

[0079] A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (e.g., a protein, RNA), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene may include accessory element sequences including, but not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. Coding sequences encode a gene product upon transcription or transcription and translation; the coding sequences of the disclosure may comprise fragments and need not contain a full-length open reading frame. A gene can include both the strand that is transcribed as well as the complementary strand containing the anticodons.

[0080] The term “downstream” as used herein, refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0081] The term “upstream” as used herein, refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0082] The term “adjacent to” with respect to polynucleotide or amino acid sequences refers to sequences that are next to, or adjoining each other in a polynucleotide or polypeptide. The skilled artisan will appreciate that two sequences can be considered to be adjacent to each other and still encompass a limited amount of intervening sequence, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or amino acids.

[0083] “Operably linked” means with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components e.g., a promoter and an encoding sequence. The skilled artisan will appreciate that the two components need not be physically linked to be operably linked.

[0084] A “heterologous coding sequence,” as used herein, refers to nucleic acid sequence present in a polynucleotide, vector, or host cell that encodes a peptide or polypeptide, or a polynucleotide that itself has a function or activity, such as an antisense or inhibitory oligonucleotide, including antisense DNA and RNA (e.g., miRNA, siRNA, and shRNA), where the heterologous coding sequence is not naturally found in the polynucleotide, vector, or host cell, i.e. is non-native.

[0085] The term “regulatory element” is used interchangeably herein with the term “regulatory sequence,” and is intended to include promoters, enhancers, and other expression regulatory elements. It will be understood that the choice of the appropriate regulatory element will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0086] The term “accessory element” is used interchangeably herein with the term “accessory sequence,” and is intended to include, inter alia, polyadenylation signals (poly(A) signal), enhancer elements, introns, posttranscriptional regulatory elements (PTREs), nuclear localization signals (NLS), deaminases, DNA glycosylase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g. in cis or in trans), selfcleaving sequences, and fusion domains, for example a fusion domain fused to a CRISPR protein. It will be understood that the choice of the appropriate accessory element or elements will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0087] The term “promoter” refers to a DNA sequence that comprises a transcription start site and additional sequences that facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as a TATA box, and / or B recognition element (BRE) and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. A promoter can be proximal or distal to the gene to be transcribed. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties. In some embodiments a promoter includes variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter can be classified according to criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. A promoter can also be classified according to its strength. As used in the context of a promoter, “strength” refers to the rate of transcription of the gene controlled by the promoter. A “strong” promoter means the rate of transcription is high, while a “weak” promoter means the rate of transcription is relatively low.

[0088] A promoter of the disclosure can be a Polymerase II (Pol II) promoter. Polymerase II transcribes all protein coding and many non-coding genes. A representative Pol II promoter includes a core promoter, which is a sequence of about 100 base pairs surrounding the transcription start site, and serves as a binding platform for the Pol II polymerase and associated general transcription factors. The promoter may contain one or more core promoter elements such as the TATA box, BRE, Initiator (INR), motif ten element (MTE), downstream core promoter element (DPE), downstream core element (DCE), although core promoters lacking these elements are known in the art.

[0089] A promoter of the disclosure can be a Polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs such as the 5S rRNA, tRNAs, and other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed section of the gene) to support transcription, although upstream elements such as the TATA box are also sometimes used. All Pol III promoters are envisaged as within the scope of the instant disclosure.

[0090] The term “enhancer” refers to regulatory DNA sequences that, when bound by specific proteins called transcription factors, regulate the expression of an associated gene. Enhancers may be located in the intron of the gene, or 5’ or 3’ of the coding sequence of the gene. Enhancers may be proximal to the gene (i.e., within a few tens or hundreds of base pairs (bp) of the promoter), or may be located distal to the gene (ie., thousands of bp, hundreds of thousands of bp, or even millions of bp away from the promoter). A single gene may be regulated by more than one enhancer, all of which are envisaged as within the scope of the instant disclosure.

[0091] As used herein, a “post-transcriptional regulatory element (PTRE),” such as a hepatitis PTRE, refers to a DNA sequence that, when transcribed creates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote expression of an associated gene operably linked thereto.

[0092] “Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “enhancers” and “promoters,” above).

[0093] The term “recombinant polynucleotide” or “recombinant nucleic acid” refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0094] Similarly, the term “recombinant polypeptide” or “recombinant protein” refers to a polypeptide or protein which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a protein that comprises a heterologous amino acid sequence is recombinant.

[0095] The term “lipoprotein” as used herein, refers to a lipid / protein particle comprising a central hydrophobic core of non-polar lipids, primarily cholesterol esters and triglycerides, surrounded by a hydrophilic membrane comprising phospholipids, free cholesterol, and apolipoproteins. Lipoproteins, such as very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL), are typically found in the serum, plasma and lymph and function in lipid transport. The chemical composition of each plasma lipoprotein differs. Accordingly, plasma lipoproteins are divided into seven classes; chylomicrons, chylomicron remnants, VLDL, VLDL remnants (IDL), LDL, HDL, and Lp(a), based on size, lipid composition, and apolipoproteins. See e.g., Feingold KR. Introduction to Lipids and Lipoproteins. [Updated 2024 Jan 14], In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet], South Dartmouth (MA): MDText.com, Inc.; 2000-. Available on the internet from: ncbi.nlm.nih.gov / books / NBK305896 / .

[0096] The term “high density lipoprotein” or “HDL” as used herein, refers to small, dense lipoproteins which transport lipids out of cells. HDL particles typically range in size from 5 to 17 nm. Thus, HDL is the smallest of the lipoprotein particles. HDL particles are also the most dense lipoprotein particles because they comprise the highest proportion of protein to lipids. Its most abundant apolipoproteins are apo A-I and apo A-II.

[0097] The term “high density lipoprotein-C” or “HDL-C” as used herein, refers to cholesterol associated with high-density lipoprotein particles. HDL-C is considered anti-atherogenic good cholesterol because the cholesterol is being transported out of cells. HDL-C shows an inverse relationship with coronary heart disease (CHD) risk. Concentration of HDL-C in serum (or plasma) is typically quantified in mg / dL or nmol / L. “Serum HDL-C” and “plasma HDL-C” mean HDL-C in serum and plasma, respectively.

[0098] The term “very low density lipoproteins” or “VLDL” as used herein, refers to Very-low-density lipoprotein (VLDL) cholesterol produced in the liver and released into the bloodstream to supply body tissues with triglycerides. About half of a VLDL particle is made up of triglycerides. High levels of VLDL cholesterol are typically associated with the development of plaque deposits on artery walls, which narrow the passage and restrict blood flow, thus contributing to the risk of coronary heart disease.

[0099] The term “low density lipoprotein” or “LDL” refers to relatively less dense lipoproteins which transport lipids into cells. LDL particles are formed when triglycerides are removed from very low density lipoproteins (VLDL) by the lipoprotein lipase enzyme (LPL) and they become smaller and denser (because they carry fewer fat molecules with same protein transport shell). Accordingly, LDL particles comprise a higher proportion of cholesterol esters 24 than VLDL particles. LDL particles typically range in size from are approximately 22 nm-27.5 nm. Thus, the term “low density lipoprotein-cholesterol (LDL-C)” as used herein, refers to cholesterol carried in low density lipoprotein particles. LDL-C is often referred to as “bad cholesterol” because high levels of LDL cholesterol raise the risk for heart disease and stroke. Concentration of LDL-C in serum (or plasma) is typically quantified in mg / dL or nmol / L. “Serum LDL-C” and “plasma LDL-C” mean LDL-C in the serum and plasma, respectively.

[00100] “Low-density lipoprotein (LDL) receptor” refers to a receptor protein of 839 amino acids (after removal of 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich LDL particles. It is a cell-surface receptor that recognizes the apoprotein Bl00 and apoE protein found in chylomicron remnants and VLDL remnants (IDL) resulting in the binding and endocytosis of LDL-cholesterol. This process occurs in all nucleated cells, but mainly in the liver which removes approximately 70% of LDL from the circulation. The human LDLR gene is described in part in the NCBI database (ncbi.nlm.nih.gov) as Reference Sequence NG_009060.1, which is incorporated by reference herein.

[00101] The term “triglyceride” or “TG” as used herein, refers to an ester derived from glycerol and three fatty acids. Triglycerides are the main constituents of body fat in humans and other vertebrates.

[00102] As used herein, the term “atherosclerosis,” refers to a thickening or hardening of the arteries caused by the buildup of fats, cholesterol, and other substances in and on the artery walls.

[00103] As used herein, the term “coronary heart disease (CHD),” refers to a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.

[00104] As used herein, the term “dyslipidemia,” refers to a disorder of lipid and / or lipoprotein metabolism, including lipid and / or lipoprotein overproduction or deficiency. Dyslipidemias may be manifest as an elevation of lipids such as chylomicron, cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.

[00105] As used herein, the term “hypercholesterolemia” refers to a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol and VLDL-cholesterol, as per the guidelines of the Expert Panel Report of the National Cholesterol Educational Program (NCEP) of Detection, Evaluation of Treatment of high cholesterol in adults (see, Arch. Int. Med. 148: 36 (1988)).

[00106] As used herein, “hyperlipidemia” or “hyperlipemia” is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests an abnormally high concentration of fats. The lipid fractions in the circulating blood are cholesterol, low-density lipoproteins, very low density lipoproteins, chylomicrons and triglycerides. The Fredrickson classification of hyperlipidemias is based on the pattern of TG and cholesterol-rich lipoprotein particles, as measured by electrophoresis or ultracentrifugation and is commonly used to characterize primary causes of hyperlipidemias such as hypertriglyceridemia.

[00107] The term “hypertriglyceridemia” refers to a condition characterized by elevated triglyceride levels. Its etiology includes primary (i.e., genetic causes) and secondary (other underlying causes such as diabetes, metabolic syndrome / insulin resistance, obesity, physical inactivity, cigarette smoking, excess alcohol, and a diet very high in carbohydrates) factors or, most often, a combination of both.

[00108] As used herein, “diabetes mellitus” or “diabetes” is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from insufficient levels of insulin or reduced insulin sensitivity. The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.

[00109] As used herein, “diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated triglycerides (TG), and elevated small, dense LDL particles.

[00110] As used herein, "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, helper phospholipids, and PEG-modified lipids), as well as cholesterol. Specific components of LNP are described more fully, below. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles of the disclosure comprise a nucleic acid. Such lipid nanoparticles typically comprise neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells; e.g., an adverse immune response.

[00111] As used herein, “lipid encapsulated” refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both. In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.

[00112] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a target nucleic acid with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid are made to share a physical connection; e.g., can hybridize if the sequences share sequence similarity.

[00113] The disclosure provides systems and methods useful for editing a target nucleic acid sequence. As used herein “editing” is used interchangeably with “modifying” and "modification" and includes but is not limited to cleaving, nicking, deleting, knocking in, knocking out, and the like.

[00114] By “cleavage” it is meant the breakage of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events.

[00115] The term "knock-down" as used herein refers to reduction in the expression of a gene or its gene product(s). As a result of a gene knock-down, the protein activity or function may be attenuated, or the protein levels may be reduced or eliminated.

[00116] The term “knock-out” as used herein, refers to the elimination of a gene or the expression of a gene. For example, a gene can be knocked out by either a deletion or an addition of a nucleotide sequence that leads to a disruption of the reading frame. As another example, a gene may be knocked out by replacing a part of the gene with an irrelevant sequence. The term "knock-down" as used herein refers to reduction in the expression of a gene or its gene product(s). As a result of a gene knock-down, the protein activity or function may be attenuated, or the protein levels may be reduced or eliminated.

[00117] As used herein, "homology-directed repair" (HDR) refers to the form of DNA repair that takes place during repair of double-strand breaks in cells. This process requires nucleotide 27 sequence homology, and uses a donor template to repair or knock-out a target DNA, leading to the transfer of genetic information from the donor to the target. Homology-directed repair can result in an alteration of the sequence of the target sequence by insertion, deletion, or mutation if the donor template differs from the target DNA sequence and part or all of the sequence of the donor template is incorporated into the target DNA.

[00118] As used herein, "non-homologous end joining" (NHEJ) refers to the repair of doublestrand breaks in DNA by direct ligation of the break ends to one another without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). NHEJ often results in the loss (deletion) or insertion of nucleotide sequence near the site of the double- strand break.

[00119] As used herein “micro-homology mediated end joining” (MMEJ) refers to a mutagenic double-strand break repair mechanism, which always associates with deletions flanking the break sites without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). MMEJ often results in the loss (deletion) of nucleotide sequence near the site of the double- strand break.

[00120] The terms “identical,” or “percent identity,” in the context of two or more polypeptide or nucleic acid molecule sequences, means two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using methods known in the art, such as a sequence comparison algorithm, by manual alignment, or by visual inspection. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Altschul et al. Nucleic Acids Res. 2007, 25, 3389-3402), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Within the context of this disclosure, it is understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” mean any set of values or parameters which originally load with the software when first initialized.

[00121] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to refer to a polymer of at least two amino acid monomers joined together through peptide bonds. Polypeptide chains can be of any length. Typically, the terms “polypeptide,” “protein,” and “peptide” refer to a molecule comprising two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the polypeptide / protein. In some embodiments, a polypeptide can be a portion of a larger protein, for example, a domain, a subdomain, or a motif of the protein. In some embodiments, a polypeptide / protein can be a variant (or mutation) of a naturally occurring or reference protein, wherein one or more amino acid residues are inserted into, deleted from, and / or substituted into the sequence of the protein relative to the naturally occurring, or reference, protein sequence. Polypeptides can be modified, for example, by the addition of carbohydrate, phosphorylation, etc. Proteins can comprise one or more polypeptides.

[00122] A protein or a variant thereof can be naturally occurring or recombinant. Methods for detection and / or measurement of polypeptides in biological material are well known in the art and include, but are not limited to, Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as e.g., an ELISA This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and / or measurement of polypeptides / proteins are described e.g., in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[00123] In the context of proteins, the term “fragment,” or equivalent term as used herein, refers to a portion of a protein that has less than the full length of the protein and optionally maintains the function of the protein.

[00124] The term “conservative amino acid substitution” as used herein, refers to the substitution of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as “conservatively modified variants.” Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. Additional conservative substitutions may be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, NY. A polypeptide or protein generated by making one or more conservative substitutions of amino acid residues in a parent polypeptide or protein is referred to as a “conservatively modified variant.”

[00125] The term “amino acid,” as used herein, refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), praline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[00126] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e., an expression cassette, may be attached so as to bring about the replication or expression of the attached segment in a cell.

[00127] The terms “naturally-occurring” or “unmodified” or “wild type” are used interchangeably herein to refer to a nucleic acid, a polypeptide, a cell, or an organism, having a structure or composition as is found in nature.

[00128] As used herein, a “mutation” refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides as compared to a wild-type or reference amino acid sequence or to a wild-type or reference nucleotide sequence.

[00129] The term “isolated” as used herein, refers to a substance that has been separated and / or recovered from its natural environment. For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all, of the co-existing materials in the natural system, is isolated. Thus, an “isolated” polynucleotide, polypeptide, or cell is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs.

[00130] A “host cell,” as used herein, denotes a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells are used as recipients for a nucleic acid (e.g., a vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., a vector.

[00131] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a biologic, alone or as a part of a composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject such as a human or an experimental animal. Such effect need not be absolute to be beneficial.

[00132] The term “administering” as used herein, refers to providing a dosage of a compound (e.g., a composition of the disclosure) or a composition (e.g., a pharmaceutical composition) to a subject.

[00133] As used herein, “treatment” or “treating,” are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can also be achieved with the eradication or amelioration of one or more of the symptoms or an improvement in one or more clinical parameters associated with the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

[00134] The term “subject” as used herein, refers to a mammalian subject. Exemplary subjects include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, and rodents e.g., mice, rats. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease that can be treated with the engineered X editor system provided herein.

[00135] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. I. General Methods

[0136] The present disclosure employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found e.g., in standard textbooks. For example, basic texts disclosing methods in recombinant genetics and molecular biology include, e.g., Sambrook et al. Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Press 4th Edition (Cold Spring Harbor, N.Y. 2012) and Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987-2016). Basic texts disclosing the general methods and terms in biochemistry include, e.g., Lehninger Principles of Biochemistry sixth edition, David L. Nelson and Michael M. Cox eds. W.H. Freeman (2012); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods, Bollag et al., John Wiley & Sons (1996); Nonviral Vectors for Gene Therapy, Wagner et al. eds., Academic Press (1999); Viral Vectors Kaplift & Loewy eds., Academic Press (1995); Immunology Methods Manual, I. Lefkovits ed., Academic Press 1997; and Cell and Tissue Culture: Laboratory Procedures in Biotechnology Doyle & Griffiths, eds., John Wiley & Sons (1998); Next Generation Sequencing and Data Analysis, Melanie Kappelmann-Fenzl ed. Springer (2021); Bioinformatics and Functional Genomics, 3rd Edition, Jonathan Pevsner, Wiley-Blackwell (2015). II. Introduction

[0137] Cardiovascular diseases (“CVD”) due to atherosclerosis and thrombosis are the largest cause of premature morbidity, mortality, disability, and financial burden, worldwide. Lipid disorders are well known to promote the development of atherosclerosis and its clinical consequences, such as CVD, including e.g., coronary heart disease (“CHD”), ischemic stroke, peripheral arterial disease (“PAD”), heart failure, and sudden cardiac death. It has been indicated that the genetic predispositions to hypertriglyceridemia can lead to excess of TG in the bloodstream, or poor capacity to remove TG from the blood. In result, both of these scenarios are correlated with increased risk for future CV events (Liu J., et al. (2013) Lipids Health Dis. 12:159.

[0138] Therefore, the prevention and treatment of dyslipidemias are key issues of cardiovascular disease (CVD) prevention and treatment. Consequently, what is needed in the art are new therapies for the effective management of lipid disorders, including hypertriglyceridemia. III. APOC3

[0139] The human APOC3 gene (HGNC: 610) encodes a 99 amino acid ApoC3 protein, apolipoprotein C3 (P02656, also known as "apolipoprotein C-III,” “apo-CIII,” “APOC3,” “Apo-C3,” or “ApoC-3”). A representative ApoC3 protein sequence is shown below: MQPRVLLWA LLALLASARA SEAEDASLLS FMQGYMKHAT KTAKDALSSV QESQVAQQAR GWVTDGFSSL KDYWSTVKDK FSEFWDLDPE VRPTSAVAA (SEQ ID NO: 342).

[0140] The AP0C3 gene (comprising a sequence of SEQ ID NO: 343) is defined as the sequence that spans chrl 1 116,700,623-116,703,788 (GRCh37 / hgl9 Ensembl version ENSG00000110245.7) of the human genome of chromosome 11. This gene and other related genes comprise an apolipoprotein gene cluster on chromosome 11. Splice variants and precursors of ApoC3 protein are also contemplated as within the scope of the instant disclosure.

[0141] Apolipoprotein C3 expression is largely restricted to the liver (hepatocytes), but is also produced in the small intestine, with smaller amounts found in cells of the kidney and cells of arterial endothelial walls. APOC3 is a component of triglyceride (TG)-rich lipoproteins (TRLs) including very low-density lipoproteins (VLDL), high density lipoproteins (HDL) and chylomicrons ) in plasma (see e.g., Yao Z., and Wang Y, (2012) Curr. Opin. Lipidol. 23:206212) and plays a multifaceted role in triglyceride homeostasis (see e.g., Chan, D.C. et al (2008) Int. J. Clin. Pract. 62:799-809). Apolipoprotein C3 plays an important role in the production, metabolism, and clearance of TG-rich lipoproteins from plasma and has been shown to promote the secretion of VLDL1, inhibit lipoprotein lipase enzyme activity, and delay catabolism of TRL remnants. Mutations in the APOC3 gene are associated with low plasma triglyceride levels and reduced risk of ischemic cardiovascular disease, and hyperalphalipoproteinemia, which is characterized by elevated levels of high density lipoprotein (HDL) and HDL cholesterol in human patients.

[0142] In humans, apolipoprotein C3 is encoded by the APOC3 gene (SEQ ID NO: 343, see also GenBank Accession NG 008949). Apolipoprotein C3 is known to be a component of triglyceride-rich very low density lipoproteins (VLDL) and high density lipoproteins (HDL IV. Systems for Genetic Editing of APOC3 Genes

[0143] In one aspect, the disclosure provides systems comprising an engineered X-editor protein and one or more guide ribonucleic acids (gRNA), as well as nucleic acids encoding the engineered X-editor proteins and gRNA, for use in modifying an APOC3 gene, inclusive of coding and non-coding regions of the APOC3 gene. As used herein, a “system,” is used interchangeably with “composition,” and may comprise an engineered X-editor protein and one or more gRNAs of the disclosure as gene editing pairs, nucleic acids encoding the engineered X- editor proteins and / or gRNA, as well as vectors or lipid nanoparticles (LNP) comprising the nucleic acids or engineered X-editor protein and one or more gRNAs the disclosure.

[0144] The present disclosure provides systems specifically designed to modify the APOC3 gene in eukaryotic cells. In some embodiments, the disclosure provides systems comprising one or more engineered X-editor proteins and one or more guide nucleic acid variants (gRNA) as a system designed to target and edit specific locations of a target nucleic acid sequence of the APOC3 gene. In some embodiments, the systems of the disclosure are designed to knock-down or knock-out the APOC3 gene such that expression of the gene is reduced or eliminated. Generally, any portion of the APOC3 gene can be targeted using the programable compositions and methods provided herein, described more fully, below.

[0145] In some embodiments, the disclosure provides vectors encoding the engineered X-editor:gRNA components, which, when delivered to the target cells, are expressed and associate as gene editing pairs capable of modifying the APOC3 gene. In some embodiments, the disclosure provides LNPs that encompass a system of the disclosure. In some embodiments, the LNP encompasses an mRNA that encodes the engineered X-editor component. In some embodiments, the LNP encompasses a guide RNA that targets the APOC3 gene. In some embodiments, the LNP encompasses an mRNA that encodes the engineered X-editor component, and one or more gRNAs. Upon delivery to the target cells, the mRNA encoding the engineered X-editor is expressed and the engineered X-editor protein then associates with the gRNA to form a gene editing pair capable of modifying the APOC3 gene. Each of these components and their use in the editing of the APOC3 gene is described herein, below. V. Engineered X-Editor Proteins for Modifying a Target Nucleic Acid of an APOC3 gene

[0146] The present disclosure provides engineered X-editor proteins that have utility for the modification of a target nucleic acid of an APOC3 gene in eukaryotic cells. The terms “engineered X-editor" and “engineered X-editor protein,” as used herein, refers to reference CasX proteins engineered with sequence modifications (interchangeably referred to herein as “CasX variants”) possessing one or more improved characteristics relative to a naturally occurring (reference) CasX protein.

[0147] The engineered X-editor proteins employed in the genome modifying systems disclosed herein, are Class 2, Type V CRISPR nucleases. Although members of Class 2, Type V 34 CRISPR-Cas systems have differences, they share some common characteristics that distinguish them from the Cas9 systems. First, the engineered X-editor proteins, are highly modified relative to wild-type, reference CasX, but retain the general characteristics of Class 2, Type V CRISPR nucleases. Thus, engineered X-editor proteins, like reference CasX proteins, possess a single RNA-guided RuvC domain-containing effector but no HNH domain, and they recognize a TC motif protospacer adjacent motif (PAM) 5' upstream to the target region on the non-targeted strand, which is different from Cas9 systems which rely on G-rich PAM at 3' side of target sequences. Like all Type V nucleases, engineered X-editor proteins generate staggered doublestranded breaks distal to the PAM sequence, unlike Cas9, which generates a blunt end in the proximal site close to the PAM. In addition, engineered X-editor proteins, being Type V nucleases, degrade ssDNA in trans when activated by target dsDNA or ssDNA binding in cis. In some embodiments, the engineered X-editor proteins disclosed herein recognize a 5'-TC PAM motif and produce staggered ends cleaved solely by the RuvC domain.

[0148] Engineered X-editor proteins of the disclosure may comprise the following domains: a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain, a helical II domain, an oligonucleotide binding domain (OBD), and a RuvC DNA cleavage domain. In some cases, domains can be further divided into subdomains, as provided in Tables 2 and 3.

[0149] In some embodiments, an engineered X-editor protein binds and modifies a target nucleic acid targeted by an associated gRNA; the gRNA has a targeting sequence that is complementary to the target nucleic acid to be modified. In some embodiments, an engineered X-editor protein binds and modifies a nucleic acid by catalyzing a single strand break (a "nickase") in a target nucleic acid targeted by an associated gRNA. In some embodiments, an engineered X-editor protein binds and modifies a nucleic acid by catalyzing a double strand break in a target nucleic acid targeted by an associated gRNA. In some embodiments, the engineered X-editor comprises a nuclease domain having double-stranded cleavage activity that generates a double-stranded break within 18-26 nucleotides 5' of a PAM site on the target strand and 10-18 nucleotides 3' on the non-target strand, resulting in overhangs that can facilitate a higher degree of editing efficiency or insertion of a donor template nucleic acid by homology-directed repair (HDR) or homology-independent targeted insertion (HITI) repair mechanisms of the host cell. a. Reference CasX Proteins

[0150] The disclosure provides naturally-occurring CasX proteins (referred to herein as a “reference CasX proteins”), which were subsequently modified to create the synthetic engineered X-editor proteins of the disclosure. Exemplary reference CasX proteins include those isolated from naturally occurring prokaryotes, such as Deltaproteobacteria, Planctomycetes, or Candidatus Sungbacteria species. A reference CasX protein (interchangeably referred to herein as a reference CasX polypeptide) is a Class 2, Type V CRISPR / Cas endonuclease belonging to the CasX (interchangeably referred to as Casl2e) family of proteins that interacts with a guide RNA to form a ribonucleoprotein (RNP) complex.

[0151] In some embodiments, the reference CasX protein is isolated or derived from Deltaproteobacter, and comprises a sequence of SEQ ID NO: 1.

[0152] In some embodiments, the reference CasX protein is isolated or derived from Planctomycetes, and comprises a sequence of SEQ ID NO: 2.

[0153] In some embodiments, the reference CasX protein is isolated or derived from Candidatus Sungbacteria, and comprises a sequence of SEQ ID NO: 3. b. Engineered X-editor Proteins

[0154] The present disclosure provides highly-modified engineered X-editor proteins having multiple mutations relative to one or more reference CasX proteins. Any changes in the amino acid sequence of a reference CasX protein that results in a protein that exhibits an improved characteristic relative to the reference CasX protein is considered an engineered X-editor protein of the disclosure, provided that the engineered X-editor retains the ability to form an RNP with a gRNA and retains nuclease activity. Exemplary improved characteristics of the engineered X-editor proteins, relative to reference CasX proteins include, but are not limited to: improved ability to utilize a greater spectrum of PAM sequences in the editing and / or binding of target nucleic acid, increased nuclease activity, improved editing efficiency, improved editing specificity for the target nucleic acid, decreased off-target editing or cleavage, increased percentage of a eukaryotic genome that can be efficiently edited, increased activity of the nuclease, improved ability to form cleavage-competent ribonucleoprotein (RNP) complexes with gRNA, and improved ribonucleoprotein (RNP) complex stability. In particular, the engineered X-editor proteins of the disclosure have an enhanced ability to efficiently edit and / or bind target DNA, when complexed with a guide RNA scaffold as an RNP, utilizing a PAM TC motif, including PAM sequences selected from TTC, ATC, GTC, or CTC, compared to an RNP of a reference CasX protein and a reference gRNA. In the foregoing embodiments, the one or more of the improved characteristics of the engineered X-editor is at least about 1.1 to about 100,000fold improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, when assayed in a comparable fashion. In other embodiments, the improvement is at least about 1.1-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0155] The engineered X-editor can comprise one or more amino acid substitutions, insertions, deletions relative to a reference CasX, swapped domains from a second reference CasX, or any combinations thereof, relative to a reference CasX protein sequence or a parental CasX from which it was derived. Mutations can be introduced in any one or more domains of the reference CasX protein or in a CasX variant to result in an engineered X-editor, and may include, for example, deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain of the reference CasX protein or the CasX variant from which it was derived.

[0156] In some embodiments, the disclosure provides an engineered X-editor wherein the engineered X-editor comprises a RuvC cleavage domain, wherein the RuvC cleavage domain comprises the sequence of amino acids 648-812 of SEQ ID NO: 2 with one or more amino acid modifications relative to the RuvC cleavage domain sequence. In some embodiments, the one or more amino acid modifications of the RuvC domain comprise a modification at a position selected from the group consisting of 1658, A708, and P793 relative to SEQ ID NO: 2.

[0157] In some embodiments, an engineered X-editor protein comprises a sequence of SEQ ID NOS: 22-25 as set forth in Table 1. In some embodiments, an engineered X-editor protein consists of a sequence of SEQ ID NOS: 22-25, as set forth in Table 1. In other embodiments, an engineered X-editor protein comprises a sequence at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical to a sequence of SEQ ID NO: 22, as set forth in Table 1, wherein the engineered X-editor protein retains the functional properties of the ability to form an RNP with a gRNA and retains nuclease activity. In other embodiments, an engineered X-editor protein comprises a sequence of SEQ ID NO: 22.

[0158] In some embodiments, an engineered X-editor protein comprises a sequence of SEQ ID NO: 23, or a sequence at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% thereto, wherein the engineered X-editor protein retains the functional properties of the ability to form an RNP with a gRNA and retains nuclease activity. In some embodiments, an engineered X-editor protein comprises a sequence of SEQ ID NO: 23.

[0159] In some embodiments, an engineered X-editor protein comprises a sequence of SEQ ID NO: 24, or a sequence at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% thereto, wherein the engineered X-editor protein retains the functional properties of the ability to form an RNP with a gRNA and retains nuclease activity. In some embodiments, an engineered X-editor protein comprises a sequence of SEQ ID NO: 24.

[0160] In some embodiments, an engineered X-editor protein comprises a sequence of SEQ ID NO: 25, or a sequence at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% thereto, wherein the engineered X-editor protein retains the functional properties of the ability to form an RNP with a gRNA and retains nuclease activity. In some embodiments, an engineered X-editor protein comprises a sequence of SEQIDNO: 25. Table 1: Engineered X-editor Protein Sequences SEQ ID NO Enginee redX-editor Amino Acid Sequence 22 491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF LSKYQDIIIEHQKWKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME DWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTIN GKELKVEGQITYYNRYKRQNWKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRF SHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET WQSFYRKKLKEVWKPAV 23 515 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF LSKYQDIIIEHQKWKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI SEQ ID NO Enginee redX-editor Amino Acid Sequence NGKELKVEGQITYYNRYKRQNWKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE TWQSFYRKKLKEVWKPAV 24 676 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPIS NTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEK GNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLIKLAQLKPEKDSD EAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALSDACMGTIAS FLSKYQDIIIEHQKWKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIA RVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKK EDGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYD EAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRC ELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGG KLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWN DLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVD RGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGY SRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRM EDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTT INGKELKVEGQITYYNRYKRQNWKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKK RFSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFV ETWQSFYRKKLKEVWKPAV 25 812 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDE AVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF LSKYQDIIIEHQKWKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIAR VRMWVNLNLWQKLKLSRDDAKPLLRLKKFPSFPLVERQANEVDWWDMVCNVKKLINEKKE DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDE AWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGK LRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVDR GENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRME DWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTI NGKELKVEGQITYYNRYKRQNWKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR FSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVE TWQSFYRKKLKEVWKPAV

[0161] Further engineered X-editor proteins contemplated for use in the systems of the disclosure are described e.g., in International Publication Nos. WO2020 / 247882, WO2022 / 120095, and WO2023 / 235818. c. Engineered X-editor Proteins with Domains from Multiple Source Proteins

[0162] As described further herein, the disclosure provides engineered X-editor proteins that are chimeric CasX proteins. As used herein, a “chimeric engineered X-editor protein" refers to both an engineered X-editor protein containing at least two domains from different sources, as well an engineered X-editor protein containing at least one domain that itself is chimeric.

[0163] In some embodiments, an engineered X-editor is a chimeric protein derived from SEQ ID NO: 2, wherein the helical I-II domain is replaced with the corresponding helical I-II domain sequence from SEQ ID NO: 1, thereby providing an engineered X-editor protein wherein the engineered X-editor is a chimeric protein comprising at least two domains derived from different reference CasX proteins.

[0164] In some embodiments, the helical I-II domain and NTSB domain of an engineered X-editor derived from a reference CasX of SEQ ID NO: 2 are replaced with the corresponding helical I-II and NTSB sequences from SEQ ID NO: 1, resulting in a chimeric engineered X-editor protein comprising at least two domains from different reference CasX proteins.

[0165] In some embodiments, an engineered X-editor protein comprises at least one chimeric domain. In some embodiments, the at least one chimeric domain can be any of the NTSB, TSL, helical I, helical II, OBD or RuvC domains as described herein. In the case of split or noncontiguous domains such as helical I, RuvC and OBD, a portion of the non-contiguous domain can be replaced with the corresponding portion from any other source. Domain sequences from reference CasX proteins, and their coordinates, are shown in Table 2. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 660 to 823 of SEQ ID NO: 1 and amino acids 921 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, a chimeric RuvC domain comprises amino acids 647 to 810 of SEQ ID NO: 2 and amino acids 934 to 986 of SEQ ID NO: 1. In some embodiments, the engineered X-editor 491 (SEQ ID NO: 22), engineered X-editor 515 (SEQ ID NO: 23), engineered X-editor 676 (SEQ ID NO: 24), and engineered X-editor 812 (SEQ ID NO: 25), have an NTSB and a portion of the helical I-II domain derived from the reference CasX sequence of SEQ ID NO: 1, and other domains are derived from the reference CasX sequence of SEQ ID NO: 2. It is understood that the engineered X-editors can have additional amino acid changes at select locations relative to the reference CasX sequences, and the resulting chimeric engineered X-editor proteins were determined to have improved characteristics relative to the reference CasX proteins. In one embodiment, engineered X-editor 515 (SEQ ID NO: 23) has an NTSB and the helical I-II domain derived from the reference CasX of SEQ ID NO: 1, and an insertion of P793 relative to the sequence of engineered X-editor 491 (SEQ ID NO: 22), wherein the resulting engineered X-editor 515 exhibits enhanced specificity for the modification of a target nucleic acid relative to engineered X-editor 491.

[0166] In one embodiment, the chimeric helical I domain of the chimeric X-editor proteins of SEQ ID NOS: 22-25 comprise amino acids 58-101 of SEQ ID NO: 2, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto (helical I-I), and comprises amino acids 191-331 of SEQ ID NO: 1, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto (helical I-II). The skilled artisan will appreciate that the domain boundaries indicated in Table 2 below are approximate, and that protein fragments whose boundaries differ from those given in the table below by 1, 2, or 3 amino acids may have the same activity as the domains described below. Table 2: Domain coordinates in Reference CasX Proteins Domain Name Coordinates in SEQ ID NO: 1 Coordinates in SEQ ID NO: 2 OBD-I 1-55 1-57 helical I-I 56-99 58-101 NTSB 100-190 102-191 helical I-II 191-331 192-332 helical II 332-508 333-500 OBD-II 509-659 501-646 RuvC-I 660-823 647-810 TSL 824-933 811-920 RuvC-II 934-986 921-978 Table 3: Exemplary Domain Sequences in Reference CasX Proteins Deltaproteobacter sp. (reference CasX of SEQ ID NO: 1) SEQ ID Domain Sequence 4 OBD-I EKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQ 5 helical I-I VISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFA 6 NTSB QPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRC NVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQ 7 helical I-II RALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIII EHQKWKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNL NLWQKLKLSRDDAKPLLRLKGFPSF 8 helical II PWERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDH KKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIERE EARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWYGDLRGNPFAV EAE 9 OBD-II NRWDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKK SGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKL ANGRVIEKTIYNKKIG RDEPALFVALTFERREWD 10 RuvC-I PSNIKPVNLIGVDRGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRA IQAAKEVEQ RRAGGYS RKFAS KS RNLADDMVRNSARDL FYHAVTHDAVLVFENL S RGF GRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTC 11 TSL SNCGFTITTADYDGMLVRLKKTSDGWATTLNNKELKAEGQITYYNRYKRQTVEKELSA ELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVH 12 RuvC-II ADEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA Planctomycetes sp. (Reference CasX of SEQ ID NO: 2) SEQ ID Domain Sequence 13 OBD-I QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQ 14 helical I-I PISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA 15 NTSB QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYFGRC NVSEHERLILLSPHKPEANDELVTYSLGKFGQ 16 helical I-II RALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIIL EHQKVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNWAQIVIWVNL NLWQKLKIGRDEAKPLQRLKGFPSF 17 helical II PLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKK GKKFARYQFGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQ S KAALTDWLRAKAS FVIEGLKEADKDEFCRCELKLQKWYGDLRGKP FAIEAE 18 OBD-II NSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYT VINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLD 19 RuvC-I SSNIKPMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRT IQAAKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGF GRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTC 20 TSL SNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNWKDLSV ELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETH 21 RuvC-II ADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV d. Engineered X-editor Fusion Proteins

[0167] The disclosure also contemplates engineered X-editor proteins that comprise a heterologous protein (i.e., a protein other than derived from a CRISPR protein) fused to the engineered X-editor. This includes engineered X-editors comprising N-terminal or C-terminal fusions of the engineered X-editor to a heterologous protein or domain thereof. In some embodiments, the engineered X-editor protein is fused to one or more proteins or domains thereof that has a different activity of interest, thereby resulting in a fusion protein.

[0168] In some embodiments, a heterologous polypeptide (a fusion partner) for use with an engineered X-editor in the systems of the disclosure provides for subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES) to escort the engineered X-editor through the nuclear pore complex, a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like.

[0169] The disclosure contemplates assembly of multiple NLS in various configurations for linkage to the engineered X-editor proteins utilized in the embodiments described herein. In some embodiments, a single NLS is linked at or near the N-terminus of the engineered X-editor protein. In some embodiments, a single NLS is linked at or near the N-terminus and at or near the C-terminus of the engineered X-editor protein. In some embodiments, the N-terminal NLS comprises one or more a c-MYC NLS. In some embodiments, the C-terminal NLS comprises one or more c-MYC NLS. In some embodiments, 2, 3, 4 or more NLS are linked by linker peptides at or near the C-terminus and / or the N-terminus of the engineered X-editor protein. A person of ordinary skill in the art will appreciate that an NLS at or near the N- or C-terminus of a protein can be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the N- or C-terminus. In some embodiments, the NLS linked to the N-terminus of the engineered X-editor protein are identical to the NLS linked to the C-terminus. In other embodiments, the NLS linked to the N-terminus of the engineered X-editor protein are different than the NLS linked to the C-terminus. In some embodiments, non-limiting examples of NLSs suitable for use with an engineered X-editor in the systems of the disclosure include sequences having at least about 80%, at least about 90%, or at least about 95% identity or are identical to sequences derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 67); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 68); the c-MYC NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 69) or RQRRNELKRSP (SEQ ID NO: 70). In some embodiments, the NLS and a short peptide linker linked to the N-terminus of the engineered X-editor protein comprises the sequence PKKKRKVSR (SEQ ID NO: 71). In some embodiments, the NLS and a short peptide linker linked to the N-terminus of the engineered X-editor protein comprise the sequence PKKKRKVSRVNGSGSGGG (SEQ ID NO: 135). In some embodiments, the NLS and a short peptide linker linked to the C-terminus of the engineered X-editor protein comprise the sequence TSPKKKRKV (SEQ ID NO: 136). Other NLS and linker peptide contemplated for use in the systems of the disclosure include the sequences of SEQ ID NOS: 71-136. The person of ordinary skill in the art will appreciate that any of the foregoing NLS sequences can be fused to, or proximal to either the N- or C-terminus of the long-term repressor fusion proteins described herein.

[0170] In some embodiments, the one or more NLSs are linked to the engineered X-editor protein or to adjacent NLS with a linker peptide wherein the linker peptide is selected from the group consisting of SR, RS, GS, GGS, GP, TS, PPP, GGS, VGS, (G)n (SEQ ID NO: 137), (GS)n (SEQ ID NO: 138), (GSGGS)n (SEQ ID NO: 139), (GGSGGS)n (SEQ ID NO: 140), (GGGS)n (SEQ ID NO: 141), GGSG (SEQ ID NO: 142), GGSGG (SEQ ID NO: 143), GSGSG (SEQ ID NO: 144), GSGGG (SEQ ID NO: 145), GGGSG (SEQ ID NO: 146), GSSSG (SEQ ID NO: 147), GPGP (SEQ ID NO: 148), GGP, PPP, PPAPPA (SEQ ID NO: 149), PPPG (SEQ ID NO: 150), PPPGPPP (SEQ ID NO: 151), PPP(GGGS)n (SEQ ID NO: 152), (GGGS)nPPP (SEQ ID NO: 153), AEAAAKEAAAKEAAAKA (SEQ ID NO: 154), TPPKTKRKVEFE (SEQ ID NO: 155), GGSGGGS (SEQ ID NO: 156), GSGSGGG (SEQ ID NO: 157), and SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 158), where n is 1 to 5.

[0171] In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of an engineered X-editor fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to an engineered X-editor fusion protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly. e. Sequences encoding engineered X-editor proteins

[0172] As described further herein, the disclosure provides nucleic acids (e.g., mRNA and DNA) comprising sequences encoding engineered X-editor proteins. In some embodiments, the sequences encoding engineered X-editors were modified using one or more parameters to enhance expression in the target cell. Non-limiting examples of such parameters include the codon usage in human host cells (e.g., utilizing the codon adaptation index (CAI)) or codonusage tables derived from biologies intended for use as therapeutics. In some embodiments, provided herein are sequences encoding engineered X-editor proteins that are codon-optimized for expression in a human cell.

[0173] In some embodiments, the disclosure provides an mRNA sequence encoding an engineered X-editor comprising a sequence selected from the group consisting of SEQ ID NOS: 22-25, or at least 70% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides an mRNA sequence encoding the engineered X-editor 491 (SEQ ID NO: 22). In some embodiments, the mRNA sequence encoding engineered X-editor 491 comprises the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 39, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides a DNA sequence encoding the amino acid sequence of SEQ ID NO: 22. In some embodiments, the DNA sequence comprises a sequence of SEQ ID NO: 29, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0174] In some embodiments, the disclosure provides an mRNA sequence encoding the engineered X-editor 515 (SEQ ID NO: 23). In some embodiments, the mRNA sequence encoding engineered X-editor 515 comprises the nucleic acid sequence of SEQ ID NO: 34 or SEQ ID NO: 40, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the mRNA sequence encoding engineered X-editor 515 comprises the nucleic acid sequence of SEQ ID NO: 34 or SEQ ID NO: 40. In some embodiments, the mRNA sequence encoding engineered X-editor 515 comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the disclosure provides a DNA sequence encoding the amino acid sequence of SEQ ID NO: 23. In some embodiments, the DNA sequence comprises a sequence of SEQ ID NO: 30, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the fulllength sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the DNA sequence comprises a sequence of SEQ ID NO: 30.

[0175] In some embodiments, the disclosure provides an mRNA sequence encoding engineered X-editor 676 (SEQ ID NO: 24). In some embodiments, the mRNA sequence encoding X-editor 676 comprises the nucleic acid sequence of SEQ ID NO: 35 or SEQ ID NO: 41, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the fulllength sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides a DNA sequence encoding the amino acid sequence of SEQ ID NO: 24. In some embodiments, the DNA sequence comprises a sequence of SEQ ID NO: 31, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0176] In some embodiments, the disclosure provides an mRNA sequence encoding engineered X-editor 812 (SEQ ID NO: 25). In some embodiments, the mRNA sequence encoding X-editor 812 comprises the nucleic acid sequence of SEQ ID NO: 36 or SEQ ID NO: 42, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the fulllength sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides a DNA sequence encoding the amino acid sequence of SEQ ID NO: 25. In some embodiments, the DNA sequence comprises a sequence of SEQ ID NO: 32, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0177] In some embodiments, the disclosure provides a composition comprising an mRNA encoding an engineered X-editor protein, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 33-54, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, and comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0178] Exemplary sequences encoding engineered X-editors, including RNA and DNA sequences encoding engineered X-editors, are provided as SEQ ID NOS: 29-66. Select sequences are provided in Table 4. Table 4: RNA and DNA sequences encoding Engineered X-editor proteins SEQ ID NO: Engineered X-editor Designation coding sequence 33 491 CAGGAGAUCAAGCGGAUUAAUAAAAUUCGGAGAAGACUGGUGAAGGAUUCUAACAC AAAGAAGGCUGGCAAGACAGGCCCUAUGAAGACACUGCUGGUGAGAGUGAUGACAC CC GAG CU GAGAGAAAGACU GGAAAAC CU GAGAAAGAAGC CU GAGAAUAU C C C C GAG CCCAUCAGCAACACAAGCCGGGCCAACCUGAAUAAGCUGCUGACCGACUACACCGA AAUGAAGAAGGCCAUCCUGCACGUGUAUUGGGAAGAGUUCCAGAAAGACCCAGUCG GCCUGAUGAGCAGAGUGGCUCAGCCUGCCAGCAAGAAGAUCGAUCAGAACAAGCUG AAGCCCGAAAUGGACGAGAAGGGGAACCUGACAACCGCCGGCUUUGCCUGUAGCCA GUGCGGCCAGCCCCUGUUUGUGUACAAACUGGAACAGGUGAGCGAAAAGGGCAAGG CUUACACGAAUUACUUCGGCAGAUGCAACGUGGCCGAGCACGAGAAGCUGAUCCUG CUGGCCCAGCUGAAGCCUGAGAAGGAUAGCGAUGAGGCAGUGACAUAUUCCCUGGG CAAGUUCGGACAGCGGGCCCUGGAUUUUUAUUCCAUUCAUGUGACCAAGGAAUCCA CCCACCCCGUCAAGCCUCUUGCCCAAAUUGCCGGCAACAGAUACGCCAGCGGCCCC GUGGGCAAGGCCCUGAGCGACGCCUGUAUGGGCACCAUCGCCAGCUUCCUGUCUAA GUACCAGGACAUUAUCAUCGAGCACCAGAAGGUGGUGAAGGGCAACCAGAAGAGAC UGGAGAGCCUGCGCGAGCUGGCCGGCAAGGAAAACCUGGAGUAUCCUAGCGUGACC CUGCCUCCUCAGCCUCAUACAAAGGAGGGCGUGGAUGCCUACAACGAAGUGAUCGC CCGGGUGCGGAUGUGGGUGAACCUGAAUCUGUGGCAGAAGCUGAAGCUGUCUAGAG SEQ ID NO: Engineered X-editor Designation coding sequence ACGACGCCAAGCCCCUGCUGAGACUGAAGGGCUUCCCCAGCUUCCCUCUGGUGGAG AGACAGGCAAAUGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCUGAU CAAUGAGAAGAAGGAGGACGGCAAAGUGUUCUGGCAGAAUCUGGCCGGCUACAAGC GUCAGGAGGCCCUGCGGCCCUACCUGAGCAGCGAGGAAGACAGAAAGAAGGGCAAG AAGUUCGCCCGGUAUCAGCUGGGGGACCUGCUGCUGCACCUCGAGAAGAAGCACGG CGAAGACUGGGGGAAGGUGUACGAUGAGGCCUGGGAGCGGAUCGAUAAGAAGGUGG AGGGCCUGAGCAAGCACAUCAAGCUGGAGGAGGAACGGAGAUCUGAGGACGCCCAG AGCAAGGCCGCCCUGACCGACUGGCUGAGAGCCAAGGCCAGCUUCGUCAUCGAGGG GCUGAAGGAGGCCGACAAGGACGAGUUCUGCCGGUGCGAACUGAAGCUGCAGAAGU GGUACGGAGAUCUGAGAGGCAAACCUUUCGCCAUCGAGGCCGAGAACAGCAUCCUG GACAUCAGCGGCUUCAGCAAGCAGUACAACUGCGCCUUUAUUUGGCAGAAGGACGG AGUGAAGAAGCUGAACCUGUACCUGAUCAUCAACUAUUUCAAGGGCGGCAAGCUGA GAUU CAAGAAGAU CAAGC CU GAAGC CUU C GAGGC CAACAGAUU CUACAC C GU GAUU AACAAGAAAAG C G GAGAGAU C GU G C CAAU G GAAGU GAACUU CAACUU C GAG GAG C C UAACCUGAUCAUCCUGCCCCUGGCAUUUGGCAAGCGGCAGGGCAGAGAGUUCAUCU GGAACGACCUGCUGUCUCUGGAGACCGGCAGCCUGAAGCUGGCCAACGGCAGAGUG AU C GAGAAGACACU GUACAACAGAC GAAC CAGACAAGAC GAGGC CGCCCUGUUUGU GGCCCUGACCUUCGAGAGAAGAGAGGUGCUGGACAGCAGCAAUAUCAAGCCUAUGA ACCUGAUCGGCGUGGACCGGGGCGAGAACAUCCCUGCCGUGAUCGCCCUUACCGAC CCCGAGGGAUGCCCUCUGAGCCGGUUUAAAGACAGCCUGGGCAACCCUACCCACAU CCUGAGAAUUGGCGAGUCCUACAAGGAGAAGCAGAGAACCAUCCAGGCCAAGAAGG AGGUGGAGCAGCGGCGGGCUGGCGGCUACUCCCGGAAGUACGCCAGCAAGGCCAAG AACCUGGCCGACGACAUGGUUAGAAAUACCGCCAGAGACCUCCUGUACUACGCUGU GACCCAGGACGCCAUGCUGAUCUUCGAGAACCUGAGCAGAGGCUUCGGCAGACAGG GCAAGAGAACCUUCAUGGCCGAGAGACAGUACACCCGGAUGGAGGACUGGCUGACC GCCAAGCUGGCCUACGAGGGCCUGUCUAAGACCUACCUGUCCAAGACCUUGGCACA GUACACCAGCAAGACAUGCUCUAACUGCGGCUUCACAAUCACGAGCGCCGACUACG ACCGGGUGCUGGAGAAACUGAAGAAGACCGCCACAGGCUGGAUGACCACCAUUAAC GGCAAGGAGCUGAAGGUGGAGGGCCAGAUCACCUACUACAACAGGUACAAACGGCA GAACGUGGUGAAGGACCUGAGCGUGGAACUGGAUAGACUGAGCGAGGAAAGCGUAA ACAAUGACAUCAGCAGCUGGACCAAGGGCCGGAGCGGCGAGGCCCUGAGCCUGCUG AAGAAGAGAUU CU C C CACAGAC CAGU GCAGGAGAAGUU C GU GU GU CU GAACU GC GG CUUCGAGACCCACGCCGACGAGCAAGCCGCCCUGAACAUCGCCCGGUCUUGGCUUU UCCUGCGGAGCCAGGAGUACAAGAAGUACCAGACAAACAAGACCACAGGCAACACA GACAAGAGAGCCUUCGUCGAGACCUGGCAGAGCUUCUACAGAAAGAAGCUGAAGGA GGUGUGGAAGCCUGCCGUG 34 515 CAGGAGAUCAAGCGGAUUAAUAAAAUUCGGAGAAGACUGGUGAAGGAUUCUAACAC AAAGAAGGCUGGCAAGACAGGCCCUAUGAAGACACUGCUGGUGAGAGUGAUGACAC CC GAC CU GAGAGAAAGACU GGAAAAC CU GAGAAAGAAGC CU GAGAAUAU C C C C CAG CCCAUCAGCAACACAAGCCGGGCCAACCUGAAUAAGCUGCUGACCGACUACACCGA AAUGAAGAAGGCCAUCCUGCACGUGUAUUGGGAAGAGUUCCAGAAAGACCCAGUCG GCCUGAUGAGCAGAGUGGCUCAGCCUGCCAGCAAGAAGAUCGAUCAGAACAAGCUG AAGCCCGAAAUGGACGAGAAGGGGAACCUGACAACCGCCGGCUUUGCCUGUAGCCA GUGCGGCCAGCCCCUGUUUGUGUACAAACUGGAACAGGUGAGCGAAAAGGGCAAGG CUUACACGAAUUACUUCGGCAGAUGCAACGUGGCCGAGCACGAGAAGCUGAUCCUG CUGGCCCAGCUGAAGCCUGAGAAGGAUAGCGAUGAGGCAGUGACAUAUUCCCUGGG CAAGUUCGGACAGCGGGCCCUGGAUUUUUAUUCCAUUCAUGUGACCAAGGAAUCCA CCCACCCCGUCAAGCCUCUUGCCCAAAUUGCCGGCAACAGAUACGCCAGCGGCCCC GUGGGCAAGGCCCUGAGCGACGCCUGUAUGGGCACCAUCGCCAGCUUCCUGUCUAA GUACCAGGACAUUAUCAUCGAGCACCAGAAGGUGGUGAAGGGCAACCAGAAGAGAC UGGAGAGCCUGCGCGAGCUGGCCGGCAAGGAAAACCUGGAGUAUCCUAGCGUGACC CUGCCUCCUCAGCCUCAUACAAAGGAGGGCGUGGAUGCCUACAACGAAGUGAUCGC CCGGGUGCGGAUGUGGGUGAACCUGAAUCUGUGGCAGAAGCUGAAGCUGUCUAGAG ACGACGCCAAGCCCCUGCUGAGACUGAAGGGCUUCCCCAGCUUCCCUCUGGUGGAG SEQ ID NO: Engineered X-editor Designation coding sequence AGACAGGCAAAUGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCUGAU CAAUGAGAAGAAGGAGGACGGCAAAGUGUUCUGGCAGAAUCUGGCCGGCUACAAGC GUCAGGAGGCCCUGCGGCCCUACCUGAGCAGCGAGGAAGACAGAAAGAAGGGCAAG AAGUUCGCCCGGUAUCAGCUGGGGGACCUGCUGCUGCACCUCGAGAAGAAGCACGG CGAAGACUGGGGGAAGGUGUACGAUGAGGCCUGGGAGCGGAUCGAUAAGAAGGUGG AGGGCCUGAGCAAGCACAUCAAGCUGGAGGAGGAACGGAGAUCUGAGGACGCCCAG AGCAAGGCCGCCCUGACCGACUGGCUGAGAGCCAAGGCCAGCUUCGUCAUCGAGGG GCUGAAGGAGGCCGACAAGGACGAGUUCUGCCGGUGCGAACUGAAGCUGCAGAAGU GGUACGGAGAUCUGAGAGGCAAACCUUUCGCCAUCGAGGCCGAGAACAGCAUCCUG GACAUCAGCGGCUUCAGCAAGCAGUACAACUGCGCCUUUAUUUGGCAGAAGGACGG AGUGAAGAAGCUGAACCUGUACCUGAUCAUCAACUAUUUCAAGGGCGGCAAGCUGA GAUU CAAGAAGAU CAAGC CU GAAGC CUU C GAGGC CAACAGAUU CUACAC C GU GAUU AACAAGAAAAG C G GAGAGAU C GU G C CAAU G GAAGU GAACUU CAACUU C GAG GAG C C UAACCUGAUCAUCCUGCCCCUGGCAUUUGGCAAGCGGCAGGGCAGAGAGUUCAUCU GGAACGACCUGCUGUCUCUGGAGACCGGCAGCCUGAAGCUGGCCAACGGCAGAGUG AU C GAGAAGACACU GUACAACAGAC GAAC CAGACAAGAC GAGGC CGCCCUGUUUGU GGCCCUGACCUUCGAGAGAAGAGAGGUGCUGGACAGCAGCAAUAUCAAGCCUAUGA ACCUGAUCGGCGUGGACCGGGGCGAGAACAUCCCUGCCGUGAUCGCCCUUACCGAC CCCGAGGGAUGCCCUCUGAGCCGGUUUAAAGACAGCCUGGGCAACCCUACCCACAU CCUGAGAAUUGGCGAGUCCUACAAGGAGAAGCAGAGAACCAUCCAGGCCAAGAAGG AGGUGGAGCAGCGGCGGGCUGGCGGCUACUCCCGGAAGUACGCCAGCAAGGCCAAG AACCUGGCCGACGACAUGGUUAGAAAUACCGCCAGAGACCUCCUGUACUACGCUGU GACCCAGGACGCCAUGCUGAUCUUCGAGAACCUGAGCAGAGGCUUCGGCAGACAGG GCAAGAGAACCUUCAUGGCCGAGAGACAGUACACCCGGAUGGAGGACUGGCUGACC GCCAAGCUGGCCUACGAGGGCCUGCCCUCUAAGACCUACCUGUCCAAGACCUUGGC ACAGUACACCAGCAAGACAUGCUCUAACUGCGGCUUCACAAUCACGAGCGCCGACU ACGACCGGGUGCUGGAGAAACUGAAGAAGACCGCCACAGGCUGGAUGACCACCAUU AACGGCAAGGAGCUGAAGGUGGAGGGCCAGAUCACCUACUACAACAGGUACAAACG GCAGAACGUGGUGAAGGACCUGAGCGUGGAACUGGAUAGACUGAGCGAGGAAAGCG UAAACAAUGACAUCAGCAGCUGGACCAAGGGCCGGAGCGGCGAGGCCCUGAGCCUG CU GAAGAAGAGAUU CU C C CACAGAC CAGU GCAGGAGAAGUU C GU GU GU CU GAACU G CGGCUUCGAGACCCACGCCGACGAGCAAGCCGCCCUGAACAUCGCCCGGUCUUGGC UUUUCCUGCGGAGCCAGGAGUACAAGAAGUACCAGACAAACAAGACCACAGGCAAC ACAGACAAGAGAGCCUUCGUCGAGACCUGGCAGAGCUUCUACAGAAAGAAGCUGAA GGAGGUGUGGAAGCCUGCCGUG 35 676 CAGGAGAUCAAGCGGAUUAAUAAAAUUCGGAGAAGACUGGUGAAGGAUUCUAACAC AAAGAAGGCUGGCAAGACACGGGGCCCUAUGAAGACACUGCUGGUGAGAGUGAUGA CAC C C GAC CU GAGAGAAAGACU GGAAAAC CU GAGAAAGAAG C CU GAGAAUAU C C C C CAGCCCAUCAGCAACACAAGCCGGGCCAACCUGAAUAAGCUGCUGACCGACUACAC CGAAAUGAAGAAGGCCAUCCUGCACGUGUAUUGGGAAGAGUUCCAGAAAGACCCAG UCGGCCUGAUGAGCAGAGUGGCUCAGCCUGCCAGCAAGAAGAUCGAUCAGAACAAG CUGAAGCCCGAAAUGGACGAGAAGGGGAACCUGACAACCGCCGGCUUUGCCUGUAG CCAGUGCGGCCAGCCCCUGUUUGUGUACAAACUGGAACAGGUGAGCGAAAAGGGCA AGGCUUACACGAAUUACUUCGGCAGAUGCAACGUGGCCGAGCACGAGAAGCUGAUC AAGCUGGCCCAGCUGAAGCCUGAGAAGGAUAGCGAUGAGGCAGUGACAUAUUCCCU GGGCAAGUUCGGACAGCGGGCCCUGGAUUUUUAUUCCAUUCAUGUGACCAAGGAAU CCACCCACCCCGUCAAGCCUCUUGCCCAAAUUGCCGGCAACAGAUACGCCUCCAGC CCCGUGGGCAAGGCCCUGAGCGACGCCUGUAUGGGCACCAUCGCCAGCUUCCUGUC UAAGUACCAGGACAUUAUCAUCGAGCACCAGAAGGUGGUGAAGGGCAACCAGAAGA GACUGGAGAGCCUGCGCGAGCUGGCCGGCAAGGAAAACCUGGAGUAUCCUAGCGUG ACCCUGCCUCCUCAGCCUCAUACAAAGGAGGGCGUGGAUGCCUACAACGAAGUGAU CGCCCGGGUGCGGAUGUGGGUGAACCUGAAUCUGUGGCAGAAGCUGAAGCUGUCUA GAGACGACGCCAAGCCCCUGCUGAGACUGAAGGGCUUCCCCAGCUUCCCUCUGGUG GAGAGACAGGCAAAUGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCU SEQ ID NO: Engineered X-editor Designation coding sequence GAUCAAUGAGAAGAAGGAGGACGGCAAAGUGUUCUGGCAGAAUCUGGCCGGCUACA AGCGUCAGGAGGCCCUGCGGCCCUACCUGAGCAGCGAGGAAGACAGAAAGAAGGGC AAGAAGUUCGCCCGGUAUCAGCUGGGGGACCUGCUGCUGCACCUCGAGAAGAAGCA CGGCGAAGACUGGGGGAAGGUGUACGAUGAGGCCUGGGAGCGGAUCGAUAAGAAGG UGGAGGGCCUGAGCAAGCACAUCAAGCUGGAGGAGGAACGGAGAUCUGAGGACGCC CAGAGCAAGGCCGCCCUGACCGACUGGCUGAGAGCCAAGGCCAGCUUCGUCAUCGA GGGGCUGAAGGAGGCCGACAAGGACGAGUUCUGCCGGUGCGAACUGAAGCUGCAGA AGUGGUACGGAGAUCUGAGAGGCAAACCUUUCGCCAUCGAGGCCGAGAACAGCAUC CUGGACAUCAGCGGCUUCAGCAAGCAGUACAACUGCGCCUUUAUUUGGCAGAAGGA CGGAGUGAAGAAGCUGAACCUGUACCUGAUCAUCAACUAUUUCAAGGGCGGCAAGC UGAGAUUCAAGAAGAUCAAGCCUGAAGCCUUCGAGGCCAACAGAUUCUACACCGUG AUUAACAAGAAAAGC GGAGAGAU C GU GC CAAU GGAAGU GAACUU CAACUU C GAG GA CCCUAACCUGAUCAUCCUGCCCCUGGCAUUUGGCAAGCGGCAGGGCAGAGAGUUCA UCUGGAACGACCUGCUGUCUCUGGAGACCGGCAGCCUGAAGCUGGCCAACGGCAGA GU GAU C GAGAAGACACU GUACAACAGAC GAAC CAGACAAGAC GAGC C CGCCCUGUU UGUGGCCCUGACCUUCGAGAGAAGAGAGGUGCUGGACAGCAGCAAUAUCAAGCCUA UGAACCUGAUCGGCGUGGACCGGGGCGAGAACAUCCCUGCCGUGAUCGCCCUUACC GACCCCGAGGGAUGCCCUCUGAGCCGGUUUAAAGACAGCCUGGGCAACCCUACCCA CAUCCUGAGAAUUGGCGAGUCCUACAAGGAGAAGCAGAGAACCAUCCAGGCCAAGA AGGAGGUGGAGCAGCGGCGGGCUGGCGGCUACUCCCGGAAGUACGCCAGCAAGGCC AAGAACCUGGCCGACGACAUGGUUAGAAAUACCGCCAGAGACCUCCUGUACUACGC UGUGACCCAGGACGCCAUGCUGAUCUUCGAGAACCUGAGCAGAGGCUUCGGCAGAC AGGGCAAGAGAACCUUCAUGGCCGAGAGACAGUACACCCGGAUGGAGGACUGGCUG ACCGCCAAGCUGGCCUACGAGGGCCUGCCCUCUAAGACCUACCUGUCCAAGACCUU GGCACAGUACACCAGCAAGACAUGCUCUAACUGCGGCUUCACAAUCACGAGCGCCG ACUACGACCGGGUGCUGGAGAAACUGAAGAAGACCGCCACAGGCUGGAUGACCACC AUUAACGGCAAGGAGCUGAAGGUGGAGGGCCAGAUCACCUACUACAACAGGUACAA ACGGCAGAACGUGGUGAAGGACCUGAGCGUGGAACUGGAUAGACUGAGCGAGGAAA GCGUAAACAAUGACAUCAGCAGCUGGACCAAGGGCCGGAGCGGCGAGGCCCUGAGC GU GCU GAAGAAGAGAUU GU C C CACAGAC CAGU GCAGGAGAAGUU C GU GU GU GU GAA CUGCGGCUUCGAGACCCACGCCGACGAGCAAGCCGCCCUGAACAUCGCCCGGUCUU GGCUUUUCCUGCGGAGCCAGGAGUACAAGAAGUACCAGACAAACAAGACCACAGGC AACACAGACAAGAGAGCCUUCGUCGAGACCUGGCAGAGCUUCUACAGAAAGAAGCU GAAGGAGGUGUGGAAGCCUGCCGUG 36 812 CAGGAGAUCAAGCGGAUUAAUAAAAUUCGGAGAAGACUGGUGAAGGAUUCUAACAC AAAGAAGGCUGGCAAGACAGGCCCUAUGAAGACACUGCUGGUGAGAGUGAUGACAC CC GAG GU GAGAGAAAGACU GGAAAAC GU GAGAAAGAAGC GU GAGAAUAU C C C C GAG CCCAUCAGCAACACAAGCCGGGCCAACCUGAAUAAGCUGCUGACCGACUACACCGA AAUGAAGAAGGCCAUCCUGCACGUGUAUUGGGAAGAGUUCCAGAAAGACCCAGUCG GCCUGAUGAGCAGAGUGGCUCAGCCUGCCAGCAAGAAGAUCGAUCAGAACAAGCUG AAGCCCGAAAUGGACGAGAAGGGGAACCUGACAACCGCCGGCUUUGCCUGUAGCCA GUGCGGCCAGCCCCUGUUUGUGUACAAACUGGAACAGGUGAGCGAAAAGGGCAAGG CUUACACGAAUUACUUCGGCAGAUGCAACGUGGCCGAGCACGAGAAGCUGAUCCUG CUGGCCCAGCUGAAGCCUGAGAAGGAUAGCGAUGAGGCAGUGACAUAUUCCCUGGG CAAGUUCGGACAGCGGGCCCUGGAUUUUUAUUCCAUUCAUGUGACCAAGGAAUCCA CCCACCCCGUCAAGCCUCUUGCCCAAAUUGCCGGCAACAGAUACGCCAGCGGCCCC GUGGGCAAGGCCCUGAGCGACGCCUGUAUGGGCACCAUCGCCAGCUUCCUGUCUAA GUACCAGGACAUUAUCAUCGAGCACCAGAAGGUGGUGAAGGGCAACCAGAAGAGAC UGGAGAGCCUGCGCGAGCUGGCCGGCAAGGAAAACCUGGAGUAUCCUAGCGUGACC CUGCCUCCUCAGCCUCAUACAAAGGAGGGCGUGGAUGCCUACAACGAAGUGAUCGC CCGGGUGCGGAUGUGGGUGAACCUGAAUCUGUGGCAGAAGCUGAAGCUGUCUAGAG ACGACGCCAAGCCCCUGCUGAGACUGAAGAAGUUCCCCAGCUUCCCUCUGGUGGAG AGACAGGCAAAUGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCUGAU CAAUGAGAAGAAGGAGGACGGCAAAGUGUUCUGGCAGAAUCUGGCCGGCUACAAGC SEQ ID NO: Engineered X-editor Designation coding sequence GUCAGGAGGCCCUGCGGCCCUACCUGAGCAGCGAGGAAGACAGAAAGAAGGGCAAG AAGUUCGCCCGGUAUCAGCUGGGGGACCUGCUGCUGCACCUCGAGAAGAAGCACGG CGAAGACUGGGGGAAGGUGUACGAUGAGGCCUGGGAGCGGAUCGAUAAGAAGGUGG AGGGCCUGAGCAAGCACAUCAAGCUGGAGGAGGAACGGAGAUCUGAGGACGCCCAG AGCAAGGCCGCCCUGACCGACUGGCUGAGAGCCAAGGCCAGCUUCGUCAUCGAGGG GCUGAAGGAGGCCGACAAGGACGAGUUCUGCCGGUGCGAACUGAAGCUGCAGAAGU GGUACGGAGAUCUGAGAGGCAAACCUUUCGCCAUCGAGGCCGAGAACAGCAUCCUG GACAUCAGCGGCUUCAGCAAGCAGUACAACUGCGCCUUUAUUUGGCAGAAGGACGG AGUGAAGAAGCUGAACCUGUACCUGAUCAUCAACUAUUUCAAGGGCGGCAAGCUGA GAUU CAAGAAGAU CAAGC CU GAAGC CUU C GAGGC CAACAGAUU CUACAC C GU GAUU AACAAGAAAAG C G GAGAGAU C GU G C CAAU G GAAGU GAACUU CAACUU C GAG GAG C C UAACCUGAUCAUCCUGCCCCUGGCAUUUGGCAAGCGGCAGGGCAGAGAGUUCAUCU GGAACGACCUGCUGUCUCUGGAGACCGGCAGCCUGAAGCUGGCCAACGGCAGAGUG AU C GAGAAGACACU GUACAACAGAC GAAC CAGACAAGAC GAGGC CGCCCUGUUUGU GGCCCUGACCUUCGAGAGAAGAGAGGUGCUGGACAGCAGCAAUAUCAAGCCUAUGA ACCUGAUCGGCGUGGACCGGGGCGAGAACAUCCCUGCCGUGAUCGCCCUUACCGAC CCCGAGGGAUGCCCUCUGAGCCGGUUUAAAGACAGCCUGGGCAACCCUACCCACAU CCUGAGAAUUGGCGAGUCCUACAAGGAGAAGCAGAGAACCAUCCAGGCCAAGAAGG AGGUGGAGCAGCGGCGGGCUGGCGGCUACUCCCGGAAGUACGCCAGCAAGGCCAAG AACCUGGCCGACGACAUGGUUAGAAAUACCGCCAGAGACCUCCUGUACUACGCUGU GACCCAGGACGCCAUGCUGAUCUUCGAGAACCUGAGCAGAGGCUUCGGCAGACAGG GCAAGAGAACCUUCAUGGCCGAGAGACAGUACACCCGGAUGGAGGACUGGCUGACC GCCAAGCUGGCCUACGAGGGCCUGCCCUCUAAGACCUACCUGUCCAAGACCUUGGC ACAGUACACCAGCAAGACAUGCUCUAACUGCGGCUUCACAAUCACGAGCGCCGACU ACGACCGGGUGCUGGAGAAACUGAAGAAGACCGCCACAGGCUGGAUGACCACCAUU AACGGCAAGGAGCUGAAGGUGGAGGGCCAGAUCACCUACUACAACAGGUACAAACG GCAGAACGUGGUGAAGGACCUGAGCGUGGAACUGGAUAGACUGAGCGAGGAAAGCG UAAACAAUGACAUCAGCAGCUGGACCAAGGGCCGGAGCGGCGAGGCCCUGAGCCUG CU GAAGAAGAGAUU CU C C CACAGAC CAGU GCAGGAGAAGUU C GU GU GU CU GAACU G CGGCUUCGAGACCCACGCCGACGAGCAAGCCGCCCUGAACAUCGCCCGGUCUUGGC UUUUCCUGCGGAGCCAGGAGUACAAGAAGUACCAGACAAACAAGACCACAGGCAAC ACAGACAAGAGAGCCUUCGUCGAGACCUGGCAGAGCUUCUACAGAAAGAAGCUGAA GGAGGUGUGGAAGCCUGCCGUG 39 Nl-methylpseudo uridine substituted RNA sequence encoding X-editor491 CAGGAGAmWCAAGCGGAmWmWAAmWAAAAmWmWCGGAGAAGACmWGGmWGAAGGAm WmWCmWAACACAAAGAAGGCmWGGCAAGACAGGCCCmWAmWGAAGACACmWGCmWG GmWGAGAGmWGAmWGACAC C C GAC CmWGAGAGAAAGACmWG GAAAAC CmWGAGAAA GAAGCCmWGAGAAmWAmWCCCCCAGCCCAmWCAGCAACACAAGCCGGGCCAACCmW GAAmWAAGCmWGCmWGACCGACmWACACCGAAAmWGAAGAAGGCCAmWCCmWGCAC GmWGmWAmWWGGGAAGAGmWmWC CAGAAAGAC C CAGmWC G GC CmWGAmWGAGCAG AGmWGGCmWCAGCCmWGCCAGCAAGAAGAmWCGAmWCAGAACAAGCmWGAAGCCCG AAAmWGGACGAGAAGGGGAACCmWGACAACCGCCGGCmWmWmWGCCmWGmWAGCCA GmWGCGGCCAGCCCCmWGmWmWmWGmWGmWACAAACmWGGAACAGGmWGAGCGAAA AGGGCAAGGCmWmWACACGAAmWmWACmWmWCGGCAGAmWGCAACGmWGGCCGAGC ACGAGAAGCmWGAmWCCmWGCmWGGCCCAGCmWGAAGCCmWGAGAAGGAmWAGCGA mWGAGGCAGmWGACAmWAmWmWCCCmWGGGCAAGmWmWCGGACAGCGGGCCCmWGG AmWWmWAmTmTCCAm^^^ CAAGGAAmWC CAC C CAC C C C GmW CAAGCCmWCmWmWGCCCAAAmWmWGCCGGCAACAGAmWACGCCAGCGGCCCCGmWG GGCAAGGCCCmWGAGCGACGCCmWGmWAmWGGGCACCAmWCGCCAGCmWmWCCmWG mWCmWAAGmWACCAGGACAmWmWAmWCAmWCGAGCACCAGAAGGmWGGmWGAAGGG CAACCAGAAGAGACmWGGAGAGCCmWGCGCGAGCmWGGCCGGCAAGGAAAACCmWG GAGmWAmWCCmWAGCGmWGACCCmWGCCmWCCmWCAGCCmWCAmWACAAAGGAGGG CGmWGGAmWGCCmWACAACGAAGmWGAmWCGCCCGGGmWGCGGAmWGmWGGGmWGA ACCmWGAAmWCmWGmWGGCAGAAGCmWGAAGCmWGmWCmWAGAGACGACGCCAAGC CCCmWGCmWGAGACmWGAAGGGCmWmWCCCCAGCmWmWCCCmWCmWGGmWGGAGAG SEQ ID NO: Engineered X-editor Designation coding sequence ACAGGCAAAmWGAAGmWGGACmWGGmWGGGACAmWGGmWGmWGmWAACGmWGAAGA AGCmWGAmWCAAmWGAGAAGAAGGAGGACGGCAAAGmWGmWmWCmWGGCAGAAmWC mWGGCCGGCmWACAAGCGmWCAGGAGGCCCmWGCGGCCCmWACCmWGAGCAGCGAG GAAGACAGAAAGAAGGGCAAGAAGmWmWCGCCCGGmWAmWCAGCmWGGGGGACCmW GCmWGCmWGCACCmWCGAGAAGAAGCACGGCGAAGACmWGGGGGAAGGmWGmWACG AmWGAGGCCmWGGGAGCGGAmWCGAmWAAGAAGGmWGGAGGGCCmWGAGCAAGCAC AmYCAAGCmYGGAGGAGGAACGGAGAmYCmYGAGGACGCCCAGAGCAAGGCCGCCC mWGACCGACmWGGCmWGAGAGCCAAGGCCAGCmWmWCGmWCAmWCGAGGGGCmWGA AGGAGGCCGACAAGGACGAGmWmWCmWGCCGGmWGCGAACmWGAAGCmWGCAGAAG mWGGmWACGGAGAmWCmWGAGAGGCAAACCmWmWmWCGCCAmWCGAGGCCGAGAAC AGCAmWCCmWGGACAmWCAGCGGCmWmWCAGCAAGCAGmWACAACmWGCGCCmWmW mWAmWmWmWGGCAGAAGGACGGAGmWGAAGAAGCmWGAACCmWGmWACCmWGAmWC AmWCAACmWAmWmWmWCAAGGGCGGCAAGCmWGAGAmWmWCAAGAAGAmWCAAGCC mWGAAGCCmWmWCGAGGCCAACAGAmWmWCmWACACCGmWGAmWmWAACAAGAAAA GC GGAGAGAmWC GmWGC CAAmWGGAAGmWGAACmWmWCAACmWmWC GAG GAG C CmW AACCmWGAmWCAmWCCmWGCCCCmWGGCAmWmWmWGGCAAGCGGCAGGGCAGAGAG mWmWCAmWCmWGGAACGACCmWGCmWGmWCmWCmWGGAGACCGGCAGCCmWGAAGC mWG G C CAAC G G CAGAGmWGAmWC GAGAAGACACmWGmWACAACAGAC GAAC CAGAC AAGACGAGCCCGCCCmWGmWmWmWGmWGGCCCmWGACCmWmWCGAGAGAAGAGAGG mWGCmWGGACAGCAGCAAmWAmWCAAGCCmWAmWGAACCmWGAmWCGGCGmWGGAC CGGGGCGAGAACAmWCCCmWGCCGmWGAmWCGCCCmWmWACCGACCCCGAGGGAmW GCCCmWCmWGAGCCGGmWmWmWAAAGACAGCCmWGGGCAACCCmWACCCACAmWCC mWGAGAAmWmWGGCGAGmWCCmWACAAGGAGAAGCAGAGAACCAmWCCAGGCCAAG AAGGAGGmWGGAGCAGCGGCGGGCmWGGCGGCmWACmWCCCGGAAGmWACGCCAGC AAGGCCAAGAACCmWGGCCGACGACAmWGGmWmWAGAAAmWACCGCCAGAGACCmW CCmWGmWACmWACGCmWGmWGACCCAGGACGCCAmWGCmWGAmWCmWmWCGAGAAC CmWGAGCAGAGGCmWmWCGGCAGACAGGGCAAGAGAACCm¥mWCAmWGGCCGAGAG ACAGmWACACCCGGAmWGGAGGACmWGGCmWGACCGCCAAGCmWGGCCmWACGAGG GC CmWGmWCmWAAGAC CmWAC CmWGmWC CAAGAC CmWmWGG CACAGmWACAC CAGC AAGACAmWGCmWCmWAACmWGCGGCmWmWCACAAmWCACGAGCGCCGACmWACGAC CGGGmWGCmWGGAGAAACmWGAAGAAGACCGCCACAGGCmWGGAmWGACCACCAmW mWAACGGCAAGGAGCmWGAAGGmWGGAGGGCCAGAmWCACCmWACmWACAACAGGm WACAAACGGCAGAACGmWGGmWGAAGGACCmWGAGCGmWGGAACmWGGAmWAGACm WGAGCGAGGAAAGCGmWAAACAAmWGACAmWCAGCAGCmWGGACCAAGGGCCGGAG CGGCGAGGCCCmWGAGCCmWGCmWGAAGAAGAGAmWmWCmWCCCACAGACCAGmWG CAGGAGAAGmWmWCGmWGmWGmWCmWGAACmWGCGGCmWmWCGAGACCCACGCCGA CGAGCAAGCCGCCCmWGAACAmWCGCCCGGmWCmWmWGGCmWmWmWmWCCmWGCGG AGCCAGGAGmWACAAGAAGmWACCAGACAAACAAGACCACAGGCAACACAGACAAG AGAGCCmWmWCGmWCGAGACCmWGGCAGAGCmWmWCmWACAGAAAGAAGCmWGAAG GAGGmWGmWGGAAGCCmWGCCGmWG 40 Nl-methylpseudo uridine substituted RNA sequence encoding X-editor 515 CAGGAGAmWCAAGCGGAmWmWAAmWAAAAmWmWCGGAGAAGACmWGGmWGAAGGAm WmWCmWAACACAAAGAAGGCmWGGCAAGACAGGCCCmWAmWGAAGACACmWGCmWG GmWGAGAGmWGAmWGACAC C C GAG CmWGAGAGAAAGACmWG GAAAAC CmWGAGAAA GAAGCCmWGAGAAmWAmWCCCCCAGCCCAmWCAGCAACACAAGCCGGGCCAACCmW GAAmWAAGCmWGCmWGACCGACmWACACCGAAAmWGAAGAAGGCCAmWCCmWGCAC GmWGmWAmWWGGGAAGAGmWmWC CAGAAAGAC C CAGmWC G GC CmWGAmWGAGCAG AGmWGGCmWCAGCCmWGCCAGCAAGAAGAmWCGAmWCAGAACAAGCmWGAAGCCCG AAAmWGGACGAGAAGGGGAACCmWGACAACCGCCGGCmWmWmWGCCmWGmWAGCCA GmWGCGGCCAGCCCCmWGmWmWmWGmWGmWACAAACmWGGAACAGGmWGAGCGAAA AGGGCAAGGCmWmWACACGAAmWmWACmWmWCGGCAGAmWGCAACGmWGGCCGAGC ACGAGAAGCmWGAmWCCmWGCmWGGCCCAGCmWGAAGCCmWGAGAAGGAmWAGCGA mWGAGGCAGmWGACAmWAmWmWCCCmWGGGCAAGmWmWCGGACAGCGGGCCCmWGG AmWWmWAmTmTCCAm^^^ CAAGGAAmWC GAG C GAG C C C GmW CAAGCCmWCmWmWGCCCAAAmWmWGCCGGCAACAGAmWACGCCAGCGGCCCCGmWG SEQ ID NO: Engineered X-editor Designation coding sequence GGCAAGGCCCmWGAGCGACGCCmWGmWAmWGGGCACCAmWCGCCAGCmWmWCCmWG mWCmWAAGmWACCAGGACAmWmWAmWCAmWCGAGCACCAGAAGGmWGGmWGAAGGG CAACCAGAAGAGACmWGGAGAGCCmWGCGCGAGCmWGGCCGGCAAGGAAAACCmWG GAGmWAmWCCmWAGCGmWGACCCmWGCCmWCCmWCAGCCmWCAmWACAAAGGAGGG CGmWGGAmWGCCmWACAACGAAGmWGAmWCGCCCGGGmWGCGGAmWGmWGGGmWGA ACCmWGAAmWCmWGmWGGCAGAAGCmWGAAGCmWGmWCmWAGAGACGACGCCAAGC CCCmYGCmYGAGACmYGAAGGGCmYmYCCCCAGCmYmYCCCmYCmYGGmYGGAGAG ACAGGCAAAmWGAAGmWGGACmWGGmWGGGACAmWGGmWGmWGmWAACGmWGAAGA AGCmWGAmWCAAmWGAGAAGAAGGAGGACGGCAAAGmWGmWmWCmWGGCAGAAmWC mWGGCCGGCmWACAAGCGmWCAGGAGGCCCmWGCGGCCCmWACCmWGAGCAGCGAG GAAGACAGAAAGAAGGGCAAGAAGmWmWCGCCCGGmWAmWCAGCmWGGGGGACCmW GCmWGCmWGCACCmWCGAGAAGAAGCACGGCGAAGACmWGGGGGAAGGmWGmWACG AmWGAGGCCmWGGGAGCGGAmWCGAmWAAGAAGGmWGGAGGGCCmWGAGCAAGCAC AmWCAAGCmWGGAGGAGGAACGGAGAmWCmWGAGGACGCCCAGAGCAAGGCCGCCC mWGACCGACmWGGCmWGAGAGCCAAGGCCAGCmWmWCGmWCAmWCGAGGGGCmWGA AGGAGGCCGACAAGGACGAGmWmWCmWGCCGGmWGCGAACmWGAAGCmWGCAGAAG mWGGmWACGGAGAmWCmWGAGAGGCAAACCmWmWmWCGCCAmWCGAGGCCGAGAAC AGCAmWCCmWGGACAmWCAGCGGCmWmWCAGCAAGCAGmWACAACmWGCGCCmWmW mWAmWmWmWGGCAGAAGGACGGAGmWGAAGAAGCmWGAACCmWGmWACCmWGAmWC AmWCAACmWAmWmWmWCAAGGGCGGCAAGCmWGAGAmWmWCAAGAAGAmWCAAGCC mWGAAGC CmWmWC GAGGC CAACAGAmWmWCmWACAC C GmWGAmWmWAACAAGAAAA GC GGAGAGAmWC GmWGC CAAmWGGAAGmWGAACmWmWCAACmWmWC GAG GAG C CmW AACCmWGAmWCAmWCCmWGCCCCmWGGCAmWmWmWGGCAAGCGGCAGGGCAGAGAG mWmWCAmWCmWGGAACGACCmWGCmWGmWCmWCmWGGAGACCGGCAGCCmWGAAGC mWG G C CAAC G G CAGAGmWGAmWC GAGAAGACACmWGmWACAACAGAC GAAC CAGAC AAGACGAGCCCGCCCmWGmWmWmWGmWGGCCCmWGACCmWmWCGAGAGAAGAGAGG mWGCmWGGACAGCAGCAAmWAmWCAAGCCmWAmWGAACCmWGAmWCGGCGmWGGAC CGGGGCGAGAACAmWCCCmWGCCGmWGAmWCGCCCmWmWACCGACCCCGAGGGAmW GCCCmWCmWGAGCCGGmWmWmWAAAGACAGCCmWGGGCAACCCmWACCCACAmWCC mWGAGAAmWmWGGCGAGmWCCmWACAAGGAGAAGCAGAGAACCAmWCCAGGCCAAG AAGGAGGmWGGAGCAGCGGCGGGCmWGGCGGCmWACmWCCCGGAAGmWACGCCAGC AAGGCCAAGAACCmWGGCCGACGACAmWGGmWmWAGAAAm¥ACCGCCAGAGACCmW CCmWGmWACmWACGCmWGmWGACCCAGGACGCCAmWGCmWGAmWCmWmWCGAGAAC CmWGAGCAGAGGCmWmWCGGCAGACAGGGCAAGAGAACCmWmWCAmWGGCCGAGAG ACAGmWACACCCGGAmWGGAGGACmWGGCmWGACCGCCAAGCmWGGCCmWACGAGG GC CmWGC C CmWCmWAAGAC CmWAC CmWGmWC CAAGAC CmWmWGGCACAGmWACAC C AGCAAGACAmWGCmWCmWAACmWGCGGCmWmWCACAAmWCACGAGCGCCGACmWAC GACCGGGmWGCmWGGAGAAACmWGAAGAAGACCGCCACAGGCmWGGAmWGACCACC AmWmWAACGGCAAGGAGCmWGAAGGmWGGAGGGCCAGAmWCACCmWACmWACAACA GGmWACAAACGGCAGAACGmWGGmWGAAGGACCmWGAGCGmWGGAACmWGGAmWAG ACmWGAGCGAGGAAAGCGmWAAACAAmWGACAmWCAGCAGCmWGGACCAAGGGCCG GAGCGGCGAGGCCCmWGAGCCmWGCmWGAAGAAGAGAmWmWCmWCCCACAGACCAG mWGCAGGAGAAGmWmWCGmWGmWGmWCmWGAACmWGCGGCmWmWCGAGACCCACGC CGACGAGCAAGCCGCCCmWGAACAmWCGCCCGGmWCmWmWGGCmWmWmWmWCCmWG CGGAGCCAGGAGmWACAAGAAGmWACCAGACAAACAAGACCACAGGCAACACAGAC AAGAGAGCCmWmWCGmWCGAGACCmWGGCAGAGCmWmWCmWACAGAAAGAAGCmWG AAGGAGGmWGmWGGAAGCCmWGCCGmWG 41 Nl-methylpseudo uridine substituted RNA sequence CAGGAGAmWCAAGCGGAmWmWAAmWAAAAmWmWCGGAGAAGACmWGGmWGAAGGAm WmWCmWAACACAAAGAAGGCmWGGCAAGACACGGGGCCCmWAmWGAAGACACmWGC mWGGmWGAGAGmWGAmWGACAC C C GAG CmWGAGAGAAAGACmWGGAAAAC CmWGAG AAAGAAGCCmWGAGAAmWAmWCCCCCAGCCCAmWCAGCAACACAAGCCGGGCCAAC CmWGAAmWAAGCmWGCmWGAC C GACmWACAC C GAAAmWGAAGAAGGC CAmWC CmWG GAG GmWGmWAmWWGGGAAGAGmWmWC CAGAAAGAC C CAGmWC GGC CmWGAmWGAG CAGAGmWGGCmWCAGCCmWGCCAGCAAGAAGAmWCGAmWCAGAACAAGCmWGAAGC SEQ ID NO: Engineered X-editor Designation coding sequence encoding X-editor 676 CCGAAAmWGGACGAGAAGGGGAACCmWGACAACCGCCGGCmWmWmWGCCmWGmWAG CCAGmWGCGGCCAGCCCCmWGmWmWmWGmWGmWACAAACmWGGAACAGGmWGAGCG AAAAGGGCAAGGCmWmWACACGAAmWmWACmWmWCGGCAGAmWGCAACGmWGGCCG AGCACGAGAAGCmWGAmWCAAGCmWGGCCCAGCmWGAAGCCmWGAGAAGGAmWAGC GAmWGAGGCAGmWGACAmWAmWmWCCCmWGGGCAAGmWmWCGGACAGCGGGCCCmW GGAmWmWmVAmWC CAmWmWCAmWGmWGAC CAAGGAAmWCCAC C GAG C C C G mYCAAGCCmYCmYmYGCCCAAAmYmYGCCGGCAACAGAmYACGCCmYCCAGCCCCG mWGGGCAAGGCCCmWGAGCGACGCCmWGmWAmWGGGCACCAmWCGCCAGCmWmWCC mWGmWCmWAAGmWACCAGGACAmWmWAmWCAmWCGAGCACCAGAAGGmWGGmWGAA GGGCAACCAGAAGAGACmWGGAGAGCCmWGCGCGAGCmWGGCCGGCAAGGAAAACC mWGGAGmWAmWCCmWAGCGmWGACCCmWGCCmWCCmWCAGCCmWCAmWACAAAGGA GGGCGmWGGAmWGCCmWACAACGAAGmWGAmWCGCCCGGGmWGCGGAmWGmWGGGm WGAACCmWGAAmWCmWGmWGGCAGAAGCmWGAAGCmWGmWCmWAGAGACGACGCCA AGCCCCmWGCmWGAGACmWGAAGGGCmWmWCCCCAGCmWmWCCCmWCmWGGmWGGA GAGACAGGCAAAmWGAAGmWGGACmWGGmWGGGACAmWGGmWGmWGmWAACGmWGA AGAAGCmWGAmWCAAmWGAGAAGAAGGAGGACGGCAAAGmWGmWmWCmWGGCAGAA mWCmWGGCCGGCmWACAAGCGmWCAGGAGGCCCmWGCGGCCCmWACCmWGAGCAGC GAGGAAGACAGAAAGAAGGGCAAGAAGmWmWCGCCCGGmWAmWCAGCmWGGGGGAC CmWGCmWGCmWGCACCmWCGAGAAGAAGCACGGCGAAGACmWGGGGGAAGGmWGmW ACGAmWGAGGCCmWGGGAGCGGAmWCGAmWAAGAAGGmWGGAGGGCCmWGAGCAAG CACAmWCAAGCmWGGAGGAGGAACGGAGAmWCmWGAGGACGCCCAGAGCAAGGCCG CCCmWGACCGACmWGGCmWGAGAGCCAAGGCCAGCmWmWCGmWCAmWCGAGGGGCm WGAAGGAGGCCGACAAGGACGAGmWmWCmWGCCGGmWGCGAACmWGAAGCmWGCAG AAGmWGGmWACGGAGAmWCmWGAGAGGCAAACCmWmWmWCGCCAmWCGAGGCCGAG AACAGCAmWCCmWGGACAmWCAGCGGCmWmWCAGCAAGCAGmWACAACmWGCGCCm WmWmWAmWmWmWGGCAGAAGGACGGAGmWGAAGAAGCmWGAACCmWGmWACCmWGA mWCAmWCAACmWAmWmWmWCAAGGGCGGCAAGCmWGAGAmWmWCAAGAAGAmWCAA G C CmWGAAG C CmWmWC GAG G C CAACAGAmWmWCmWACAC C GmWGAmWmWAACAAGA AAAGC GGAGAGAmWC GmWGC CAAmWGGAAGmWGAACmWWCAACmWmWC GAC GAC C CmWAACCmWGAmWCAmWCCmWGCCCCmWGGCAmWmWmWGGCAAGCGGCAGGGCAGA GAGmWmWCAmWCmWGGAACGACCmWGCmWGmWCmWCmWGGAGACCGGCAGCCmWGA AGCmWGGCCAACGGCAGAGmWGAmWCGAGAAGACACmWGmWACAACAGACGAACCA GACAAGAC GAGC C C GC C CmWGmWmWmWGmWGGC C CmWGAC CmWmWC GAGAGAAGAG AGGmWGCmWGGACAGCAGCAAmWAmWCAAGCCmWAmWGAACCmWGAmWCGGCGmWG GACCGGGGCGAGAACAmWCCCmWGCCGmWGAmWCGCCCmWmWACCGACCCCGAGGG AmWGCCCmWCmWGAGCCGGmWmWmWAAAGACAGCCmWGGGCAACCCmWACCCACAm WCCmWGAGAAmWmWGGCGAGmWCCmWACAAGGAGAAGCAGAGAACCAmWCCAGGCC AAGAAGGAGGmWGGAGCAGCGGCGGGCmWGGCGGCmWACmWCCCGGAAGmWACGCC AGCAAGGCCAAGAACCmWGGCCGACGACAmWGGmWmWAGAAAmWACCGCCAGAGAC CmWC CmWGmWACmWAC GCmWGmWGAC C CAGGAC GC CAmWGCmWGAmWCmWmWC GAG AACCmWGAGCAGAGGCmWmWCGGCAGACAGGGCAAGAGAACCmWmWCAmWGGCCGA GAGACAGmWACACCCGGAmWGGAGGACmWGGCmWGACCGCCAAGCmWGGCCmWACG AGGGCCmWGCCCmWCmWAAGACCmWACCmWGmWCCAAGACCmWmWGGCACAGmWAC ACCAGCAAGACAmWGCmWCmWAACmWGCGGCmWmWCACAAmWCACGAGCGCCGACm WACGACCGGGmWGCmWGGAGAAACmWGAAGAAGACCGCCACAGGCmWGGAmWGACC ACCAmWmWAACGGCAAGGAGCmWGAAGGmWGGAGGGCCAGAmWCACCmWACmWACA ACAGGmWACAAACGGCAGAACGmWGGmWGAAGGACCmWGAGCGmWGGAACmWGGAm WAGACmWGAGCGAGGAAAGCGmWAAACAAmWGACAmWCAGCAGCmWGGACCAAGGG CCGGAGCGGCGAGGCCCmWGAGCCmWGCmWGAAGAAGAGAmWmWCmWCCCACAGAC CAGmWGCAGGAGAAGmWmWC GmWGmWGmWCmWGAACmWGC G GCmWmWC GAGAC C CA CGCCGACGAGCAAGCCGCCCmWGAACAmWCGCCCGGmWCm¥mWGGCmWmWmWmWCC mWGCGGAGCCAGGAGmWACAAGAAGmWACCAGACAAACAAGACCACAGGCAACACA GACAAGAGAGCCmWmWCGmWCGAGACCmWGGCAGAGCmWmWCmWACAGAAAGAAGC mWGAAGGAGGmWGmWGGAAGCCmWGCCGmWG SEQ ID NO: Engineered X-editor Designation coding sequence 42 Nl-methylpseudo uridine substituted RNA sequence encoding X-editor 812 CAGGAGAmWCAAGCGGAmWmWAAmWAAAAmWmWCGGAGAAGACmWGGmWGAAGGAm. WmWCmWAACACAAAGAAGGCmWGGCAAGACAGGCCCmWAmWGAAGACACmWGCmWG GmWGAGAGmWGAmWGACAC C C GAG CmWGAGAGAAAGACmWG GAAAAC CmWGAGAAA GAAGCCmWGAGAAmWAmWCCCCCAGCCCAmWCAGCAACACAAGCCGGGCCAACCmW GAAmWAAGCmWGCmWGACCGACmWACACCGAAAmWGAAGAAGGCCAmWCCmWGCAC GmWGmWAmWWGGGAAGAGmWmWC CAGAAAGAC C CAGmWC G GC CmWGAmWGAGCAG AGmYGGCmYCAGCCmYGCCAGCAAGAAGAmYCGAmYCAGAACAAGCmYGAAGCCCG AAAmWGGACGAGAAGGGGAACCmWGACAACCGCCGGCmWmWmWGCCmWGmWAGCCA GmWGCGGCCAGCCCCmWGmWmWmWGmWGmWACAAACmWGGAACAGGmWGAGCGAAA AGGGCAAGGCmWmWACACGAAmWmWACmWmWCGGCAGAmWGCAACGmWGGCCGAGC ACGAGAAGCmWGAmWCCmWGCmWGGCCCAGCmWGAAGCCmWGAGAAGGAmWAGCGA mWGAGGCAGmWGACAmWAmWmWCCCmWGGGCAAGmWmWCGGACAGCGGGCCCmWGG AmWWmWAmTmTCCAm^^^ CAAGGAAmWC GAG C GAG C C C GmW CAAGCCmWCmWmWGCCCAAAmWmWGCCGGCAACAGAmWACGCCAGCGGCCCCGmWG GGCAAGGCCCmWGAGCGACGCCmWGmWAmWGGGCACCAmWCGCCAGCmWmWCCmWG mWCmWAAGmWACCAGGACAmWmWAmWCAmWCGAGCACCAGAAGGmWGGmWGAAGGG CAACCAGAAGAGACmWGGAGAGCCmWGCGCGAGCmWGGCCGGCAAGGAAAACCmWG GAGmWAmWCCmWAGCGmWGACCCmWGCCmWCCmWCAGCCmWCAmWACAAAGGAGGG CGmWGGAmWGCCmWACAACGAAGmWGAmWCGCCCGGGmWGCGGAmWGmWGGGmWGA ACCmWGAAmWCmWGmWGGCAGAAGCmWGAAGCmWGmWCmWAGAGACGACGCCAAGC C C CmWGCmWGAGACmWGAAGAAGmWmWC C C CAGCmWmWC C CmWCmWGGmWGGAGAG ACAGGCAAAmWGAAGmWGGACmWGGmWGGGACAmWGGmWGmWGmWAACGmWGAAGA AGCmWGAmWCAAmWGAGAAGAAGGAGGACGGCAAAGmWGmWmWCmWGGCAGAAmWC mWGGCCGGCmWACAAGCGmWCAGGAGGCCCmWGCGGCCCmWACCmWGAGCAGCGAG GAAGACAGAAAGAAGGGCAAGAAGmWmWCGCCCGGmWAmWCAGCmWGGGGGACCmW GCmWGCmWGCACCmWCGAGAAGAAGCACGGCGAAGACmWGGGGGAAGGmWGmWACG AmWGAGGCCmWGGGAGCGGAmWCGAmWAAGAAGGmWGGAGGGCCmWGAGCAAGCAC AmWCAAGCmWGGAGGAGGAACGGAGAmWCmWGAGGACGCCCAGAGCAAGGCCGCCC mWGACCGACmWGGCmWGAGAGCCAAGGCCAGCmWmWCGmWCAmWCGAGGGGCmWGA AGGAGGCCGACAAGGACGAGmWmWCmWGCCGGmWGCGAACmWGAAGCmWGCAGAAG mWGGmWACGGAGAmWCmWGAGAGGCAAACCmWmWmWCGCCAmWCGAGGCCGAGAAC AGCAmWCCmWGGACAmWCAGCGGCmWmWCAGCAAGCAGmWACAACmWGCGCCmWmW mWAmWmWmWGGCAGAAGGACGGAGmWGAAGAAGCmWGAACCmWGmWACCmWGAmWC AmWCAACmWAmWmWmWCAAGGGCGGCAAGCmWGAGAmWmWCAAGAAGAmWCAAGCC mWGAAGC CmWmWC GAGGC CAACAGAmWmWCmWACAC C GmWGAmWmWAACAAGAAAA GC GGAGAGAmWC GmWGC CAAmWGGAAGmWGAACmWmWCAACmWmWC GAG GAG C CmW AACCmWGAmWCAmWCCmWGCCCCmWGGCAmWmWmWGGCAAGCGGCAGGGCAGAGAG mWmWCAmWCmWGGAACGACCmWGCmWGmWCmWCmWGGAGACCGGCAGCCmWGAAGC mWG G C CAAC G G CAGAGmWGAmWC GAGAAGACACmWGmWACAACAGAC GAAC CAGAC AAGACGAGCCCGCCCmWGmWmWmWGmWGGCCCmWGACCmWmWCGAGAGAAGAGAGG mWGCmWGGACAGCAGCAAmWAmWCAAGCCmWAmWGAACCmWGAmWCGGCGmWGGAC CGGGGCGAGAACAmWCCCmWGCCGmWGAmWCGCCCmWmWACCGACCCCGAGGGAmW GCCCmWCmWGAGCCGGmWmWmWAAAGACAGCCmWGGGCAACCCmWACCCACAmWCC mWGAGAAmWmWGGCGAGmWCCmWACAAGGAGAAGCAGAGAACCAmWCCAGGCCAAG AAGGAGGmWGGAGCAGCGGCGGGCmWGGCGGCmWACmWCCCGGAAGmWACGCCAGC AAGGCCAAGAACCmWGGCCGACGACAmWGGmWmWAGAAAmWACCGCCAGAGACCmW C CmWGmWACmWAC GCmWGmWGAC C CAGGAC GC CAmWGCmWGAmWCmWmWC GAGAAC CmWGAGCAGAGGCmWmWCGGCAGACAGGGCAAGAGAACCmWmWCAmWGGCCGAGAG ACAGmWACACCCGGAmWGGAGGACmWGGCmWGACCGCCAAGCmWGGCCmWACGAGG GC CmWGC C CmWCmWAAGAC CmWAC CmWGmWC CAAGAC CmWmWGGCACAGmWACAC C AGCAAGACAmWGCmWCmWAACmWGCGGCmWmWCACAAmWCACGAGCGCCGACmWAC GACCGGGmWGCmWGGAGAAACmWGAAGAAGACCGCCACAGGCmWGGAmWGACCACC AmWmWAACGGCAAGGAGCmWGAAGGmWGGAGGGCCAGAmWCACCmWACmWACAACA GGmWACAAACGGCAGAACGmWGGmWGAAGGACCmWGAGCGmWGGAACmWGGAmWAG SEQ ID NO: Engineered X-editor Designation coding sequence ACmTGAGCGAGGAAAGCGmTAAACAAmTGACAmTCAGCAGCmTGGACCAAGGGCCG GAGCGGCGAGGCCCmTGAGCCmTGCmTGAAGAAGAGAmTmTCmTCCCACAGACCAG mWGCAGGAGAAGmWmWCGmWGmWGmWCmWGAACmWGCGGCmWmWCGAGACCCACGC CGACGAGCAAGCCGCCCmTGAACAmTCGCCCGGmTCmTmTGGCmTmTmTmTCCmTG CGGAGCCAGGAGmTACAAGAAGmTACCAGACAAACAAGACCACAGGCAACACAGAC AAGAGAGCCmTmTCGmTCGAGACCmTGGCAGAGCmTmTCmTACAGAAAGAAGCmTG AAGGAGGmTGmTGGAAGCCmTGCCGmTG 29 DNA sequence encoding X-editor491 CAGGAGATCAAGCGGATTAATAAAATTCGGAGAAGACTGGTGAAGGATTCTAACAC AAAGAAGGCTGGCAAGACAGGCCCTATGAAGACACTGCTGGTGAGAGTGATGACAC CCGACCTGAGAGAAAGACTGGAAAACCTGAGAAAGAAGCCTGAGAATATCCCCCAG CCCATCAGCAACACAAGCCGGGCCAACCTGAATAAGCTGCTGACCGACTACACCGA AATGAAGAAGGCCATCCTGCACGTGTATTGGGAAGAGTTCCAGAAAGACCCAGTCG GCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAAGATCGATCAGAACAAGCTG AAGCCCGAAATGGACGAGAAGGGGAACCTGACAACCGCCGGCTTTGCCTGTAGCCA GTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGAGCGAAAAGGGCAAGG CTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGAGAAGCTGATCCTG CTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGACATATTCCCTGGG CAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCAAGGAATCCA CCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCAGCGGCCCC GTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTGTCTAA GTACCAGGACATTATCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGAGAC TGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTGACC CTGCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGATCGC CCGGGTGCGGATGTGGGTGAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTAGAG ACGACGCCAAGCCCCTGCTGAGACTGAAGGGCTTCCCCAGCTTCCCTCTGGTGGAG AGACAGGCAAATGAAGTGGACTGGTGGGACATGGTGTGTAACGTGAAGAAGCTGAT CAATGAGAAGAAGGAGGACGGCAAAGTGTTCTGGCAGAATCTGGCCGGCTACAAGC GTCAGGAGGCCCTGCGGCCCTACCTGAGCAGCGAGGAAGACAGAAAGAAGGGCAAG AAGTTCGCCCGGTATCAGCTGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCACGG CGAAGACTGGGGGAAGGTGTACGATGAGGCCTGGGAGCGGATCGATAAGAAGGTGG AGGGCCTGAGCAAGCACATCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCCCAG AGCAAGGCCGCCCTGACCGACTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGAGGG GCTGAAGGAGGCCGACAAGGACGAGTTCTGCCGGTGCGAACTGAAGCTGCAGAAGT GGTACGGAGATCTGAGAGGCAAACCTTTCGCCATCGAGGCCGAGAACAGCATCCTG GACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTTATTTGGCAGAAGGACGG AGTGAAGAAGCTGAACCTGTACCTGATCATCAACTATTTCAAGGGCGGCAAGCTGA GATTCAAGAAGATCAAGCCTGAAGCCTTCGAGGCCAACAGATTCTACACCGTGATT AACAAGAAAAGCGGAGAGATCGTGCCAATGGAAGTGAACTTCAACTTCGACGACCC TAACCTGATCATCCTGCCCCTGGCATTTGGCAAGCGGCAGGGCAGAGAGTTCATCT GGAACGACCTGCTGTCTCTGGAGACCGGCAGCCTGAAGCTGGCCAACGGCAGAGTG AT C GAGAAGACAC T GTACAACAGAC GAAC CAGACAAGAC GAGCCCGCCCTGTTTGT GGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAATATCAAGCCTATGA ACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCGTGATCGCCCTTACCGAC CCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGGCAACCCTACCCACAT CCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATCCAGGCCAAGAAGG AGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCAGCAAGGCCAAG AACCTGGCCGACGACATGGTTAGAAATACCGCCAGAGACCTCCTGTACTACGCTGT GACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGCAGACAGG GCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGCTGACC GCCAAGCTGGCCTACGAGGGCCTGTCTAAGACCTACCTGTCCAAGACCTTGGCACA GTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCGACTACG ACCGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACCATTAAC GGCAAGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACAGGTACAAACGGCA GAACGTGGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAAGCGTAA SEQ ID NO: Engineered X-editor Designation coding sequence ACAATGACATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGCCTGCTG AAGAAGAGATTCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAACTGCGG CTTCGAGACCCACGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTTGGCTTT TCCTGCGGAGCCAGGAGTACAAGAAGTACCAGACAAACAAGACCACAGGCAACACA GACAAGAGAGCCTTCGTCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCTGAAGGA GGTGTGGAAGCCTGCCGTG 30 DNA sequence encoding X-editor 515 CAGGAGATCAAGCGGATTAATAAAATTCGGAGAAGACTGGTGAAGGATTCTAACAC AAAGAAGGCTGGCAAGACAGGCCCTATGAAGACACTGCTGGTGAGAGTGATGACAC CCGACCTGAGAGAAAGACTGGAAAACCTGAGAAAGAAGCCTGAGAATATCCCCCAG CCCATCAGCAACACAAGCCGGGCCAACCTGAATAAGCTGCTGACCGACTACACCGA AATGAAGAAGGCCATCCTGCACGTGTATTGGGAAGAGTTCCAGAAAGACCCAGTCG GCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAAGATCGATCAGAACAAGCTG AAGCCCGAAATGGACGAGAAGGGGAACCTGACAACCGCCGGCTTTGCCTGTAGCCA GTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGAGCGAAAAGGGCAAGG CTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGAGAAGCTGATCCTG CTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGACATATTCCCTGGG CAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCAAGGAATCCA CCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCAGCGGCCCC GTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTGTCTAA GTACCAGGACATTATCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGAGAC TGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTGACC CTGCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGATCGC CCGGGTGCGGATGTGGGTGAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTAGAG ACGACGCCAAGCCCCTGCTGAGACTGAAGGGCTTCCCCAGCTTCCCTCTGGTGGAG AGACAGGCAAATGAAGTGGACTGGTGGGACATGGTGTGTAACGTGAAGAAGCTGAT CAATGAGAAGAAGGAGGACGGCAAAGTGTTCTGGCAGAATCTGGCCGGCTACAAGC GTCAGGAGGCCCTGCGGCCCTACCTGAGCAGCGAGGAAGACAGAAAGAAGGGCAAG AAGTTCGCCCGGTATCAGCTGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCACGG CGAAGACTGGGGGAAGGTGTACGATGAGGCCTGGGAGCGGATCGATAAGAAGGTGG AGGGCCTGAGCAAGCACATCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCCCAG AGCAAGGCCGCCCTGACCGACTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGAGGG GCTGAAGGAGGCCGACAAGGACGAGTTCTGCCGGTGCGAACTGAAGCTGCAGAAGT GGTACGGAGATCTGAGAGGCAAACCTTTCGCCATCGAGGCCGAGAACAGCATCCTG GACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTTATTTGGCAGAAGGACGG AGTGAAGAAGCTGAACCTGTACCTGATCATCAACTATTTCAAGGGCGGCAAGCTGA GATTCAAGAAGATCAAGCCTGAAGCCTTCGAGGCCAACAGATTCTACACCGTGATT AACAAGAAAAGCGGAGAGATCGTGCCAATGGAAGTGAACTTCAACTTCGACGACCC TAACCTGATCATCCTGCCCCTGGCATTTGGCAAGCGGCAGGGCAGAGAGTTCATCT GGAACGACCTGCTGTCTCTGGAGACCGGCAGCCTGAAGCTGGCCAACGGCAGAGTG AT C GAGAAGACAC T GTACAACAGAC GAAC CAGACAAGAC GAGCCCGCCCTGTTTGT GGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAATATCAAGCCTATGA ACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCGTGATCGCCCTTACCGAC CCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGGCAACCCTACCCACAT CCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATCCAGGCCAAGAAGG AGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCAGCAAGGCCAAG AACCTGGCCGACGACATGGTTAGAAATACCGCCAGAGACCTCCTGTACTACGCTGT GACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGCAGACAGG GCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGCTGACC GCCAAGCTGGCCTACGAGGGCCTGCCCTCTAAGACCTACCTGTCCAAGACCTTGGC ACAGTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCGACT ACGACCGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACCATT AACGGCAAGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACAGGTACAAACG GCAGAACGTGGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAAGCG TAAACAATGACATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGCCTG SEQ ID NO: Engineered X-editor Designation coding sequence CTGAAGAAGAGATTCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAACTG CGGCTTCGAGACCCACGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTTGGC TTTTCCTGCGGAGCCAGGAGTACAAGAAGTACCAGACAAACAAGACCACAGGCAAC ACAGACAAGAGAGCCTTCGTCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCTGAA GGAGGTGTGGAAGCCTGCCGTG 31 DNA sequence encoding X-editor 676 CAGGAGATCAAGCGGATTAATAAAATTCGGAGAAGACTGGTGAAGGATTCTAACAC AAAGAAGGCTGGCAAGACACGGGGCCCTATGAAGACACTGCTGGTGAGAGTGATGA CACCCGACCTGAGAGAAAGACTGGAAAACCTGAGAAAGAAGCCTGAGAATATCCCC CAGCCCATCAGCAACACAAGCCGGGCCAACCTGAATAAGCTGCTGACCGACTACAC CGAAATGAAGAAGGCCATCCTGCACGTGTATTGGGAAGAGTTCCAGAAAGACCCAG TCGGCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAAGATCGATCAGAACAAG CTGAAGCCCGAAATGGACGAGAAGGGGAACCTGACAACCGCCGGCTTTGCCTGTAG CCAGTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGAGCGAAAAGGGCA AGGCTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGAGAAGCTGATC AAGCTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGACATATTCCCT GGGCAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCAAGGAAT CCACCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCTCCAGC CCCGTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTGTC TAAGTACCAGGACATTATCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGA GACTGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTG ACCCTGCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGAT CGCCCGGGTGCGGATGTGGGTGAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTA GAGACGACGCCAAGCCCCTGCTGAGACTGAAGGGCTTCCCCAGCTTCCCTCTGGTG GAGAGACAGGCAAATGAAGTGGACTGGTGGGACATGGTGTGTAACGTGAAGAAGCT GATCAATGAGAAGAAGGAGGACGGCAAAGTGTTCTGGCAGAATCTGGCCGGCTACA AGCGTCAGGAGGCCCTGCGGCCCTACCTGAGCAGCGAGGAAGACAGAAAGAAGGGC AAGAAGTTCGCCCGGTATCAGCTGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCA CGGCGAAGACTGGGGGAAGGTGTACGATGAGGCCTGGGAGCGGATCGATAAGAAGG TGGAGGGCCTGAGCAAGCACATCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCC CAGAGCAAGGCCGCCCTGACCGACTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGA GGGGCTGAAGGAGGCCGACAAGGACGAGTTCTGCCGGTGCGAACTGAAGCTGCAGA AGTGGTACGGAGATCTGAGAGGCAAACCTTTCGCCATCGAGGCCGAGAACAGCATC CTGGACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTTATTTGGCAGAAGGA CGGAGTGAAGAAGCTGAACCTGTACCTGATCATCAACTATTTCAAGGGCGGCAAGC TGAGATTCAAGAAGATCAAGCCTGAAGCCTTCGAGGCCAACAGATTCTACACCGTG ATTAACAAGAAAAGCGGAGAGATCGTGCCAATGGAAGTGAACTTCAACTTCGACGA CCCTAACCTGATCATCCTGCCCCTGGCATTTGGCAAGCGGCAGGGCAGAGAGTTCA TCTGGAACGACCTGCTGTCTCTGGAGACCGGCAGCCTGAAGCTGGCCAACGGCAGA GT GAT C GAGAAGACAC T GTACAACAGAC GAAC CAGACAAGAC GAGCCCGCCCTGTT TGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAATATCAAGCCTA TGAACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCGTGATCGCCCTTACC GACCCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGGCAACCCTACCCA CATCCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATCCAGGCCAAGA AGGAGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCAGCAAGGCC AAGAACCTGGCCGACGACATGGTTAGAAATACCGCCAGAGACCTCCTGTACTACGC TGTGACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGCAGAC AGGGCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGCTG ACCGCCAAGCTGGCCTACGAGGGCCTGCCCTCTAAGACCTACCTGTCCAAGACCTT GGCACAGTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCG ACTACGACCGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACC ATTAACGGCAAGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACAGGTACAA ACGGCAGAACGTGGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAA GCGTAAACAATGACATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGC CTGCTGAAGAAGAGATTCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAA SEQ ID NO: Engineered X-editor Designation coding sequence CTGCGGCTTCGAGACCCACGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTT GGCTTTTCCTGCGGAGCCAGGAGTACAAGAAGTACCAGACAAACAAGACCACAGGC AACACAGACAAGAGAGCCTTCGTCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCT GAAGGAGGTGTGGAAGCCTGCCGTG 32 DNA sequence encoding X-editor 812 CAGGAGATCAAGCGGATTAATAAAATTCGGAGAAGACTGGTGAAGGATTCTAACAC AAAGAAGGCTGGCAAGACAGGCCCTATGAAGACACTGCTGGTGAGAGTGATGACAC CCGACCTGAGAGAAAGACTGGAAAACCTGAGAAAGAAGCCTGAGAATATCCCCCAG CCCATCAGCAACACAAGCCGGGCCAACCTGAATAAGCTGCTGACCGACTACACCGA AATGAAGAAGGCCATCCTGCACGTGTATTGGGAAGAGTTCCAGAAAGACCCAGTCG GCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAAGATCGATCAGAACAAGCTG AAGCCCGAAATGGACGAGAAGGGGAACCTGACAACCGCCGGCTTTGCCTGTAGCCA GTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGAGCGAAAAGGGCAAGG CTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGAGAAGCTGATCCTG CTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGACATATTCCCTGGG CAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCAAGGAATCCA CCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCAGCGGCCCC GTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTGTCTAA GTACCAGGACATTATCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGAGAC TGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTGACC CTGCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGATCGC CCGGGTGCGGATGTGGGTGAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTAGAG ACGACGCCAAGCCCCTGCTGAGACTGAAGAAGTTCCCCAGCTTCCCTCTGGTGGAG AGACAGGCAAATGAAGTGGACTGGTGGGACATGGTGTGTAACGTGAAGAAGCTGAT CAATGAGAAGAAGGAGGACGGCAAAGTGTTCTGGCAGAATCTGGCCGGCTACAAGC GTCAGGAGGCCCTGCGGCCCTACCTGAGCAGCGAGGAAGACAGAAAGAAGGGCAAG AAGTTCGCCCGGTATCAGCTGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCACGG CGAAGACTGGGGGAAGGTGTACGATGAGGCCTGGGAGCGGATCGATAAGAAGGTGG AGGGCCTGAGCAAGCACATCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCCCAG AGCAAGGCCGCCCTGACCGACTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGAGGG GCTGAAGGAGGCCGACAAGGACGAGTTCTGCCGGTGCGAACTGAAGCTGCAGAAGT GGTACGGAGATCTGAGAGGCAAACCTTTCGCCATCGAGGCCGAGAACAGCATCCTG GACATCAGCGGCTTCAGCAAGCAGTACAACTGCGCCTTTATTTGGCAGAAGGACGG AGTGAAGAAGCTGAACCTGTACCTGATCATCAACTATTTCAAGGGCGGCAAGCTGA GATTCAAGAAGATCAAGCCTGAAGCCTTCGAGGCCAACAGATTCTACACCGTGATT AACAAGAAAAGCGGAGAGATCGTGCCAATGGAAGTGAACTTCAACTTCGACGACCC TAACCTGATCATCCTGCCCCTGGCATTTGGCAAGCGGCAGGGCAGAGAGTTCATCT GGAACGACCTGCTGTCTCTGGAGACCGGCAGCCTGAAGCTGGCCAACGGCAGAGTG AT C GAGAAGACAC T GTACAACAGAC GAAC CAGACAAGAC GAGCCCGCCCTGTTTGT GGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGCAATATCAAGCCTATGA ACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCGTGATCGCCCTTACCGAC CCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGGCAACCCTACCCACAT CCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATCCAGGCCAAGAAGG AGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCAGCAAGGCCAAG AACCTGGCCGACGACATGGTTAGAAATACCGCCAGAGACCTCCTGTACTACGCTGT GACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGCAGACAGG GCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGCTGACC GCCAAGCTGGCCTACGAGGGCCTGCCCTCTAAGACCTACCTGTCCAAGACCTTGGC ACAGTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCGACT ACGACCGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACCATT AACGGCAAGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACAGGTACAAACG GCAGAACGTGGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAAGCG TAAACAATGACATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGCCTG CTGAAGAAGAGATTCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAACTG CGGCTTCGAGACCCACGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTTGGC SEQ ID NO: Engineered X-editor Designation coding sequence TTTTCCTGCGGAGCCAGGAGTACAAGAAGTACCAGACAAACAAGACCACAGGCAAC ACAGACAAGAGAGCCTTCGTCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCTGAA GGAGGTGTGGAAGCCTGCCGTG = Nl-methyl-pseudouridine

[0179] As described further herein, the disclosure provides mRNA modified to improve certain properties of the mRNA molecules, as well as inclusion of non-encoding regions to improve translation and stability, described more fully below. In some cases, the mRNA are designed for use in certain delivery formulations; e.g., nanoparticles such as synthetic nanoparticles or lipid nanoparticles (LNP) for delivery to a population of cells.

[0180] Various naturally-occurring or modified nucleosides may be used to produce the modified mRNA according to the present disclosure. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-l-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases such as 2'-O-methylation); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine (“5 mC”), pseudouridine (“\| / U”), and / or 2-thio-uridine (“2sU”). In a particular embodiment, one or more of the uridine residues of the mRNA of the disclosure are replaced with 1-methyl-pseudouridine. See, e.g., U.S. Pat. No. 8,278,036 or WO2011012316, for a discussion of such residues and their incorporation into mRNA. In some embodiments, the mRNA encoding engineered X-editor 515 (SEQ ID NO: 23) has pseudouridine nucleosides replacing one or more, or all uridines in the sequence. In one embodiment, the mRNA encoding engineered X-editor 515 with pseudouridine nucleosides replacing all uridines in the sequence comprises the sequence of SEQ ID NO: 40, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or having at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In another embodiment, the mRNA encoding engineered X-editor 515 with pseudouridine nucleosides replacing all uridines in the sequence comprises the sequence of SEQ ID NO: 40. In some embodiments, the mRNA encoding X-editor 812 (SEQ ID NO: 25) has pseudouridine nucleosides replacing one or more, or all uridines in the sequence. In one embodiment, the mRNA encoding engineered X-editor 491 with pseudouridine nucleosides replacing all uridines in the sequence comprises the sequence of SEQ ID NO: 42. In some embodiments, the mRNA encoding X-editor 491 (SEQ ID NO: 22) has pseudouridine nucleosides replacing one or more, or all uridines in the sequence. In one embodiment, the mRNA encoding engineered X-editor 491 with pseudouridine nucleosides replacing all uridines in the sequence comprises the sequence of SEQ ID NO: 39. In some embodiments, the mRNA encoding X-editor 676 (SEQ ID NO: 24) has pseudouridine nucleosides replacing one or more, or all uridines in the sequence. In one embodiment, the mRNA encoding engineered X-editor 491 with pseudouridine nucleosides replacing all uridines in the sequence comprises the sequence of SEQ ID NO: 41.

[0181] Accordingly, in some embodiments, the mRNA encoding an engineered X-editor is chemically modified. Thus, in some embodiments, the chemically mRNA sequence encoding the engineered X-editor 491 protein comprises the sequence of SEQ ID NO: 39, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the mRNA sequence encoding the engineered X-editor protein comprises the sequence of SEQ ID NO: 39. In some embodiments, the mRNA sequence encoding the engineered X-editor protein consists of the sequence of SEQ ID NO: 39.

[0182] In some embodiments, the chemically modified mRNA sequence encoding the engineered X-editor 515 protein comprises the sequence of SEQ ID NO: 40, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the mRNA sequence encoding the engineered X-editor protein comprises the sequence of SEQ ID NO: 40. In some embodiments, the mRNA sequence encoding the engineered X-editor protein consists of the sequence of SEQ ID NO: 40.

[0183] In some embodiments, the chemically modified mRNA sequence encoding the engineered X-editor 676 protein comprises the sequence of SEQ ID NO: 41 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the mRNA sequence encoding the engineered X-editor protein comprises the sequence of SEQ ID NO: 41. In some embodiments, the mRNA sequence encoding the engineered X-editor protein consists of the sequence of SEQ ID NO: 41.

[0184] In some embodiments, the chemically modified mRNA sequence encoding the engineered X-editor 812 protein comprises the sequence of SEQ ID NO: 42 or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the mRNA sequence encoding the engineered X-editor protein comprises the sequence of SEQ ID NO: 42. In some embodiments, the mRNA sequence encoding the engineered X-editor protein consists of the sequence of SEQ ID NO: 42.

[0185] In any of the foregoing embodiments, percent identity can be determined with respect to the full-length RNA sequence encoding the engineered X-editor. The person of ordinary skill in the art will appreciate that RNA sequences can be varied significantly, while at the same time preserving the amino acid sequences of the encoded protein. .

[0186] In some embodiments, a DNA sequence encoding the engineered X-editor protein is selected from the group consisting of SEQ ID NOS: 29-32. f. S' untranslated region (UTR)

[0187] The 5' UTR of an mRNA molecule may influence both the stability of the mRNA and how efficiently it is translated into protein. Specifically, the 5' UTR, in conjunction with the 5' cap structure, serves as a binding site and recruitment platform for the translation pre-initiation complex as well as additional regulatory proteins that may positively or negatively affect translation. Structures within the 5' UTR can enhance translation by recruiting initiation factors or other protein or RNA factors, reduce translation by physically blocking ribosome binding and scanning, and contribute to the stability of the mRNA by affecting both hydrolysis and nuclease digestion. An exemplary 5’ UTR sequence for use in the mRNA of the disclosure is provided in Table 5. Table 5 lists the RNA sequence, RNA sequence with N1 -methylpseudouridine substituted in place of uridine, and DNA sequence of the 5’ UTR. Table 5: 5’ UTR sequences SEQ ID NO Nucleic Acid Sequence* Description 320 AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA RNA sequence of 5' UTR 339 AAAmWAAGAGAGAAAAGAAGAGmWAAGAAGAAAmWAmWAAGA N1 -methylpseudouridine substituted RNA sequence of 5 ’ UTR 301 AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA DNA sequence encoding 5’ UTR = Nl-methyl-pseudouridine g. 5’ cap

[0188] In some embodiments of the mRNA of the disclosure, the mRNA comprises a 5’ cap linked 5’ to the 5’ UTR of the mRNA sequence of any of the embodiments described herein. In some embodiments, the 5’ cap is a 7-methylguanylate cap. In some embodiments, the 5’ cap has the nucleic acid sequence of m7G(5’)ppp(5’)mAG. In other embodiments, the 5' cap has the nucleic acid sequence of m7G(5')ppp (5'(A,G(5')ppp(5')A or G(5')ppp(5')G. Exemplary caps are known in the art, and described, for example, in WO 2017 / 053297, the contents of which are incorporated by reference herein. h. 3’UTR

[0189] 3’ UTR sequences can have an impact on mRNA stability and translation efficiency and can determine both subcellular localization and tissue-specific expression. Factors influencing these properties include microRNA binding sites, AU-rich elements that recruit an array of RNA-binding proteins, Pumilio binding elements, and other binding sites for RNA-binding proteins. While many of these interactions with the 3' UTR are known to negatively impact stability or expression, some can enhance translation. The effects of a 3' UTR sequence can be highly cell-type specific due to differential expression of microRNAs and RNA binding proteins, which provides opportunities for engineering tissue-specific expression into a therapeutic mRNA. In some embodiments, the 3' UTR for use in the mRNA of the disclosure is a mouse 3' UTR. In some embodiments, the 3' UTR is a mouse HBA gene 3' UTR.

[0190] Exemplary 3’ UTR sequences for use in the mRNA of the disclosure are provided in Table 6. Table 6 lists the RNA sequence, RNA sequence with N1 -methylpseudouridine substituted in place of uridine, and DNA sequence of the 3’ UTR. Table 6: 3’ UTR sequences SEQ ID NO Nucleic Acid Sequence* Description 309 GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCC CUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG RNA sequence of 3' UTR 328 GCmTOCCmWTCmWGCGGGGCmWiWGCCmWiWCmWGGCCAmWGCCCm WmWCmWmWCmWCmWCCCmWmWGCACCmWGmWACCmWCmWmWGGmWCm WWWGAAmWAAAGCCmWGAGmWAGGAAG Nl- methylpseudouridin e substituted RNA sequence of 3 ’ UTR 291 GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCC CTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG DNA sequence encoding 3 ’ UTR = h> 1 -methyl -p seudouri dine

[0191] In some embodiments, the 3’ UTR for use in the mRNA of the disclosure comprises the sequence of SEQ ID NO: 309 or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% identity thereto. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 309. In some embodiments, the 3’ UTR consists of the sequence of SEQ ID NO: 309. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 328, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% identity thereto. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 328. In some embodiments, the 3’ UTR consists of the sequence of SEQ ID NO: 328. i. Poly(A) sequence

[0192] Inclusion of a 3' poly(A) tail in an mRNA can contribute to the stability and translation efficiency. Generally, longer poly(A) tails are associated with increased mRNA stability, thereby allowing their translation and promoting high protein expression.

[0193] In some embodiments, the mRNAs of the disclosure comprise a poly(A) sequence. In some embodiments, the poly(A) sequence comprises at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides. In some embodiments, the poly(A) sequence comprises about 40-190 adenine nucleotides. In some embodiments, the poly(A) sequence comprises about 50-150 adenine nucleotides. In some embodiments, the poly(A) sequence comprises about 70-90 adenine nucleotides. In some embodiments, the poly(A) sequence comprises about 78-82 adenine nucleotides. In some embodiments, the poly(A) sequence comprises 80 adenine nucleotides.

[0194] In some embodiments, the poly(A) sequence comprises the nucleic acid sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 294). In some embodiments, the poly(A) sequence comprises 79 adenine nucleotides. In some embodiments, the poly(A) sequence comprises the nucleic acid sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 295). j. mRNA sequences of the systems

[0195] In some embodiments, the disclosure provides systems comprising mRNAs comprising the following components in 5’ to 3’ orientation: a 5' UTR; a start codon; an NLS; a sequence encoding an engineered X-editor protein; an NLS; stop codon; and a 3' UTR. In some embodiments, the mRNA comprises the following components in 5’ to 3’ orientation: a 5’ cap; a 5' UTR; a start codon; an NLS; a sequence encoding an engineered X-editor protein; a stop codon; a 3' UTR; and a poly(A) sequence. In some embodiments, the mRNA comprises the following components in 5’ to 3’ orientation: a 5’ cap; a 5' UTR; a start codon; a sequence encoding an engineered X-editor protein; an NLS; a stop codon; a 3' UTR; and a poly(A) sequence. In some embodiments, the mRNA comprises the following components in 5’ to 3’ orientation: a 5’ cap; a 5' UTR; a start codon; a sequence encoding an engineered X-editor protein; a stop codon; a 3' UTR; and a poly(A) sequence. In some embodiments, the mRNA has pseudouridine nucleosides replacing one or more, uridines in the sequence. In some embodiments, the mRNA has pseudouridine nucleosides replacing all uridines in the mRNA.

[0196] In some embodiments, the disclosure provides an mRNA sequence comprising the sequence of SEQ ID NO: 47 or SEQ ID NO: 51, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides an mRNA sequence comprising the sequence of SEQ ID NO: 47 or SEQ ID NO: 51. In some embodiments, the disclosure provides an mRNA sequence consisting of the sequence of SEQ ID NO: 47 or SEQ ID NO: 51.

[0197] In some embodiments, the disclosure provides an mRNA sequence comprising the sequence of SEQ ID NO: 48 or SEQ ID NO: 52, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides an mRNA sequence comprising the sequence of SEQ ID NO: 48 or SEQ ID NO: 52. In some embodiments, the disclosure provides an mRNA sequence consisting of the sequence of SEQ ID NO: 48 or SEQ ID NO: 52.

[0198] In some embodiments, the disclosure provides an mRNA sequence comprising the sequence of SEQ ID NO: 49 or SEQ ID NO: 53, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides an mRNA sequence comprising the sequence of SEQ ID NO: 49 or SEQ ID NO: 53. In some embodiments, the disclosure provides an mRNA sequence consisting of the sequence of SEQ ID NO: 49 or SEQ ID NO: 53.

[0199] In some embodiments, the disclosure provides an mRNA sequence comprising the sequence of SEQ ID NO: 50 or SEQ ID NO: 54, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides an mRNA sequence comprising the sequence of SEQ ID NO: 50 or SEQ ID NO: 54. In some embodiments, the disclosure provides an mRNA sequence consisting of the sequence of SEQ ID NO: 50 or SEQ ID NO: 54.

[0200] Exemplary full-length mRNA sequences are provided in Table 7. Table 7 lists exemplary RNA sequences, RNA sequences with N1-methylpseudouridine substituted in place of uridine, and DNA sequences encoding the mRNAs. The sequences in Table 7 include, from 5’ to 3’, AGG nucleotides 5’ of the 5’ UTR, a 5’ UTR, GCCACC nucleotides, a start codon, a sequence encoding a c-MYC NLS, a sequence encoding a linker, a sequence encoding CasX, a sequence encoding a linker, a sequence encoding a c-MYC NLS, a stop codon, a 3’ UTR, and a sequence corresponding to a partial Xbal restriction site (UCUAG, m\| / Cm\| / AG,; or TCTAG, for RNA, N1 -methylpseudouridine substituted RNA, and DNA sequences, respectively). Table 7: Exemplary full-length mRNA sequences SEQ ID NO Nucleotide Sequence* Description 47 AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCCCUGC UGCCAAGAGAGUGAAGCUGGAUAGCAGACAGGAGAUCAAGCGGAUUAAUAAAAUUCGG AGAAGACUGGUGAAGGAUUCUAACACAAAGAAGGCUGGCAAGACAGGCCCUAUGAAGA CACU GCU GGU GAGAGU GAU GACAC C C GAG CU GAGAGAAAGACU GGAAAAC CU GAGAAA GAAGCCUGAGAAUAUCCCCCAGCCCAUCAGCAACACAAGCCGGGCCAACCUGAAUAAG CUGCUGACCGACUACACCGAAAUGAAGAAGGCCAUCCUGCACGUGUAUUGGGAAGAGU UCCAGAAAGACCCAGUCGGCCUGAUGAGCAGAGUGGCUCAGCCUGCCAGCAAGAAGAU CGAUCAGAACAAGCUGAAGCCCGAAAUGGACGAGAAGGGGAACCUGACAACCGCCGGC UUUGCCUGUAGCCAGUGCGGCCAGCCCCUGUUUGUGUACAAACUGGAACAGGUGAGCG AAAAGGGCAAGGCUUACACGAAUUACUUCGGCAGAUGCAACGUGGCCGAGCACGAGAA GCUGAUCCUGCUGGCCCAGCUGAAGCCUGAGAAGGAUAGCGAUGAGGCAGUGACAUAU UCCCUGGGCAAGUUCGGACAGCGGGCCCUGGAUUUUUAUUCCAUUCAUGUGACCAAGG AAUCCACCCACCCCGUCAAGCCUCUUGCCCAAAUUGCCGGCAACAGAUACGCCAGCGG CCCCGUGGGCAAGGCCCUGAGCGACGCCUGUAUGGGCACCAUCGCCAGCUUCCUGUCU AAGUACCAGGACAUUAUCAUCGAGCACCAGAAGGUGGUGAAGGGCAACCAGAAGAGAC UGGAGAGCCUGCGCGAGCUGGCCGGCAAGGAAAACCUGGAGUAUCCUAGCGUGACCCU GCCUCCUCAGCCUCAUACAAAGGAGGGCGUGGAUGCCUACAACGAAGUGAUCGCCCGG GUGCGGAUGUGGGUGAACCUGAAUCUGUGGCAGAAGCUGAAGCUGUCUAGAGACGACG CCAAGCCCCUGCUGAGACUGAAGGGCUUCCCCAGCUUCCCUCUGGUGGAGAGACAGGC AAAUGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCUGAUCAAUGAGAAG AAGGAGGACGGCAAAGUGUUCUGGCAGAAUCUGGCCGGCUACAAGCGUCAGGAGGCCC UGCGGCCCUACCUGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGUUCGCCCGGUA UCAGCUGGGGGACCUGCUGCUGCACCUCGAGAAGAAGCACGGCGAAGACUGGGGGAAG GUGUACGAUGAGGCCUGGGAGCGGAUCGAUAAGAAGGUGGAGGGCCUGAGCAAGCACA UCAAGCUGGAGGAGGAACGGAGAUCUGAGGACGCCCAGAGCAAGGCCGCCCUGACCGA CUGGCUGAGAGCCAAGGCCAGCUUCGUCAUCGAGGGGCUGAAGGAGGCCGACAAGGAC GAGUUCUGCCGGUGCGAACUGAAGCUGCAGAAGUGGUACGGAGAUCUGAGAGGCAAAC CUUUCGCCAUCGAGGCCGAGAACAGCAUCCUGGACAUCAGCGGCUUCAGCAAGCAGUA CAACUGCGCCUUUAUUUGGCAGAAGGACGGAGUGAAGAAGCUGAACCUGUACCUGAUC AUCAACUAUUUCAAGGGCGGCAAGCUGAGAUUCAAGAAGAUCAAGCCUGAAGCCUUCG AGGCCAACAGAUUCUACACCGUGAUUAACAAGAAAAGCGGAGAGAUCGUGCCAAUGGA AGUGAACUUCAACUUCGACGACCCUAACCUGAUCAUCCUGCCCCUGGCAUUUGGCAAG CGGCAGGGCAGAGAGUUCAUCUGGAACGACCUGCUGUCUCUGGAGACCGGCAGCCUGA AGCUGGCCAACGGCAGAGUGAUCGAGAAGACACUGUACAACAGACGAACCAGACAAGA CGAGCCCGCCCUGUUUGUGGCCCUGACCUUCGAGAGAAGAGAGGUGCUGGACAGCAGC AAUAUCAAGCCUAUGAACCUGAUCGGCGUGGACCGGGGCGAGAACAUCCCUGCCGUGA UCGCCCUUACCGACCCCGAGGGAUGCCCUCUGAGCCGGUUUAAAGACAGCCUGGGCAA C C CUAC C CACAU C CU GAGAAUU G G C GAGU C CUACAAG GAGAAG CAGAGAAC CAU C CAG GCCAAGAAGGAGGUGGAGCAGCGGCGGGCUGGCGGCUACUCCCGGAAGUACGCCAGCA Full-length RNA sequence of mRNA encoding X-editor 491 SEQ ID NO Nucleotide Sequence* Description AGGCCAAGAACCUGGCCGACGACAUGGUUAGAAAUACCGCCAGAGACCUCCUGUACUA CGCUGUGACCCAGGACGCCAUGCUGAUCUUCGAGAACCUGAGCAGAGGCUUCGGCAGA CAGGGCAAGAGAACCUUCAUGGCCGAGAGACAGUACACCCGGAUGGAGGACUGGCUGA CCGCCAAGCUGGCCUACGAGGGCCUGUCUAAGACCUACCUGUCCAAGACCUUGGCACA GUACACCAGCAAGACAUGCUCUAACUGCGGCUUCACAAUCACGAGCGCCGACUACGAC CGGGUGCUGGAGAAACUGAAGAAGACCGCCACAGGCUGGAUGACCACCAUUAACGGCA AGGAGCUGAAGGUGGAGGGCCAGAUCACCUACUACAACAGGUACAAACGGCAGAACGU GGUGAAGGACCUGAGCGUGGAACUGGAUAGACUGAGCGAGGAAAGCGUAAACAAUGAC AUCAGCAGCUGGACCAAGGGCCGGAGCGGCGAGGCCCUGAGCCUGCUGAAGAAGAGAU UCUCCCACAGACCAGUGCAGGAGAAGUUCGUGUGUCUGAACUGCGGCUUCGAGACCCA CGCCGACGAGCAAGCCGCCCUGAACAUCGCCCGGUCUUGGCUUUUCCUGCGGAGCCAG GAGUACAAGAAGUAC CAGACAAACAAGAC CACAG G CAACACAGACAAGAGAG C CUU C G UCGAGACCUGGCAGAGCUUCUACAGAAAGAAGCUGAAGGAGGUGUGGAAGCCUGCCGU GGGAAGCCCCGCUGCCAAGAGAGUGAAGCUGGACUAAGCUGCCUUCUGCGGGGCUUGC CUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAA G C CU GAGUAG GAAGU CUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 48 AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCCCUGC UGCCAAGAGAGUGAAGCUGGAUAGCAGACAGGAGAUCAAGCGGAUUAAUAAAAUUCGG AGAAGACUGGUGAAGGAUUCUAACACAAAGAAGGCUGGCAAGACAGGCCCUAUGAAGA CACU GCU GGU GAGAGU GAU GACAC C C GAC CU GAGAGAAAGACU GGAAAAC CU GAGAAA GAAGCCUGAGAAUAUCCCCCAGCCCAUCAGCAACACAAGCCGGGCCAACCUGAAUAAG CUGCUGACCGACUACACCGAAAUGAAGAAGGCCAUCCUGCACGUGUAUUGGGAAGAGU UCCAGAAAGACCCAGUCGGCCUGAUGAGCAGAGUGGCUCAGCCUGCCAGCAAGAAGAU CGAUCAGAACAAGCUGAAGCCCGAAAUGGACGAGAAGGGGAACCUGACAACCGCCGGC UUUGCCUGUAGCCAGUGCGGCCAGCCCCUGUUUGUGUACAAACUGGAACAGGUGAGCG AAAAGGGCAAGGCUUACACGAAUUACUUCGGCAGAUGCAACGUGGCCGAGCACGAGAA GCUGAUCCUGCUGGCCCAGCUGAAGCCUGAGAAGGAUAGCGAUGAGGCAGUGACAUAU UCCCUGGGCAAGUUCGGACAGCGGGCCCUGGAUUUUUAUUCCAUUCAUGUGACCAAGG AAUCCACCCACCCCGUCAAGCCUCUUGCCCAAAUUGCCGGCAACAGAUACGCCAGCGG CCCCGUGGGCAAGGCCCUGAGCGACGCCUGUAUGGGCACCAUCGCCAGCUUCCUGUCU AAGUACCAGGACAUUAUCAUCGAGCACCAGAAGGUGGUGAAGGGCAACCAGAAGAGAC UGGAGAGCCUGCGCGAGCUGGCCGGCAAGGAAAACCUGGAGUAUCCUAGCGUGACCCU GCCUCCUCAGCCUCAUACAAAGGAGGGCGUGGAUGCCUACAACGAAGUGAUCGCCCGG GUGCGGAUGUGGGUGAACCUGAAUCUGUGGCAGAAGCUGAAGCUGUCUAGAGACGACG CCAAGCCCCUGCUGAGACUGAAGGGCUUCCCCAGCUUCCCUCUGGUGGAGAGACAGGC AAAUGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCUGAUCAAUGAGAAG AAGGAGGACGGCAAAGUGUUCUGGCAGAAUCUGGCCGGCUACAAGCGUCAGGAGGCCC UGCGGCCCUACCUGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGUUCGCCCGGUA UCAGCUGGGGGACCUGCUGCUGCACCUCGAGAAGAAGCACGGCGAAGACUGGGGGAAG GUGUACGAUGAGGCCUGGGAGCGGAUCGAUAAGAAGGUGGAGGGCCUGAGCAAGCACA UCAAGCUGGAGGAGGAACGGAGAUCUGAGGACGCCCAGAGCAAGGCCGCCCUGACCGA CUGGCUGAGAGCCAAGGCCAGCUUCGUCAUCGAGGGGCUGAAGGAGGCCGACAAGGAC GAGUUCUGCCGGUGCGAACUGAAGCUGCAGAAGUGGUACGGAGAUCUGAGAGGCAAAC CUUUCGCCAUCGAGGCCGAGAACAGCAUCCUGGACAUCAGCGGCUUCAGCAAGCAGUA CAACUGCGCCUUUAUUUGGCAGAAGGACGGAGUGAAGAAGCUGAACCUGUACCUGAUC AUCAACUAUUUCAAGGGCGGCAAGCUGAGAUUCAAGAAGAUCAAGCCUGAAGCCUUCG AGGCCAACAGAUUCUACACCGUGAUUAACAAGAAAAGCGGAGAGAUCGUGCCAAUGGA AGUGAACUUCAACUUCGACGACCCUAACCUGAUCAUCCUGCCCCUGGCAUUUGGCAAG CGGCAGGGCAGAGAGUUCAUCUGGAACGACCUGCUGUCUCUGGAGACCGGCAGCCUGA AGCUGGCCAACGGCAGAGUGAUCGAGAAGACACUGUACAACAGACGAACCAGACAAGA CGAGCCCGCCCUGUUUGUGGCCCUGACCUUCGAGAGAAGAGAGGUGCUGGACAGCAGC AAUAUCAAGCCUAUGAACCUGAUCGGCGUGGACCGGGGCGAGAACAUCCCUGCCGUGA UCGCCCUUACCGACCCCGAGGGAUGCCCUCUGAGCCGGUUUAAAGACAGCCUGGGCAA C C CUAC C CACAU C CU GAGAAUU G G C GAGU C CUACAAG GAGAAG CAGAGAAC CAU C CAG GCCAAGAAGGAGGUGGAGCAGCGGCGGGCUGGCGGCUACUCCCGGAAGUACGCCAGCA Full-length RNA sequence of mRNA encoding X-editor 515 SEQ ID NO Nucleotide Sequence* Description AGGCCAAGAACCUGGCCGACGACAUGGUUAGAAAUACCGCCAGAGACCUCCUGUACUA CGCUGUGACCCAGGACGCCAUGCUGAUCUUCGAGAACCUGAGCAGAGGCUUCGGCAGA CAGGGCAAGAGAACCUUCAUGGCCGAGAGACAGUACACCCGGAUGGAGGACUGGCUGA CCGCCAAGCUGGCCUACGAGGGCCUGCCCUCUAAGACCUACCUGUCCAAGACCUUGGC ACAGUACACCAGCAAGACAUGCUCUAACUGCGGCUUCACAAUCACGAGCGCCGACUAC GACCGGGUGCUGGAGAAACUGAAGAAGACCGCCACAGGCUGGAUGACCACCAUUAACG GCAAGGAGCUGAAGGUGGAGGGCCAGAUCACCUACUACAACAGGUACAAACGGCAGAA CGUGGUGAAGGACCUGAGCGUGGAACUGGAUAGACUGAGCGAGGAAAGCGUAAACAAU GACAUCAGCAGCUGGACCAAGGGCCGGAGCGGCGAGGCCCUGAGCCUGCUGAAGAAGA GAUUCUCCCACAGACCAGUGCAGGAGAAGUUCGUGUGUCUGAACUGCGGCUUCGAGAC CCACGCCGACGAGCAAGCCGCCCUGAACAUCGCCCGGUCUUGGCUUUUCCUGCGGAGC CAGGAGUACAAGAAGUACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCU UCGUCGAGACCUGGCAGAGCUUCUACAGAAAGAAGCUGAAGGAGGUGUGGAAGCCUGC CGUGGGAAGCCCCGCUGCCAAGAGAGUGAAGCUGGACUAAGCUGCCUUCUGCGGGGCU UGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAU AAAG C CU GAGUAG GAAGU CUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 49 AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCCCUGC UGCCAAGAGAGUGAAGCUGGAUAGCAGACAGGAGAUCAAGCGGAUUAAUAAAAUUCGG AGAAGACUGGUGAAGGAUUCUAACACAAAGAAGGCUGGCAAGACACGGGGCCCUAUGA AGACACU GCU GGU GAGAGU GAU GACAC C C GAG CU GAGAGAAAGACU GGAAAAC CU GAG AAAGAAGCCUGAGAAUAUCCCCCAGCCCAUCAGCAACACAAGCCGGGCCAACCUGAAU AAGCUGCUGACCGACUACACCGAAAUGAAGAAGGCCAUCCUGCACGUGUAUUGGGAAG AGUUCCAGAAAGACCCAGUCGGCCUGAUGAGCAGAGUGGCUCAGCCUGCCAGCAAGAA GAUCGAUCAGAACAAGCUGAAGCCCGAAAUGGACGAGAAGGGGAACCUGACAACCGCC GGCUUUGCCUGUAGCCAGUGCGGCCAGCCCCUGUUUGUGUACAAACUGGAACAGGUGA GCGAAAAGGGCAAGGCUUACACGAAUUACUUCGGCAGAUGCAACGUGGCCGAGCACGA GAAGCUGAUCAAGCUGGCCCAGCUGAAGCCUGAGAAGGAUAGCGAUGAGGCAGUGACA UAUUCCCUGGGCAAGUUCGGACAGCGGGCCCUGGAUUUUUAUUCCAUUCAUGUGACCA AGGAAUCCACCCACCCCGUCAAGCCUCUUGCCCAAAUUGCCGGCAACAGAUACGCCUC CAGCCCCGUGGGCAAGGCCCUGAGCGACGCCUGUAUGGGCACCAUCGCCAGCUUCCUG UCUAAGUACCAGGACAUUAUCAUCGAGCACCAGAAGGUGGUGAAGGGCAACCAGAAGA GACUGGAGAGCCUGCGCGAGCUGGCCGGCAAGGAAAACCUGGAGUAUCCUAGCGUGAC CCUGCCUCCUCAGCCUCAUACAAAGGAGGGCGUGGAUGCCUACAACGAAGUGAUCGCC CGGGUGCGGAUGUGGGUGAACCUGAAUCUGUGGCAGAAGCUGAAGCUGUCUAGAGACG ACGCCAAGCCCCUGCUGAGACUGAAGGGCUUCCCCAGCUUCCCUCUGGUGGAGAGACA GGCAAAUGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCUGAUCAAUGAG AAGAAGGAGGACGGCAAAGUGUUCUGGCAGAAUCUGGCCGGCUACAAGCGUCAGGAGG CCCUGCGGCCCUACCUGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGUUCGCCCG GUAUCAGCUGGGGGACCUGCUGCUGCACCUCGAGAAGAAGCACGGCGAAGACUGGGGG AAGGUGUACGAUGAGGCCUGGGAGCGGAUCGAUAAGAAGGUGGAGGGCCUGAGCAAGC ACAUCAAGCUGGAGGAGGAACGGAGAUCUGAGGACGCCCAGAGCAAGGCCGCCCUGAC CGACUGGCUGAGAGCCAAGGCCAGCUUCGUCAUCGAGGGGCUGAAGGAGGCCGACAAG GACGAGUUCUGCCGGUGCGAACUGAAGCUGCAGAAGUGGUACGGAGAUCUGAGAGGCA AACCUUUCGCCAUCGAGGCCGAGAACAGCAUCCUGGACAUCAGCGGCUUCAGCAAGCA GUACAACUGCGCCUUUAUUUGGCAGAAGGACGGAGUGAAGAAGCUGAACCUGUACCUG AUCAUCAACUAUUUCAAGGGCGGCAAGCUGAGAUUCAAGAAGAUCAAGCCUGAAGCCU UCGAGGCCAACAGAUUCUACACCGUGAUUAACAAGAAAAGCGGAGAGAUCGUGCCAAU GGAAGUGAACUUCAACUUCGACGACCCUAACCUGAUCAUCCUGCCCCUGGCAUUUGGC AAGCGGCAGGGCAGAGAGUUCAUCUGGAACGACCUGCUGUCUCUGGAGACCGGCAGCC UGAAGCUGGCCAACGGCAGAGUGAUCGAGAAGACACUGUACAACAGACGAACCAGACA AGACGAGCCCGCCCUGUUUGUGGCCCUGACCUUCGAGAGAAGAGAGGUGCUGGACAGC AGCAAUAUCAAGCCUAUGAACCUGAUCGGCGUGGACCGGGGCGAGAACAUCCCUGCCG UGAUCGCCCUUACCGACCCCGAGGGAUGCCCUCUGAGCCGGUUUAAAGACAGCCUGGG CAAC C CUAC C CACAU C CU GAGAAUU G G C GAGU C CUACAAG GAGAAG CAGAGAAC CAU C CAGGCCAAGAAGGAGGUGGAGCAGCGGCGGGCUGGCGGCUACUCCCGGAAGUACGCCA Full-length RNA sequence encoding X-editor 676 SEQ ID NO Nucleotide Sequence* Description GCAAGGCCAAGAACCUGGCCGACGACAUGGUUAGAAAUACCGCCAGAGACCUCCUGUA CUACGCUGUGACCCAGGACGCCAUGCUGAUCUUCGAGAACCUGAGCAGAGGCUUCGGC AGACAGGGCAAGAGAACCUUCAUGGCCGAGAGACAGUACACCCGGAUGGAGGACUGGC UGACCGCCAAGCUGGCCUACGAGGGCCUGCCCUCUAAGACCUACCUGUCCAAGACCUU GGCACAGUACACCAGCAAGACAUGCUCUAACUGCGGCUUCACAAUCACGAGCGCCGAC UACGACCGGGUGCUGGAGAAACUGAAGAAGACCGCCACAGGCUGGAUGACCACCAUUA ACGGCAAGGAGCUGAAGGUGGAGGGCCAGAUCACCUACUACAACAGGUACAAACGGCA GAACGUGGUGAAGGACCUGAGCGUGGAACUGGAUAGACUGAGCGAGGAAAGCGUAAAC AAUGACAUCAGCAGCUGGACCAAGGGCCGGAGCGGCGAGGCCCUGAGCCUGCUGAAGA AGAGAUUCUCCCACAGACCAGUGCAGGAGAAGUUCGUGUGUCUGAACUGCGGCUUCGA GACCCACGCCGACGAGCAAGCCGCCCUGAACAUCGCCCGGUCUUGGCUUUUCCUGCGG AGCCAGGAGUACAAGAAGUACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAG CCUUCGUCGAGACCUGGCAGAGCUUCUACAGAAAGAAGCUGAAGGAGGUGUGGAAGCC UGCCGUGGGAAGCCCCGCUGCCAAGAGAGUGAAGCUGGACUAAGCUGCCUUCUGCGGG GCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUG AAUAAAG C CU GAGUAG GAAGU CUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 50 AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCCCUGC UGCCAAGAGAGUGAAGCUGGAUAGCAGACAGGAGAUCAAGCGGAUUAAUAAAAUUCGG AGAAGACUGGUGAAGGAUUCUAACACAAAGAAGGCUGGCAAGACAGGCCCUAUGAAGA GAGU GCU GGU GAGAGU GAU GACAC C C GAG CU GAGAGAAAGACU GGAAAAC CU GAGAAA GAAGCCUGAGAAUAUCCCCCAGCCCAUCAGCAACACAAGCCGGGCCAACCUGAAUAAG CUGCUGACCGACUACACCGAAAUGAAGAAGGCCAUCCUGCACGUGUAUUGGGAAGAGU UCCAGAAAGACCCAGUCGGCCUGAUGAGCAGAGUGGCUCAGCCUGCCAGCAAGAAGAU CGAUCAGAACAAGCUGAAGCCCGAAAUGGACGAGAAGGGGAACCUGACAACCGCCGGC UUUGCCUGUAGCCAGUGCGGCCAGCCCCUGUUUGUGUACAAACUGGAACAGGUGAGCG AAAAGGGCAAGGCUUACACGAAUUACUUCGGCAGAUGCAACGUGGCCGAGCACGAGAA GCUGAUCCUGCUGGCCCAGCUGAAGCCUGAGAAGGAUAGCGAUGAGGCAGUGACAUAU UCCCUGGGCAAGUUCGGACAGCGGGCCCUGGAUUUUUAUUCCAUUCAUGUGACCAAGG AAUCCACCCACCCCGUCAAGCCUCUUGCCCAAAUUGCCGGCAACAGAUACGCCAGCGG CCCCGUGGGCAAGGCCCUGAGCGACGCCUGUAUGGGCACCAUCGCCAGCUUCCUGUCU AAGUACCAGGACAUUAUCAUCGAGCACCAGAAGGUGGUGAAGGGCAACCAGAAGAGAC UGGAGAGCCUGCGCGAGCUGGCCGGCAAGGAAAACCUGGAGUAUCCUAGCGUGACCCU GCCUCCUCAGCCUCAUACAAAGGAGGGCGUGGAUGCCUACAACGAAGUGAUCGCCCGG GUGCGGAUGUGGGUGAACCUGAAUCUGUGGCAGAAGCUGAAGCUGUCUAGAGACGACG CCAAGCCCCUGCUGAGACUGAAGAAGUUCCCCAGCUUCCCUCUGGUGGAGAGACAGGC AAAUGAAGUGGACUGGUGGGACAUGGUGUGUAACGUGAAGAAGCUGAUCAAUGAGAAG AAGGAGGACGGCAAAGUGUUCUGGCAGAAUCUGGCCGGCUACAAGCGUCAGGAGGCCC UGCGGCCCUACCUGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGUUCGCCCGGUA UCAGCUGGGGGACCUGCUGCUGCACCUCGAGAAGAAGCACGGCGAAGACUGGGGGAAG GUGUACGAUGAGGCCUGGGAGCGGAUCGAUAAGAAGGUGGAGGGCCUGAGCAAGCACA UCAAGCUGGAGGAGGAACGGAGAUCUGAGGACGCCCAGAGCAAGGCCGCCCUGACCGA CUGGCUGAGAGCCAAGGCCAGCUUCGUCAUCGAGGGGCUGAAGGAGGCCGACAAGGAC GAGUUCUGCCGGUGCGAACUGAAGCUGCAGAAGUGGUACGGAGAUCUGAGAGGCAAAC CUUUCGCCAUCGAGGCCGAGAACAGCAUCCUGGACAUCAGCGGCUUCAGCAAGCAGUA CAACUGCGCCUUUAUUUGGCAGAAGGACGGAGUGAAGAAGCUGAACCUGUACCUGAUC AUCAACUAUUUCAAGGGCGGCAAGCUGAGAUUCAAGAAGAUCAAGCCUGAAGCCUUCG AGGCCAACAGAUUCUACACCGUGAUUAACAAGAAAAGCGGAGAGAUCGUGCCAAUGGA AGUGAACUUCAACUUCGACGACCCUAACCUGAUCAUCCUGCCCCUGGCAUUUGGCAAG CGGCAGGGCAGAGAGUUCAUCUGGAACGACCUGCUGUCUCUGGAGACCGGCAGCCUGA AGCUGGCCAACGGCAGAGUGAUCGAGAAGACACUGUACAACAGACGAACCAGACAAGA CGAGCCCGCCCUGUUUGUGGCCCUGACCUUCGAGAGAAGAGAGGUGCUGGACAGCAGC AAUAUCAAGCCUAUGAACCUGAUCGGCGUGGACCGGGGCGAGAACAUCCCUGCCGUGA UCGCCCUUACCGACCCCGAGGGAUGCCCUCUGAGCCGGUUUAAAGACAGCCUGGGCAA C C CUAC C CACAU C CU GAGAAUU G G C GAGU C CUACAAG GAGAAG CAGAGAAC CAU C CAG GCCAAGAAGGAGGUGGAGCAGCGGCGGGCUGGCGGCUACUCCCGGAAGUACGCCAGCA Full-length RNA sequence of mRNA encoding X-editor 812 SEQ ID NO Nucleotide Sequence* Description AGGCCAAGAACCUGGCCGACGACAUGGUUAGAAAUACCGCCAGAGACCUCCUGUACUA CGCUGUGACCCAGGACGCCAUGCUGAUCUUCGAGAACCUGAGCAGAGGCUUCGGCAGA CAGGGCAAGAGAACCUUCAUGGCCGAGAGACAGUACACCCGGAUGGAGGACUGGCUGA CCGCCAAGCUGGCCUACGAGGGCCUGCCCUCUAAGACCUACCUGUCCAAGACCUUGGC ACAGUACACCAGCAAGACAUGCUCUAACUGCGGCUUCACAAUCACGAGCGCCGACUAC GACCGGGUGCUGGAGAAACUGAAGAAGACCGCCACAGGCUGGAUGACCACCAUUAACG GCAAGGAGCUGAAGGUGGAGGGCCAGAUCACCUACUACAACAGGUACAAACGGCAGAA CGUGGUGAAGGACCUGAGCGUGGAACUGGAUAGACUGAGCGAGGAAAGCGUAAACAAU GACAUCAGCAGCUGGACCAAGGGCCGGAGCGGCGAGGCCCUGAGCCUGCUGAAGAAGA GAUUCUCCCACAGACCAGUGCAGGAGAAGUUCGUGUGUCUGAACUGCGGCUUCGAGAC CCACGCCGACGAGCAAGCCGCCCUGAACAUCGCCCGGUCUUGGCUUUUCCUGCGGAGC CAGGAGUACAAGAAGUACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCU UCGUCGAGACCUGGCAGAGCUUCUACAGAAAGAAGCUGAAGGAGGUGUGGAAGCCUGC CGUGGGAAGCCCCGCUGCCAAGAGAGUGAAGCUGGACUAAGCUGCCUUCUGCGGGGCU UGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAU AAAG C CU GAGUAG GAAGU CUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 51 AGGAAAm4rAAGAGAGAAAAGAAGAGm4rAAGAAGAAAm4rAm4rAAGAGCCACCAm4rGGCC CCm4rGCm4rGCCAAGAGAGm4rGAAGCm4rGGAm4rAGCAGACAGGAGAm4rCAAGCGGAm4rm 4rAAm4rAAAAm4rm4rCGGAGAAGACm4rGGm4rGAAGGAm4rm4rCm4rAACACAAAGAAGGCm4r GGCAAGACAGGCCCm4rAm4rGAAGACACm4rGCm4rGGm4rGAGAGm4rGAm4rGACACCCGAC Cm4rGAGAGAAAGACm4rGGAAAACCm4rGAGAAAGAAGCCm4rGAGAAm4rAm4rCCCCCAGC CCAm4rCAGCAACACAAGCCGGGCCAACCm4rGAAm4rAAGCm4rGCm4rGACCGACm4rACAC CGAAAm4rGAAGAAGGCCAm4rCCm4rGCACGm4rGm4rAm4rm4rGGGAAGAGm4rm4rCCAGAAA GACCCAGm4rCGGCCm4rGAm4rGAGCAGAGm4rGGCm4rCAGCCm4rGCCAGCAAGAAGAm4rC GAm4rCAGAACAAGCm4rGAAGCCCGAAAm4rGGACGAGAAGGGGAACCm4rGACAACCGCC GGCm4rm4rm4rGCCm4rGm4rAGCCAGm4rGCGGCCAGCCCCm4rGm4rm4rm4rGm4rGm4rACAAAC m4rGGAACAGGm4rGAGCGAAAAGGGCAAGGCm4rm4rACACGAAm4rm4rACm4rm4rCGGCAGA m4rGCAACGm4rGGCCGAGCACGAGAAGCm4rGAm4rCCm4rGCm4rGGCCCAGCm4rGAAGCCm 4rGAGAAGGAm4rAGCGAm4rGAGGCAGm4rGACAm4rAm4rm4rCCCm4rGGGCAAGm4rm4rCGGA CAGCGGGCCCm4rGGAm4rm4rm4rm4rm4rAm4rm4rCCAm4rm4rCAm4rGm4rGACCAAGGAAm4rCC ACCCACCCCGm4rCAAGCCm4rCm4rm4rGCCCAAAm4rm4rGCCGGCAACAGAm4rACGCCAGC GGCCCCGm4rGGGCAAGGCCCm4rGAGCGACGCCm4rGm4rAm4rGGGCACCAm4rCGCCAGCm 4rm4rCCm4rGm4rCm4rAAGm4rACCAGGACAm4rm4rAm4rCAm4rCGAGCACCAGAAGGm4rGGm4r GAAGGGCAACCAGAAGAGACm4rGGAGAGCCm4rGCGCGAGCm4rGGCCGGCAAGGAAAAC Cm4rGGAGm4rAm4rCCm4rAGCGm4rGACCCm4rGCCm4rCCm4rCAGCCm4rCAm4rACAAAGGAG GGCGm4rGGAm4rGCCm4rACAACGAAGm4rGAm4rCGCCCGGGm4rGCGGAm4rGm4rGGGm4rGA ACCm4rGAAm4rCm4rGm4rGGCAGAAGCm4rGAAGCm4rGm4rCm4rAGAGACGACGCCAAGCCC Cm4rGCm4rGAGACm4rGAAGGGCm4rm4rCCCCAGCm4rm4rCCCm4rCm4rGGm4rGGAGAGACAG GCAAAm4rGAAGm4rGGACm4rGGm4rGGGACAm4rGGm4rGm4rGm4rAACGm4rGAAGAAGCm4rG Am4rCAAm4rGAGAAGAAGGAGGACGGCAAAGm4rGm4rm4rCm4rGGCAGAAm4rCm4rGGCCGG Cm4rACAAGCGm4rCAGGAGGCCCm4rGCGGCCCm4rACCm4rGAGCAGCGAGGAAGACAGAA AGAAGGGCAAGAAGm4rm4rCGCCCGGm4rAm4rCAGCm4rGGGGGACCm4rGCm4rGCm4rGCAC Cm4rCGAGAAGAAGCACGGCGAAGACm4rGGGGGAAGGm4rGm4rACGAm4rGAGGCCm4jGGG AGCGGAm4rCGAm4rAAGAAGGm4rGGAGGGCCm4rGAGCAAGCACAm4rCAAGCm4rGGAGGA GGAACGGAGAm4rCm4rGAGGACGCCCAGAGCAAGGCCGCCCm4rGACCGACm4rGGCm4rGA GAGCCAAGGCCAGCm4rm4rCGm4rCAm4rCGAGGGGCm4rGAAGGAGGCCGACAAGGACGAG m4rm4rCm4rGCCGGm4rGCGAACm4rGAAGCm4rGCAGAAGm4rGGm4rACGGAGAm4rCm4rGAGA GGCAAACCm4rm4rm4rCGCCAm4rCGAGGCCGAGAACAGCAm4rCCm4rGGACAm4rCAGCGGC m4rm4rCAGCAAGCAGm4rACAACm4rGCGCCm4rm4rm4rAm4rm4rm4rGGCAGAAGGACGGAGm4r GAAGAAGCm4rGAACCm4rGm4rACCm4rGAm4rCAm4rCAACm4rAm4rm4rm4rCAAGGGCGGCAA GCm4rGAGAm4rm4rCAAGAAGAm4rCAAGCCm4rGAAGCCm4rm4rCGAGGCCAACAGAm4m4rC m4rACACCGm4rGAm4rm4rAACAAGAAAAGCGGAGAGAm4rCGm4rGCCAAm4rGGAAGm4jGAA Cm4rm4rCAACm4rm4rCGACGACCCm4rAACCm4rGAm4rCAm4rCCm4rGCCCCm4rGGCAm4m4rm 4rGGCAAGCGGCAGGGCAGAGAGm4rm4rCAm4rCm4rGGAACGACCm4rGCm4rGm4rCm4rCm4rG GAGACCGGCAGCCm4rGAAGCm4rGGCCAACGGCAGAGm4rGAm4rCGAGAAGACACm4jGm4r Full-length Nl-methylpseud ouridine substituted RNA sequence of mRNA encoding X-editor 491 SEQ ID NO Nucleotide Sequence* Description ACAACAGACGAACCAGACAAGACGAGCCCGCCCm4rGm4rm4rm4rGm4rGGCCCm4rGACCm4r m4rCGAGAGAAGAGAGGm4rGCm4rGGACAGCAGCAAm4rAm4rCAAGCCm4rAm4rGAACCm4rG Am4rCGGCGm4rGGACCGGGGCGAGAACAm4rCCCm4rGCCGm4rGAm4rCGCCCm4rm4rACCGA CCCCGAGGGAm4rGCCCm4rCm4rGAGCCGGm4rm4rm4rAAAGACAGCCm4rGGGCAACCCm4rA CCCACAm4rCCm4rGAGAAm4rm4rGGCGAGm4rCCm4rACAAGGAGAAGCAGAGAACCAm4rCC AGGCCAAGAAGGAGGm4rGGAGCAGCGGCGGGCm4rGGCGGCm4rACm4rCCCGGAAGm4rAC GCCAGCAAGGCCAAGAACCm4rGGCCGACGACAm4rGGm4rm4rAGAAAm4rACCGCCAGAGA CCm4rCCm4rGm4rACm4rACGCm4rGm4rGACCCAGGACGCCAm4rGCm4rGAm4rCm4rm4rCGAGA ACCm4rGAGCAGAGGCm4rm4rCGGCAGACAGGGCAAGAGAACCm4rm4rCAm4rGGCCGAGAG ACAGm4rACACCCGGAm4rGGAGGACm4rGGCm4rGACCGCCAAGCm4rGGCCm4rACGAGGGC Cm4rGm4rCm4rAAGACCm4rACCm4rGm4rCCAAGACCm4rm4rGGCACAGm4rACACCAGCAAGA CAm4rGCm4rCm4rAACm4rGCGGCm4rm4rCACAAm4rCACGAGCGCCGACm4rACGACCGGGm4r GCm4rGGAGAAACm4rGAAGAAGACCGCCACAGGCm4rGGAm4rGACCACCAm4rm4rAACGGC AAGGAGCm4rGAAGGm4rGGAGGGCCAGAm4rCACCm4rACm4rACAACAGGm4rACAAACGGC AGAACGm4rGGm4rGAAGGACCm4rGAGCGm4rGGAACm4rGGAm4rAGACm4rGAGCGAGGAAA GCGm4rAAACAAm4rGACAm4rCAGCAGCm4rGGACCAAGGGCCGGAGCGGCGAGGCCCm4rG AGCCm4rGCm4rGAAGAAGAGAm4rm4rCm4rCCCACAGACCAGm4rGCAGGAGAAGm4rm4jCGm 4rGm4rGm4rCm4rGAACm4rGCGGCm4rm4rCGAGACCCACGCCGACGAGCAAGCCGCCCm4rGA ACAm4rCGCCCGGm4rCm4rm4rGGCm4rm4rm4rm4rCCm4rGCGGAGCCAGGAGm4rACAAGAAGm 4rACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCm4rm4rCGm4rCGAGACCm 4rGGCAGAGCm4rm4rCm4rACAGAAAGAAGCm4rGAAGGAGGm4rGm4rGGAAGCCm4rGCCGm4r GGGAAGCCCCGCm4rGCCAAGAGAGm4rGAAGCm4rGGACm4rAAGCm4rGCCm4rm4rCm4jGCG G G G Crrulrnulr G C Crrulrrmlr Cmilr G G C C Anu|r G C C Crrulrrmlr Crrulrrmlr Cmilr Cmilr C C Crrulrrmlr G GAG Cmilr Gm4rACCm4rCm4rm4rGGm4rCm4rm4rm4rGAAm4rAAAGCCm4rGAGm4rAGGAAGm4rCm4rAGAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA 52 AGGAAAm4rAAGAGAGAAAAGAAGAGm4rAAGAAGAAAm4rAm4rAAGAGCCACCAm4rGGCC CCm4rGCm4rGCCAAGAGAGm4rGAAGCm4rGGAm4rAGCAGACAGGAGAm4rCAAGCGGAm4rm 4rAAm4rAAAAm4rm4rCGGAGAAGACm4rGGm4rGAAGGAm4rm4rCm4rAACACAAAGAAGGCm4r GGCAAGACAGGCCCm4rAm4rGAAGACACm4rGCm4rGGm4rGAGAGm4rGAm4rGACACCCGAC Cm4rGAGAGAAAGACm4rGGAAAACCm4rGAGAAAGAAGCCm4rGAGAAm4rAm4rCCCCCAGC CCAm4rCAGCAACACAAGCCGGGCCAACCm4rGAAm4rAAGCm4rGCm4rGACCGACm4rACAC CGAAAm4rGAAGAAGGCCAm4rCCm4rGCACGm4rGm4rAm4rm4rGGGAAGAGm4rm4rCCAGAAA GACCCAGm4rCGGCCm4rGAm4rGAGCAGAGm4rGGCm4rCAGCCm4rGCCAGCAAGAAGAm4rC GAm4rCAGAACAAGCm4rGAAGCCCGAAAm4rGGACGAGAAGGGGAACCm4rGACAACCGCC GGCm4rm4rm4rGCCm4rGm4rAGCCAGm4rGCGGCCAGCCCCm4rGm4rm4rm4rGm4rGm4rACAAAC m4rGGAACAGGm4rGAGCGAAAAGGGCAAGGCm4rm4rACACGAAm4rm4rACm4rm4rCGGCAGA m4rGCAACGm4rGGCCGAGCACGAGAAGCm4rGAm4rCCm4rGCm4rGGCCCAGCm4rGAAGCCm 4rGAGAAGGAm4rAGCGAm4rGAGGCAGm4rGACAm4rAm4rm4rCCCm4rGGGCAAGm4rm4rCGGA CAGCGGGCCCm4rGGAm4rm4rm4rm4rm4rAm4rm4rCCAm4rm4rCAm4rGm4rGACCAAGGAAm4rCC ACCCACCCCGm4rCAAGCCm4rCm4rm4rGCCCAAAm4rm4rGCCGGCAACAGAm4rACGCCAGC GGCCCCGm4rGGGCAAGGCCCm4rGAGCGACGCCm4rGm4rAm4rGGGCACCAm4rCGCCAGCm 4rm4rCCm4rGm4rCm4rAAGm4rACCAGGACAm4rm4rAm4rCAm4rCGAGCACCAGAAGGm4rGGm4r GAAGGGCAACCAGAAGAGACm4rGGAGAGCCm4rGCGCGAGCm4rGGCCGGCAAGGAAAAC Cm4rGGAGm4rAm4rCCm4rAGCGm4rGACCCm4rGCCm4rCCm4rCAGCCm4rCAm4rACAAAGGAG GGCGm4rGGAm4rGCCm4rACAACGAAGm4rGAm4rCGCCCGGGm4rGCGGAm4rGm4rGGGm4rGA ACCm4rGAAm4rCm4rGm4rGGCAGAAGCm4rGAAGCm4rGm4rCm4rAGAGACGACGCCAAGCCC Cm4rGCm4rGAGACm4rGAAGGGCm4rm4rCCCCAGCm4rm4rCCCm4rCm4rGGm4rGGAGAGACAG GCAAAm4rGAAGm4rGGACm4rGGm4rGGGACAm4rGGm4rGm4rGm4rAACGm4rGAAGAAGCm4rG Am4rCAAm4rGAGAAGAAGGAGGACGGCAAAGm4rGm4rm4rCm4rGGCAGAAm4rCm4rGGCCGG Cm4rACAAGCGm4rCAGGAGGCCCm4rGCGGCCCm4rACCm4rGAGCAGCGAGGAAGACAGAA AGAAGGGCAAGAAGm4rm4rCGCCCGGm4rAm4rCAGCm4rGGGGGACCm4rGCm4rGCm4rGCAC Cm4rCGAGAAGAAGCACGGCGAAGACm4rGGGGGAAGGm4rGm4rACGAm4rGAGGCCm4jGGG AGCGGAm4rCGAm4rAAGAAGGm4rGGAGGGCCm4rGAGCAAGCACAm4rCAAGCm4rGGAGGA GGAACGGAGAm4rCm4rGAGGACGCCCAGAGCAAGGCCGCCCm4rGACCGACm4rGGCm4rGA GAGCCAAGGCCAGCm4rm4rCGm4rCAm4rCGAGGGGCm4rGAAGGAGGCCGACAAGGACGAG Full-length Nl-methylpseud ouridine substituted RNA sequence of mRNA encoding X-editor 515 SEQ ID NO Nucleotide Sequence* Description m4rm4rCm4rGCCGGm4rGCGAACm4rGAAGCm4rGCAGAAGm4rGGm4rACGGAGAm4rCm4rGAGA GGCAAACCm4rm4rm4rCGCCAm4rCGAGGCCGAGAACAGCAm4rCCm4rGGACAm4rCAGCGGC m4rm4rCAGCAAGCAGm4rACAACm4rGCGCCm4rm4rm4rAm4rm4rm4rGGCAGAAGGACGGAGm4r GAAGAAGCm4rGAACCm4rGm4rACCm4rGAm4rCAm4rCAACm4rAm4rm4rm4rCAAGGGCGGCAA GCm4rGAGAm4rm4rCAAGAAGAm4rCAAGCCm4rGAAGCCm4rm4rCGAGGCCAACAGAm4m4rC m4rACACCGm4rGAm4rm4rAACAAGAAAAGCGGAGAGAm4rCGm4rGCCAAm4rGGAAGm4jGAA Cm4rm4rCAACm4rm4rCGACGACCCm4rAACCm4rGAm4rCAm4rCCm4rGCCCCm4rGGCAm4m4rm 4rGGCAAGCGGCAGGGCAGAGAGm4rm4rCAm4rCm4rGGAACGACCm4rGCm4rGm4rCm4rCm4rG GAGACCGGCAGCCm4rGAAGCm4rGGCCAACGGCAGAGm4rGAm4rCGAGAAGACACm4jGm4r ACAACAGACGAACCAGACAAGACGAGCCCGCCCm4rGm4rm4rm4rGm4rGGCCCm4rGACCm4r m4rCGAGAGAAGAGAGGm4rGCm4rGGACAGCAGCAAm4rAm4rCAAGCCm4rAm4rGAACCm4rG Am4rCGGCGm4rGGACCGGGGCGAGAACAm4rCCCm4rGCCGm4rGAm4rCGCCCm4rm4rACCGA CCCCGAGGGAm4rGCCCm4rCm4rGAGCCGGm4rm4rm4rAAAGACAGCCm4rGGGCAACCCm4rA CCCACAm4rCCm4rGAGAAm4rm4rGGCGAGm4rCCm4rACAAGGAGAAGCAGAGAACCAm4rCC AGGCCAAGAAGGAGGm4rGGAGCAGCGGCGGGCm4rGGCGGCm4rACm4rCCCGGAAGm4rAC GCCAGCAAGGCCAAGAACCm4rGGCCGACGACAm4rGGm4rm4rAGAAAm4rACCGCCAGAGA CCm4rCCm4rGm4rACm4rACGCm4rGm4rGACCCAGGACGCCAm4rGCm4rGAm4rCm4rm4rCGAGA ACCm4rGAGCAGAGGCm4rm4rCGGCAGACAGGGCAAGAGAACCm4rm4rCAm4rGGCCGAGAG ACAGm4rACACCCGGAm4rGGAGGACm4rGGCm4rGACCGCCAAGCm4rGGCCm4rACGAGGGC Cm4rGCCCm4rCm4rAAGACCm4rACCm4rGm4rCCAAGACCm4rm4rGGCACAGm4rACACCAGCA AGACAm4rGCm4rCm4rAACm4rGCGGCm4rm4rCACAAm4rCACGAGCGCCGACm4rACGACCGG Gm4rGCm4rGGAGAAACm4rGAAGAAGACCGCCACAGGCm4rGGAm4rGACCACCAm4rm4jAAC GGCAAGGAGCm4rGAAGGm4rGGAGGGCCAGAm4rCACCm4rACm4rACAACAGGm4rACAAAC GGCAGAACGm4rGGm4rGAAGGACCm4rGAGCGm4rGGAACm4rGGAm4rAGACm4rGAGCGAGG AAAGCGm4rAAACAAm4rGACAm4rCAGCAGCm4rGGACCAAGGGCCGGAGCGGCGAGGCCC m4rGAGCCm4rGCm4rGAAGAAGAGAm4rm4rCm4rCCCACAGACCAGm4rGCAGGAGAAGm4rm4r CGm4rGm4rGm4rCm4rGAACm4rGCGGCm4rm4rCGAGACCCACGCCGACGAGCAAGCCGCCCm 4rGAACAm4rCGCCCGGm4rCm4rm4rGGCm4rm4rm4rm4rCCm4rGCGGAGCCAGGAGm4rACAAGA AGm4rACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCm4rm4rCGm4rCGAGA CCm4rGGCAGAGCm4rm4rCm4rACAGAAAGAAGCm4rGAAGGAGGm4rGm4rGGAAGCCm4jGCC Gm4rGGGAAGCCCCGCm4rGCCAAGAGAGm4rGAAGCm4rGGACm4rAAGCm4rGCCm4rm4jCm4r GCGGGGCm4rm4rGCCm4rm4rCm4rGGCCAm4rGCCCm4rm4rCm4rm4rCm4rCm4rCCCm4rm4rGCAC Cmilr GmilrAC Cmilr Crrulrrmlr G Gmi|r Cm4rm4rm4r GAAmilrAAAG C Cmilr GAGmi|rAG GAAGmilr CmilrAG AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA 53 AGGAAAm4rAAGAGAGAAAAGAAGAGm4rAAGAAGAAAm4rAm4rAAGAGCCACCAm4rGGCC CCm4rGCm4rGCCAAGAGAGm4rGAAGCm4rGGAm4rAGCAGACAGGAGAm4rCAAGCGGAm4rm 4rAAm4rAAAAm4rm4rCGGAGAAGACm4rGGm4rGAAGGAm4rm4rCm4rAACACAAAGAAGGCm4r GGCAAGACACGGGGCCCm4rAm4rGAAGACACm4rGCm4rGGm4rGAGAGm4rGAm4rGACACCC GACCm4rGAGAGAAAGACm4rGGAAAACCm4rGAGAAAGAAGCCm4rGAGAAm4rAm4rCCCCC AGCCCAm4rCAGCAACACAAGCCGGGCCAACCm4rGAAm4rAAGCm4rGCm4rGACCGACm4rA CACCGAAAm4rGAAGAAGGCCAm4rCCm4rGCACGm4rGm4rAm4rm4rGGGAAGAGm4rm4rCCAG AAAGACCCAGm4rCGGCCm4rGAm4rGAGCAGAGm4rGGCm4rCAGCCm4rGCCAGCAAGAAGA m4rCGAm4rCAGAACAAGCm4rGAAGCCCGAAAm4rGGACGAGAAGGGGAACCm4rGACAACC GCCGGCm4rm4rm4rGCCm4rGm4rAGCCAGm4rGCGGCCAGCCCCm4rGm4rm4rm4rGm4rGm4jACA AACm4rGGAACAGGm4rGAGCGAAAAGGGCAAGGCm4rm4rACACGAAm4rm4rACm4rm4rCGGC AGAm4rGCAACGm4rGGCCGAGCACGAGAAGCm4rGAm4rCAAGCm4rGGCCCAGCm4rGAAGC Cm4rGAGAAGGAm4rAGCGAm4rGAGGCAGm4rGACAm4rAm4rm4rCCCm4rGGGCAAGm4rm4rCG GACAGCGGGCCCm4rGGAm4rm4rm4rm4rm4rAm4rm4rCCAm4rm4rCAm4rGm4rGACCAAGGAAm^ CCACCCACCCCGm4rCAAGCCm4rCm4rm4rGCCCAAAm4rm4rGCCGGCAACAGAm4rACGCCm 4rCCAGCCCCGm4rGGGCAAGGCCCm4rGAGCGACGCCm4rGm4rAm4rGGGCACCAm4rCGCCA GCm4rm4rCCm4rGm4rCm4rAAGm4rACCAGGACAm4rm4rAm4rCAm4rCGAGCACCAGAAGGm4rG Gm4rGAAGGGCAACCAGAAGAGACm4rGGAGAGCCm4rGCGCGAGCm4rGGCCGGCAAGGAA AACCm4rGGAGm4rAm4rCCm4rAGCGm4rGACCCm4rGCCm4rCCm4rCAGCCm4rCAm4rACAAAG GAGGGCGm4rGGAm4rGCCm4rACAACGAAGm4rGAm4rCGCCCGGGm4rGCGGAm4rGm4rGGGm 4rGAACCm4rGAAm4rCm4rGm4rGGCAGAAGCm4rGAAGCm4rGm4rCm4rAGAGACGACGCCAAG Full-length Nl-methylpseud ouridine substituted RNA sequence of mRNA encoding X-editor 676 SEQ ID NO Nucleotide Sequence* Description CCCCm4rGCm4rGAGACm4rGAAGGGCm4rm4rCCCCAGCm4rm4rCCCm4rCm4rGGm4rGGAGAGA CAGGCAAAm4rGAAGm4rGGACm4rGGm4rGGGACAm4rGGm4rGm4rGm4rAACGm4rGAAGAAGC m4rGAm4rCAAm4rGAGAAGAAGGAGGACGGCAAAGm4rGm4rm4rCm4rGGCAGAAm4rCm4jGGC CGGCm4rACAAGCGm4rCAGGAGGCCCm4rGCGGCCCm4rACCm4rGAGCAGCGAGGAAGACA GAAAGAAGGGCAAGAAGm4rm4rCGCCCGGm4rAm4rCAGCm4rGGGGGACCm4rGCm4rGCm4rG CACCm4rCGAGAAGAAGCACGGCGAAGACm4rGGGGGAAGGm4rGm4rACGAm4rGAGGCCm4r GGGAGCGGAm4rCGAm4rAAGAAGGm4rGGAGGGCCm4rGAGCAAGCACAm4rCAAGCm4jGGA GGAGGAACGGAGAm4rCm4rGAGGACGCCCAGAGCAAGGCCGCCCm4rGACCGACm4rGGCm 4rGAGAGCCAAGGCCAGCm4rm4rCGm4rCAm4rCGAGGGGCm4rGAAGGAGGCCGACAAGGAC GAGm4rm4rCm4rGCCGGm4rGCGAACm4rGAAGCm4rGCAGAAGm4rGGm4rACGGAGAm4rCm4rG AGAGGCAAACCm4rm4rm4rCGCCAm4rCGAGGCCGAGAACAGCAm4rCCm4rGGACAm4rCAGC GGCm4rm4rCAGCAAGCAGm4rACAACm4rGCGCCm4rm4rm4rAm4rm4rm4rGGCAGAAGGACGGA Gm4rGAAGAAGCm4rGAACCm4rGm4rACCm4rGAm4rCAm4rCAACm4rAm4rm4rm4rCAAGGGCGG CAAGCm4rGAGAm4rm4rCAAGAAGAm4rCAAGCCm4rGAAGCCm4rm4rCGAGGCCAACAGAm4r m4rCm4rACACCGm4rGAm4rm4rAACAAGAAAAGCGGAGAGAm4rCGm4rGCCAAm4rGGAAGm4r GAACm4rm4rCAACm4rm4rCGACGACCCm4rAACCm4rGAm4rCAm4rCCm4rGCCCCm4rGGCAm4r m4rm4rGGCAAGCGGCAGGGCAGAGAGm4rm4rCAm4rCm4rGGAACGACCm4rGCm4rGm4rCm4rC m4rGGAGACCGGCAGCCm4rGAAGCm4rGGCCAACGGCAGAGm4rGAm4rCGAGAAGACACm4r Gm4rACAACAGACGAACCAGACAAGACGAGCCCGCCCm4rGm4rm4rm4rGm4rGGCCCm4jGAC Cm4rm4rCGAGAGAAGAGAGGm4rGCm4rGGACAGCAGCAAm4rAm4rCAAGCCm4rAm4rGAACC m4rGAm4rCGGCGm4rGGACCGGGGCGAGAACAm4rCCCm4rGCCGm4rGAm4rCGCCCm4rm4rAC CGACCCCGAGGGAm4rGCCCm4rCm4rGAGCCGGm4rm4rm4rAAAGACAGCCm4rGGGCAACCC m4rACCCACAm4rCCm4rGAGAAm4rm4rGGCGAGm4rCCm4rACAAGGAGAAGCAGAGAACCAm 4rCCAGGCCAAGAAGGAGGm4rGGAGCAGCGGCGGGCm4rGGCGGCm4rACm4rCCCGGAAGm 4rACGCCAGCAAGGCCAAGAACCm4rGGCCGACGACAm4rGGm4rm4rAGAAAm4rACCGCCAG AGACCm4rCCm4rGm4rACm4rACGCm4rGm4rGACCCAGGACGCCAm4rGCm4rGAm4rCm4rm4rCG AGAACCm4rGAGCAGAGGCm4rm4rCGGCAGACAGGGCAAGAGAACCm4rm4rCAm4rGGCCGA GAGACAGm4rACACCCGGAm4rGGAGGACm4rGGCm4rGACCGCCAAGCm4rGGCCm4rACGAG GGCCm4rGCCCm4rCm4rAAGACCm4rACCm4rGm4rCCAAGACCm4rm4rGGCACAGm4rACACCA GCAAGACAm4rGCm4rCm4rAACm4rGCGGCm4rm4rCACAAm4rCACGAGCGCCGACm4rACGAC CGGGm4rGCm4rGGAGAAACm4rGAAGAAGACCGCCACAGGCm4rGGAm4rGACCACCAm4rm4r AACGGCAAGGAGCm4rGAAGGm4rGGAGGGCCAGAm4rCACCm4rACm4rACAACAGGm4jACA AACGGCAGAACGm4rGGm4rGAAGGACCm4rGAGCGm4rGGAACm4rGGAm4rAGACm4rGAGCG AGGAAAGCGm4rAAACAAm4rGACAm4rCAGCAGCm4rGGACCAAGGGCCGGAGCGGCGAGG CCCm4rGAGCCm4rGCm4rGAAGAAGAGAm4rm4rCm4rCCCACAGACCAGm4rGCAGGAGAAGm 4rm4rCGm4rGm4rGm4rCm4rGAACm4rGCGGCm4rm4rCGAGACCCACGCCGACGAGCAAGCCGC CCm4rGAACAm4rCGCCCGGm4rCm4rm4rGGCm4rm4rm4rm4rCCm4rGCGGAGCCAGGAGm4jACA AGAAGm4rACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCm4rm4rCGm4rCG AGACCm4rGGCAGAGCm4rm4rCm4rACAGAAAGAAGCm4rGAAGGAGGm4rGm4rGGAAGCCm4r GCCGm4rGGGAAGCCCCGCm4rGCCAAGAGAGm4rGAAGCm4rGGACm4rAAGCm4rGCCm4rm4r Cm4rGCGGGGCm4rm4rGCCm4rm4rCm4rGGCCAm4rGCCCm4rm4rCm4rm4rCm4rCm4rCCCm4m4rG CACCm4rGm4rACCm4rCm4rm4rGGm4rCm4rm4rm4rGAAm4rAAAGCCm4rGAGm4rAGGAAGm4rCm i|rAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA 54 AGGAAAm4rAAGAGAGAAAAGAAGAGm4rAAGAAGAAAm4rAm4rAAGAGCCACCAm4rGGCC CCm4rGCm4rGCCAAGAGAGm4rGAAGCm4rGGAm4rAGCAGACAGGAGAm4rCAAGCGGAm4rm 4rAAm4rAAAAm4rm4rCGGAGAAGACm4rGGm4rGAAGGAm4rm4rCm4rAACACAAAGAAGGCm4r GGCAAGACAGGCCCm4rAm4rGAAGACACm4rGCm4rGGm4rGAGAGm4rGAm4rGACACCCGAC Cm4rGAGAGAAAGACm4rGGAAAACCm4rGAGAAAGAAGCCm4rGAGAAm4rAm4rCCCCCAGC CCAm4rCAGCAACACAAGCCGGGCCAACCm4rGAAm4rAAGCm4rGCm4rGACCGACm4rACAC CGAAAm4rGAAGAAGGCCAm4rCCm4rGCACGm4rGm4rAm4rm4rGGGAAGAGm4rm4rCCAGAAA GACCCAGm4rCGGCCm4rGAm4rGAGCAGAGm4rGGCm4rCAGCCm4rGCCAGCAAGAAGAm4rC GAm4rCAGAACAAGCm4rGAAGCCCGAAAm4rGGACGAGAAGGGGAACCm4rGACAACCGCC GGCm4rm4rm4rGCCm4rGm4rAGCCAGm4rGCGGCCAGCCCCm4rGm4rm4rm4rGm4rGm4rACAAAC m4rGGAACAGGm4rGAGCGAAAAGGGCAAGGCm4rm4rACACGAAm4rm4rACm4rm4rCGGCAGA m4rGCAACGm4rGGCCGAGCACGAGAAGCm4rGAm4rCCm4rGCm4rGGCCCAGCm4rGAAGCCm Full-length Nl-methylpseud ouridine substituted RNA sequence of mRNA encoding X-editor 812 SEQ ID NO Nucleotide Sequence* Description 4rGAGAAGGAm4rAGCGAm4rGAGGCAGm4rGACAm4rAm4rm4rCCCm4rGGGCAAGm4rm4rCGGA CAGCGGGCCCm4rGGAm4rm4rm4rm4rm4rAm4rm4rCCAm4rm4rCAm4rGm4rGACCAAGGAAm4rCC ACCCACCCCGm4rCAAGCCm4rCm4rm4rGCCCAAAm4rm4rGCCGGCAACAGAm4rACGCCAGC GGCCCCGm4rGGGCAAGGCCCm4rGAGCGACGCCm4rGm4rAm4rGGGCACCAm4rCGCCAGCm 4rm4rCCm4rGm4rCm4rAAGm4rACCAGGACAm4rm4rAm4rCAm4rCGAGCACCAGAAGGm4rGGm4r GAAGGGCAACCAGAAGAGACm4rGGAGAGCCm4rGCGCGAGCm4rGGCCGGCAAGGAAAAC Cm4rGGAGm4rAm4rCCm4rAGCGm4rGACCCm4rGCCm4rCCm4rCAGCCm4rCAm4rACAAAGGAG GGCGm4rGGAm4rGCCm4rACAACGAAGm4rGAm4rCGCCCGGGm4rGCGGAm4rGm4rGGGm4rGA ACCm4rGAAm4rCm4rGm4rGGCAGAAGCm4rGAAGCm4rGm4rCm4rAGAGACGACGCCAAGCCC Cm4rGCm4rGAGACm4rGAAGAAGm4rm4rCCCCAGCm4rm4rCCCm4rCm4rGGm4rGGAGAGACAG GCAAAm4rGAAGm4rGGACm4rGGm4rGGGACAm4rGGm4rGm4rGm4rAACGm4rGAAGAAGCm4rG Am4rCAAm4rGAGAAGAAGGAGGACGGCAAAGm4rGm4rm4rCm4rGGCAGAAm4rCm4rGGCCGG Cm4rACAAGCGm4rCAGGAGGCCCm4rGCGGCCCm4rACCm4rGAGCAGCGAGGAAGACAGAA AGAAGGGCAAGAAGm4rm4rCGCCCGGm4rAm4rCAGCm4rGGGGGACCm4rGCm4rGCm4rGCAC Cm4rCGAGAAGAAGCACGGCGAAGACm4rGGGGGAAGGm4rGm4rACGAm4rGAGGCCm4jGGG AGCGGAm4rCGAm4rAAGAAGGm4rGGAGGGCCm4rGAGCAAGCACAm4rCAAGCm4rGGAGGA GGAACGGAGAm4rCm4rGAGGACGCCCAGAGCAAGGCCGCCCm4rGACCGACm4rGGCm4rGA GAGCCAAGGCCAGCm4rm4rCGm4rCAm4rCGAGGGGCm4rGAAGGAGGCCGACAAGGACGAG m4rm4rCm4rGCCGGm4rGCGAACm4rGAAGCm4rGCAGAAGm4rGGm4rACGGAGAm4rCm4rGAGA GGCAAACCm4rm4rm4rCGCCAm4rCGAGGCCGAGAACAGCAm4rCCm4rGGACAm4rCAGCGGC m4rm4rCAGCAAGCAGm4rACAACm4rGCGCCm4rm4rm4rAm4rm4rm4rGGCAGAAGGACGGAGm4r GAAGAAGCm4rGAACCm4rGm4rACCm4rGAm4rCAm4rCAACm4rAm4rm4rm4rCAAGGGCGGCAA GCm4rGAGAm4rm4rCAAGAAGAm4rCAAGCCm4rGAAGCCm4rm4rCGAGGCCAACAGAm4m4rC m4rACACCGm4rGAm4rm4rAACAAGAAAAGCGGAGAGAm4rCGm4rGCCAAm4rGGAAGm4jGAA Cm4rm4rCAACm4rm4rCGACGACCCm4rAACCm4rGAm4rCAm4rCCm4rGCCCCm4rGGCAm4m4rm 4rGGCAAGCGGCAGGGCAGAGAGm4rm4rCAm4rCm4rGGAACGACCm4rGCm4rGm4rCm4rCm4rG GAGACCGGCAGCCm4rGAAGCm4rGGCCAACGGCAGAGm4rGAm4rCGAGAAGACACm4jGm4r ACAACAGACGAACCAGACAAGACGAGCCCGCCCm4rGm4rm4rm4rGm4rGGCCCm4rGACCm4r m4rCGAGAGAAGAGAGGm4rGCm4rGGACAGCAGCAAm4rAm4rCAAGCCm4rAm4rGAACCm4rG Am4rCGGCGm4rGGACCGGGGCGAGAACAm4rCCCm4rGCCGm4rGAm4rCGCCCm4rm4rACCGA CCCCGAGGGAm4rGCCCm4rCm4rGAGCCGGm4rm4rm4rAAAGACAGCCm4rGGGCAACCCm4rA CCCACAm4rCCm4rGAGAAm4rm4rGGCGAGm4rCCm4rACAAGGAGAAGCAGAGAACCAm4rCC AGGCCAAGAAGGAGGm4rGGAGCAGCGGCGGGCm4rGGCGGCm4rACm4rCCCGGAAGm4rAC GCCAGCAAGGCCAAGAACCm4rGGCCGACGACAm4rGGm4rm4rAGAAAm4rACCGCCAGAGA CCm4rCCm4rGm4rACm4rACGCm4rGm4rGACCCAGGACGCCAm4rGCm4rGAm4rCm4rm4rCGAGA ACCm4rGAGCAGAGGCm4rm4rCGGCAGACAGGGCAAGAGAACCm4rm4rCAm4rGGCCGAGAG ACAGm4rACACCCGGAm4rGGAGGACm4rGGCm4rGACCGCCAAGCm4rGGCCm4rACGAGGGC Cm4rGCCCm4rCm4rAAGACCm4rACCm4rGm4rCCAAGACCm4rm4rGGCACAGm4rACACCAGCA AGACAm4rGCm4rCm4rAACm4rGCGGCm4rm4rCACAAm4rCACGAGCGCCGACm4rACGACCGG Gm4rGCm4rGGAGAAACm4rGAAGAAGACCGCCACAGGCm4rGGAm4rGACCACCAm4rm4jAAC GGCAAGGAGCm4rGAAGGm4rGGAGGGCCAGAm4rCACCm4rACm4rACAACAGGm4rACAAAC GGCAGAACGm4rGGm4rGAAGGACCm4rGAGCGm4rGGAACm4rGGAm4rAGACm4rGAGCGAGG AAAGCGm4rAAACAAm4rGACAm4rCAGCAGCm4rGGACCAAGGGCCGGAGCGGCGAGGCCC m4rGAGCCm4rGCm4rGAAGAAGAGAm4rm4rCm4rCCCACAGACCAGm4rGCAGGAGAAGm4rm4r CGm4rGm4rGm4rCm4rGAACm4rGCGGCm4rm4rCGAGACCCACGCCGACGAGCAAGCCGCCCm 4rGAACAm4rCGCCCGGm4rCm4rm4rGGCm4rm4rm4rm4rCCm4rGCGGAGCCAGGAGm4rACAAGA AGm4rACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCm4rm4rCGm4rCGAGA CCm4rGGCAGAGCm4rm4rCm4rACAGAAAGAAGCm4rGAAGGAGGm4rGm4rGGAAGCCm4jGCC Gm4rGGGAAGCCCCGCm4rGCCAAGAGAGm4rGAAGCm4rGGACm4rAAGCm4rGCCm4rm4jCm4r GCGGGGCm4rm4rGCCm4rm4rCm4rGGCCAm4rGCCCm4rm4rCm4rm4rCm4rCm4rCCCm4rm4rGCAC Cmilr GmilrAC Cmilr Crrulrrmlr G Gmi|r Cm4rm4rm4r GAAmilrAAAG C Cmilr GAGmi|rAG GAAGmilr CmilrAG AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA 55 AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCTGC TGCCAAGAGAGTGAAGCTGGATAGCAGACAGGAGATCAAGCGGATTAATAAAATTCGG AGAAGACTGGTGAAGGATTCTAACACAAAGAAGGCTGGCAAGACAGGCCCTATGAAGA Full-length DNA SEQ ID NO Nucleotide Sequence* Description CACTGCTGGTGAGAGTGATGACACCCGACCTGAGAGAAAGACTGGAAAACCTGAGAAA GAAGCCTGAGAATATCCCCCAGCCCATCAGCAACACAAGCCGGGCCAACCTGAATAAG CTGCTGACCGACTACACCGAAATGAAGAAGGCCATCCTGCACGTGTATTGGGAAGAGT TCCAGAAAGACCCAGTCGGCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAAGAT CGATCAGAACAAGCTGAAGCCCGAAATGGACGAGAAGGGGAACCTGACAACCGCCGGC TTTGCCTGTAGCCAGTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGAGCG AAAAGGGCAAGGCTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGAGAA GCTGATCCTGCTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGACATAT TCCCTGGGCAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCAAGG AATCCACCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCAGCGG CCCCGTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTGTCT AAGTACCAGGACATTATCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGAGAC TGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTGACCCT GCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGATCGCCCGG GTGCGGATGTGGGTGAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTAGAGACGACG CCAAGCCCCTGCTGAGACTGAAGGGCTTCCCCAGCTTCCCTCTGGTGGAGAGACAGGC AAAT GAAGT GGACT GGT GGGACAT GGT GT GTAACGT GAAGAAGCT GAT CAAT GAGAAG AAGGAGGACGGCAAAGTGTTCTGGCAGAATCTGGCCGGCTACAAGCGTCAGGAGGCCC TGCGGCCCTACCTGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGTTCGCCCGGTA TCAGCTGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCACGGCGAAGACTGGGGGAAG GTGTACGATGAGGCCTGGGAGCGGATCGATAAGAAGGTGGAGGGCCTGAGCAAGCACA TCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCCCAGAGCAAGGCCGCCCTGACCGA CTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGAGGGGCTGAAGGAGGCCGACAAGGAC GAGTTCTGCCGGTGCGAACTGAAGCTGCAGAAGTGGTACGGAGATCTGAGAGGCAAAC CTTTCGCCATCGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTA CAACTGCGCCTTTATTTGGCAGAAGGACGGAGTGAAGAAGCTGAACCTGTACCTGATC ATCAACTATTTCAAGGGCGGCAAGCTGAGATTCAAGAAGATCAAGCCTGAAGCCTTCG AGGCCAACAGATTCTACACCGTGATTAACAAGAAAAGCGGAGAGATCGTGCCAATGGA AGTGAACTTCAACTTCGACGACCCTAACCTGATCATCCTGCCCCTGGCATTTGGCAAG CGGCAGGGCAGAGAGTTCATCTGGAACGACCTGCTGTCTCTGGAGACCGGCAGCCTGA AGCTGGCCAACGGCAGAGTGATCGAGAAGACACTGTACAACAGACGAACCAGACAAGA CGAGCCCGCCCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGC AATATCAAGCCTATGAACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCGTGA TCGCCCTTACCGACCCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGGCAA CCCTACCCACATCCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATCCAG GCCAAGAAGGAGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCAGCA AGGCCAAGAACCTGGCCGACGACATGGTTAGAAATACCGCCAGAGACCTCCTGTACTA CGCTGTGACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGCAGA CAGGGCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGCTGA CCGCCAAGCTGGCCTACGAGGGCCTGTCTAAGACCTACCTGTCCAAGACCTTGGCACA GTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCGACTACGAC CGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACCATTAACGGCA AGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACAGGTACAAACGGCAGAACGT GGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAAGCGTAAACAATGAG ATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGCCTGCTGAAGAAGAGAT TCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAACTGCGGCTTCGAGACCCA CGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTTGGCTTTTCCTGCGGAGCCAG GAGTACAAGAAGTAC CAGACAAACAAGAC CACAG G CAACACAGACAAGAGAG C C T T C G TCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCTGAAGGAGGTGTGGAAGCCTGCCGT GGGAAGCCCCGCTGCCAAGAGAGTGAAGCTGGACTAAGCTGCCTTCTGCGGGGCTTGC CTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAA GCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA sequence of mRNA encoding X-editor 491 56 AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCTGC TGCCAAGAGAGTGAAGCTGGATAGCAGACAGGAGATCAAGCGGATTAATAAAATTCGG AGAAGACTGGTGAAGGATTCTAACACAAAGAAGGCTGGCAAGACAGGCCCTATGAAGA Full-length DNA SEQ ID NO Nucleotide Sequence* Description CACTGCTGGTGAGAGTGATGACACCCGACCTGAGAGAAAGACTGGAAAACCTGAGAAA GAAGCCTGAGAATATCCCCCAGCCCATCAGCAACACAAGCCGGGCCAACCTGAATAAG CTGCTGACCGACTACACCGAAATGAAGAAGGCCATCCTGCACGTGTATTGGGAAGAGT TCCAGAAAGACCCAGTCGGCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAAGAT CGATCAGAACAAGCTGAAGCCCGAAATGGACGAGAAGGGGAACCTGACAACCGCCGGC TTTGCCTGTAGCCAGTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGAGCG AAAAGGGCAAGGCTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGAGAA GCTGATCCTGCTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGACATAT TCCCTGGGCAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCAAGG AATCCACCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCAGCGG CCCCGTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTGTCT AAGTACCAGGACATTATCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGAGAC TGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTGACCCT GCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGATCGCCCGG GTGCGGATGTGGGTGAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTAGAGACGACG CCAAGCCCCTGCTGAGACTGAAGGGCTTCCCCAGCTTCCCTCTGGTGGAGAGACAGGC AAAT GAAGT GGACT GGT GGGACAT GGT GT GTAACGT GAAGAAGCT GAT CAAT GAGAAG AAGGAGGACGGCAAAGTGTTCTGGCAGAATCTGGCCGGCTACAAGCGTCAGGAGGCCC TGCGGCCCTACCTGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGTTCGCCCGGTA TCAGCTGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCACGGCGAAGACTGGGGGAAG GTGTACGATGAGGCCTGGGAGCGGATCGATAAGAAGGTGGAGGGCCTGAGCAAGCACA TCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCCCAGAGCAAGGCCGCCCTGACCGA CTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGAGGGGCTGAAGGAGGCCGACAAGGAC GAGTTCTGCCGGTGCGAACTGAAGCTGCAGAAGTGGTACGGAGATCTGAGAGGCAAAC CTTTCGCCATCGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTA CAACTGCGCCTTTATTTGGCAGAAGGACGGAGTGAAGAAGCTGAACCTGTACCTGATC ATCAACTATTTCAAGGGCGGCAAGCTGAGATTCAAGAAGATCAAGCCTGAAGCCTTCG AGGCCAACAGATTCTACACCGTGATTAACAAGAAAAGCGGAGAGATCGTGCCAATGGA AGTGAACTTCAACTTCGACGACCCTAACCTGATCATCCTGCCCCTGGCATTTGGCAAG CGGCAGGGCAGAGAGTTCATCTGGAACGACCTGCTGTCTCTGGAGACCGGCAGCCTGA AGCTGGCCAACGGCAGAGTGATCGAGAAGACACTGTACAACAGACGAACCAGACAAGA CGAGCCCGCCCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGC AATATCAAGCCTATGAACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCGTGA TCGCCCTTACCGACCCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGGCAA CCCTACCCACATCCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATCCAG GCCAAGAAGGAGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCAGCA AGGCCAAGAACCTGGCCGACGACATGGTTAGAAATACCGCCAGAGACCTCCTGTACTA CGCTGTGACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGCAGA CAGGGCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGCTGA CCGCCAAGCTGGCCTACGAGGGCCTGCCCTCTAAGACCTACCTGTCCAAGACCTTGGC ACAGTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCGACTAC GACCGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACCATTAACG GCAAGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACAGGTACAAACGGCAGAA CGTGGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAAGCGTAAACAAT GACATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGCCTGCTGAAGAAGA GATTCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAACTGCGGCTTCGAGAC CCACGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTTGGCTTTTCCTGCGGAGC CAGGAGTACAAGAAGTACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCT TCGTCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCTGAAGGAGGTGTGGAAGCCTGC CGTGGGAAGCCCCGCTGCCAAGAGAGTGAAGCTGGACTAAGCTGCCTTCTGCGGGGCT TGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAAT AAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA sequence encoding X-editor 515 57 AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCTGC TGCCAAGAGAGTGAAGCTGGATAGCAGACAGGAGATCAAGCGGATTAATAAAATTCGG AGAAGACTGGTGAAGGATTCTAACACAAAGAAGGCTGGCAAGACACGGGGCCCTATGA Full-length DNA SEQ ID NO Nucleotide Sequence* Description AGACACTGCTGGTGAGAGTGATGACACCCGACCTGAGAGAAAGACTGGAAAACCTGAG AAAGAAGCCTGAGAATATCCCCCAGCCCATCAGCAACACAAGCCGGGCCAACCTGAAT AAGCTGCTGACCGACTACACCGAAATGAAGAAGGCCATCCTGCACGTGTATTGGGAAG AGTTCCAGAAAGACCCAGTCGGCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAA GATCGATCAGAACAAGCTGAAGCCCGAAATGGACGAGAAGGGGAACCTGACAACCGCC GGCTTTGCCTGTAGCCAGTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGA GCGAAAAGGGCAAGGCTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGA GAAGCTGATCAAGCTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGAGA TATTCCCTGGGCAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCA AGGAATCCACCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCTC CAGCCCCGTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTG TCTAAGTACCAGGACATTATCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGA GACTGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTGAC CCTGCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGATCGCC CGGGTGCGGATGTGGGTGAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTAGAGACG ACGCCAAGCCCCTGCTGAGACTGAAGGGCTTCCCCAGCTTCCCTCTGGTGGAGAGACA GGCAAATGAAGTGGACTGGTGGGACATGGTGTGTAACGTGAAGAAGCTGATCAATGAG AAGAAGGAGGACGGCAAAGTGTTCTGGCAGAATCTGGCCGGCTACAAGCGTCAGGAGG CCCTGCGGCCCTACCTGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGTTCGCCCG GTATCAGCTGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCACGGCGAAGACTGGGGG AAGGTGTACGATGAGGCCTGGGAGCGGATCGATAAGAAGGTGGAGGGCCTGAGCAAGC ACATCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCCCAGAGCAAGGCCGCCCTGAC CGACTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGAGGGGCTGAAGGAGGCCGACAAG GACGAGTTCTGCCGGTGCGAACTGAAGCTGCAGAAGTGGTACGGAGATCTGAGAGGCA AACCTTTCGCCATCGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCA GTACAACTGCGCCTTTATTTGGCAGAAGGACGGAGTGAAGAAGCTGAACCTGTACCTG ATCATCAACTATTTCAAGGGCGGCAAGCTGAGATTCAAGAAGATCAAGCCTGAAGCCT TCGAGGCCAACAGATTCTACACCGTGATTAACAAGAAAAGCGGAGAGATCGTGCCAAT GGAAGTGAACTTCAACTTCGACGACCCTAACCTGATCATCCTGCCCCTGGCATTTGGC AAGCGGCAGGGCAGAGAGTTCATCTGGAACGACCTGCTGTCTCTGGAGACCGGCAGCC TGAAGCTGGCCAACGGCAGAGTGATCGAGAAGACACTGTACAACAGACGAACCAGACA AGACGAGCCCGCCCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGC AGCAATATCAAGCCTATGAACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCG TGATCGCCCTTACCGACCCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGG CAACCCTACCCACATCCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATC CAGGCCAAGAAGGAGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCA GCAAGGCCAAGAACCTGGCCGACGACATGGTTAGAAATACCGCCAGAGACCTCCTGTA CTACGCTGTGACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGC AGACAGGGCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGC TGACCGCCAAGCTGGCCTACGAGGGCCTGCCCTCTAAGACCTACCTGTCCAAGACCTT GGCACAGTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCGAC TACGACCGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACCATTA ACGGCAAGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACAGGTACAAACGGCA GAACGTGGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAAGCGTAAAC AATGACATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGCCTGCTGAAGA AGAGATTCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAACTGCGGCTTCGA GACCCACGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTTGGCTTTTCCTGCGG AGCCAGGAGTACAAGAAGTACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAG CCTTCGTCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCTGAAGGAGGTGTGGAAGCC TGCCGTGGGAAGCCCCGCTGCCAAGAGAGTGAAGCTGGACTAAGCTGCCTTCTGCGGG GCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTG AATAAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA sequence encoding X-editor 676 58 AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCTGC TGCCAAGAGAGTGAAGCTGGATAGCAGACAGGAGATCAAGCGGATTAATAAAATTCGG AGAAGACTGGTGAAGGATTCTAACACAAAGAAGGCTGGCAAGACAGGCCCTATGAAGA Full-length DNA SEQ ID NO Nucleotide Sequence* Description CACTGCTGGTGAGAGTGATGACACCCGACCTGAGAGAAAGACTGGAAAACCTGAGAAA GAAGCCTGAGAATATCCCCCAGCCCATCAGCAACACAAGCCGGGCCAACCTGAATAAG CTGCTGACCGACTACACCGAAATGAAGAAGGCCATCCTGCACGTGTATTGGGAAGAGT TCCAGAAAGACCCAGTCGGCCTGATGAGCAGAGTGGCTCAGCCTGCCAGCAAGAAGAT CGATCAGAACAAGCTGAAGCCCGAAATGGACGAGAAGGGGAACCTGACAACCGCCGGC TTTGCCTGTAGCCAGTGCGGCCAGCCCCTGTTTGTGTACAAACTGGAACAGGTGAGCG AAAAGGGCAAGGCTTACACGAATTACTTCGGCAGATGCAACGTGGCCGAGCACGAGAA GCTGATCCTGCTGGCCCAGCTGAAGCCTGAGAAGGATAGCGATGAGGCAGTGACATAT TCCCTGGGCAAGTTCGGACAGCGGGCCCTGGATTTTTATTCCATTCATGTGACCAAGG AATCCACCCACCCCGTCAAGCCTCTTGCCCAAATTGCCGGCAACAGATACGCCAGCGG CCCCGTGGGCAAGGCCCTGAGCGACGCCTGTATGGGCACCATCGCCAGCTTCCTGTCT AAGTACCAGGACATTATCATCGAGCACCAGAAGGTGGTGAAGGGCAACCAGAAGAGAC TGGAGAGCCTGCGCGAGCTGGCCGGCAAGGAAAACCTGGAGTATCCTAGCGTGACCCT GCCTCCTCAGCCTCATACAAAGGAGGGCGTGGATGCCTACAACGAAGTGATCGCCCGG GTGCGGATGTGGGTGAACCTGAATCTGTGGCAGAAGCTGAAGCTGTCTAGAGACGACG CCAAGCCCCTGCTGAGACTGAAGAAGTTCCCCAGCTTCCCTCTGGTGGAGAGACAGGC AAAT GAAGT GGACT GGT GGGACAT GGT GT GTAACGT GAAGAAGCT GAT CAAT GAGAAG AAGGAGGACGGCAAAGTGTTCTGGCAGAATCTGGCCGGCTACAAGCGTCAGGAGGCCC TGCGGCCCTACCTGAGCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGTTCGCCCGGTA TCAGCTGGGGGACCTGCTGCTGCACCTCGAGAAGAAGCACGGCGAAGACTGGGGGAAG GTGTACGATGAGGCCTGGGAGCGGATCGATAAGAAGGTGGAGGGCCTGAGCAAGCACA TCAAGCTGGAGGAGGAACGGAGATCTGAGGACGCCCAGAGCAAGGCCGCCCTGACCGA CTGGCTGAGAGCCAAGGCCAGCTTCGTCATCGAGGGGCTGAAGGAGGCCGACAAGGAC GAGTTCTGCCGGTGCGAACTGAAGCTGCAGAAGTGGTACGGAGATCTGAGAGGCAAAC CTTTCGCCATCGAGGCCGAGAACAGCATCCTGGACATCAGCGGCTTCAGCAAGCAGTA CAACTGCGCCTTTATTTGGCAGAAGGACGGAGTGAAGAAGCTGAACCTGTACCTGATC ATCAACTATTTCAAGGGCGGCAAGCTGAGATTCAAGAAGATCAAGCCTGAAGCCTTCG AGGCCAACAGATTCTACACCGTGATTAACAAGAAAAGCGGAGAGATCGTGCCAATGGA AGTGAACTTCAACTTCGACGACCCTAACCTGATCATCCTGCCCCTGGCATTTGGCAAG CGGCAGGGCAGAGAGTTCATCTGGAACGACCTGCTGTCTCTGGAGACCGGCAGCCTGA AGCTGGCCAACGGCAGAGTGATCGAGAAGACACTGTACAACAGACGAACCAGACAAGA CGAGCCCGCCCTGTTTGTGGCCCTGACCTTCGAGAGAAGAGAGGTGCTGGACAGCAGC AATATCAAGCCTATGAACCTGATCGGCGTGGACCGGGGCGAGAACATCCCTGCCGTGA TCGCCCTTACCGACCCCGAGGGATGCCCTCTGAGCCGGTTTAAAGACAGCCTGGGCAA CCCTACCCACATCCTGAGAATTGGCGAGTCCTACAAGGAGAAGCAGAGAACCATCCAG GCCAAGAAGGAGGTGGAGCAGCGGCGGGCTGGCGGCTACTCCCGGAAGTACGCCAGCA AGGCCAAGAACCTGGCCGACGACATGGTTAGAAATACCGCCAGAGACCTCCTGTACTA CGCTGTGACCCAGGACGCCATGCTGATCTTCGAGAACCTGAGCAGAGGCTTCGGCAGA CAGGGCAAGAGAACCTTCATGGCCGAGAGACAGTACACCCGGATGGAGGACTGGCTGA CCGCCAAGCTGGCCTACGAGGGCCTGCCCTCTAAGACCTACCTGTCCAAGACCTTGGC ACAGTACACCAGCAAGACATGCTCTAACTGCGGCTTCACAATCACGAGCGCCGACTAC GACCGGGTGCTGGAGAAACTGAAGAAGACCGCCACAGGCTGGATGACCACCATTAACG GCAAGGAGCTGAAGGTGGAGGGCCAGATCACCTACTACAACAGGTACAAACGGCAGAA CGTGGTGAAGGACCTGAGCGTGGAACTGGATAGACTGAGCGAGGAAAGCGTAAACAAT GACATCAGCAGCTGGACCAAGGGCCGGAGCGGCGAGGCCCTGAGCCTGCTGAAGAAGA GATTCTCCCACAGACCAGTGCAGGAGAAGTTCGTGTGTCTGAACTGCGGCTTCGAGAC CCACGCCGACGAGCAAGCCGCCCTGAACATCGCCCGGTCTTGGCTTTTCCTGCGGAGC CAGGAGTACAAGAAGTACCAGACAAACAAGACCACAGGCAACACAGACAAGAGAGCCT TCGTCGAGACCTGGCAGAGCTTCTACAGAAAGAAGCTGAAGGAGGTGTGGAAGCCTGC CGTGGGAAGCCCCGCTGCCAAGAGAGTGAAGCTGGACTAAGCTGCCTTCTGCGGGGCT TGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAAT AAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA sequence encoding X-editor 812 VI. Guide Nucleic Acids of the Systems for Genetic Editing of Target Nucleic Acid

[0201] In another aspect, the present disclosure provides guide ribonucleic acids (gRNA) for use in modifying an APOC3 target nucleic acid in a cell. As used herein, the term “gRNA” covers naturally-occurring gRNA molecules and gRNA variants, including chimeric gRNA variants comprising domains from different gRNA. gRNAs of the disclosure comprise a scaffold and a targeting sequence complementary to an APOC3 target nucleic acid in a cell.

[0202] In some embodiments, the disclosure provides systems comprising one or more gRNA and an mRNA encoding an engineered X-editor protein as an engineered X-editor:gRNA system designed, upon expression of the engineered X-editor protein in a cell, to form a ribonucleoprotein (RNP) complex with the gRNA, to target and edit specific locations in the APOC3 target nucleic acid sequence of the cell. The gRNA provides target specificity to the complex by including a targeting sequence (or “spacer”) having a nucleotide sequence that is complementary to a sequence of the APOC3 target nucleic acid sequence while the engineered X-editor protein of the system provides the site-specific activity such as cleavage or nicking of the target sequence that is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence by virtue of its association with the gRNA. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events by the engineered X-editor protein. In some embodiments, the editing event may be a cleavage event followed by the introduction of random insertions or deletions (“indels”) or other mutations (e.g., a substitution, duplication, or inversion of one or more nucleotides). In some embodiments, indels are introduced in the target nucleic acid by the engineered X-editor:gRNA systems of the embodiments described herein and cellular repair systems that can disrupt the protein reading frame of the targeted gene.

[0203] Embodiments of gRNAs and formulations of mRNAs and gRNAs for use in the editing of target nucleic acids are described herein, below. a. Reference gRNA and gRNA variants

[0204] As used herein, a “reference gRNA” refers to a CRISPR guide ribonucleic acid comprising a wild-type sequence of a naturally-occurring gRNA. In some embodiments, a gRNA scaffold of the disclosure may be subjected to one or more mutagenesis methods, such as the mutagenesis methods described in WO2022 / 120095 and WO2020 / 247882, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, domain swapping, or chemical modification to generate one or more gRNA variants with enhanced or varied properties relative to the gRNA scaffold that was modified. The activity of the gRNA scaffold from which a gRNA variant was derived may be used as a benchmark against which the activity of the gRNA variant is compared, thereby measuring improvements in function or other characteristics of the gRNA scaffold.

[0205] Table 8 provides the sequences of reference gRNA tracr and scaffold sequences. In some embodiments, the disclosure provides gRNA variant sequences wherein the gRNA has a scaffold comprising a sequence having one or more nucleotide modifications relative to a reference gRNA sequence of any one of SEQ ID NOS: 344-356 of Table 8. Table 8: Reference gRNA tracr and scaffold sequences SEQ ID NO. Nucleotide Sequence 344 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAG C GCUUAUUUAU C GGAGAGAAAC C GAUAAGUAAAAC GCAU CAAAG 345 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGC GCUUAUUUAU C GGAGAGAAAU C C GAUAAAUAAGAAGCAU CAAAG 346 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAG CGCUUAUUUAUCGGAGA 347 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAG CGCUUAUUUAUCGG 348 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGC GCUUAUUUAUCGGAGA 349 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGC GCUUAUUUAUCGG 350 GUUUACACACU C C CU CU CAUAGGGU 351 GUUUACACACU C C CU CU CAU GAGGU 352 UUUUACAUAC C C C CU CU CAU GGGAU 353 GUUUACACACUCCCUCUCAUGGGGG 354 CCAGCGACUAUGUCGUAUGG 355 GC GCUUAUUUAU C GGAGAGAAAU C C GAUAAAUAAGAAGC 356 GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUU AUUUAUCGGA b. gRNA Domains and their Function

[0206] The gRNAs of the systems of the disclosure comprise two segments: a targeting sequence and a protein-binding segment, with the targeting sequence linked to the 3' end of the protein-binding segment, described more fully, below. The targeting segment of a gRNA includes a nucleotide sequence (referred to interchangeably as a guide sequence, a spacer, a targeter, or a targeting sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within the target nucleic acid sequence (e.g., a strand of a double stranded target DNA, a target ssRNA, a target ssDNA, etc.), described more fully below. The targeting sequence of a gRNA is capable of binding to a target nucleic acid sequence, including, in the context of the present disclosure, a coding sequence, a complement of a coding sequence, a non-coding sequence, and to accessory elements. The protein-binding segment (or “activator” or “protein-binding sequence”) interacts with (e.g., binds to) an engineered X-editor protein as a complex, forming an RNP (described more fully below). The protein-binding segment is alternatively referred to herein as a “scaffold,” which is comprised of several regions, described more fully, below. The properties and characteristics of CasX gRNA, both wild-type and variants, are described in WO / 2020247882, US20220220508, and WO2022 / 120095.

[0207] In the case of a reference gRNA, the gRNA occurs naturally as a dual guide RNA (dgRNA), wherein the targeter and the activator portions each have a duplex-forming segment that have complementarity with one another and hybridize to one another to form a double stranded duplex (dsRNA duplex for a gRNA). The term “targeter” or “targeter RNA” is used herein to refer to a crRNA-like molecule (crRNA: "CRISPR RNA") of a CasX dual guide RNA (and therefore of a CasX single guide RNA when the “activator" and the "targeter” are linked together, e.g., by intervening nucleotides). The crRNA has a 5' region that anneals with the tracrRNA followed by the nucleotides of the targeting sequence. In the case of the gRNA variants for use in the systems of the disclosure, the scaffolds are designed such that the activator and targeter portions are covalently linked to one another (rather than hybridizing to one another) and comprise a single molecule, and can be referred to as a “single-molecule gRNA,” “single guide RNA,” a “single-molecule guide RNA,” a “one-molecule guide RNA,” or a “sgRNA”. The gRNA variants of the disclosure are all single molecule versions.

[0208] Collectively, the assembled gRNAs of the disclosure comprise distinct structured regions, or domains: the RNA triplex, the scaffold stem loop, the extended stem loop, the pseudoknot, and the targeting sequence that, in the embodiments of the disclosure is specific for a target nucleic acid and is located on the 3’ end of the gRNA. The RNA triplex, the scaffold stem loop, the pseudoknot and the extended stem loop, together with the unstructured triplex loop that bridges portions of the triplex, together, are referred to as the “scaffold” of the gRNA. In some cases, the scaffold stem further comprises a bubble. In other cases, the scaffold further comprises a triplex loop region. In still other cases, the scaffold further comprises a 5’ unstructured region. In some embodiments, the gRNA scaffolds of the disclosure for use in the engineered X-editor:gRNA systems comprise a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 435), or a sequence having 1, 2, 3, 4, or 5 mismatches thereto.

[0209] Each of the structured domains contribute to establish the global RNA fold of the guide and retain functionality of the guide; particularly the ability to properly complex with the engineered X-editor protein. For example, the guide scaffold stem interacts with the helical I domain of the engineered X-editor protein, while residues within the triplex, triplex loop, and pseudoknot stem interact with the OBD of the engineered X-editor protein. Together, these interactions confer the ability of the guide to bind and form an RNP with the CasX that retains stability, while the spacer (or targeting sequence) directs and defines the specificity of the RNP for binding a specific sequence of DNA.

[0210] Site-specific binding and / or cleavage of a target nucleic acid sequence (e.g., genomic DNA) by the engineered X-editor protein can occur at one or more locations (e.g., a sequence of a target nucleic acid) determined by base-pairing complementarity between the targeting sequence of the gRNA and the target nucleic acid sequence. Thus, for example, the gRNAs of the disclosure have sequences complementary to and therefore can hybridize with the target nucleic acid that is adjacent to a sequence complementary to a TC protospacer adjacent motif (PAM) motif or a PAM sequence, such as ATC, CTC, GTC, or TTC.

[0211] Because the targeting sequence of a guide sequence hybridizes with a sequence of a target nucleic acid sequence, a targeting sequence can be modified by a user to hybridize with a specific target nucleic acid sequence, so long as the location of the PAM sequence is considered.

[0212] By selection of the targeting sequences of the gRNA, defined regions of the target nucleic acid sequence or sequences bracketing a particular location within the target nucleic acid can be modified or edited using the engineered X-editor:gRNA systems described herein. In some embodiments, the targeting sequence of the gRNA has between 15 and 20 consecutive nucleotides. In some embodiments, the targeting sequence comprises 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 21 consecutive nucleotides. In some embodiments, the targeting sequence consists of 20 consecutive nucleotides. In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides.

[0213] By selection of the targeting sequences of the gRNA, defined regions of the target nucleic acid sequence can be modified or edited using the engineered X-editor:gRNA systems described herein. In some embodiments, the target nucleic acid comprises a PAM sequence located 5’ of the targeting sequence with at least a single nucleotide separating the PAM from the first nucleotide of the targeting sequence. In some embodiments, the PAM is located on the non-targeted strand of the target region, i.e. the strand that is complementary to the target nucleic acid.

[0214] As described herein in Example 1, a computational analysis was performed to identify a set of targeting sequences with desirable characteristics based on their predicted activity and specificity. In some embodiments, the targeting sequence is complementary to a nucleic acid sequence that is unique within the APOC3 locus. The use of a targeting sequence complementary to a single and / or unique sequence in the APOC3 locus avoids the introduction of multiple breaks in the APOC3 gene.

[0215] In some embodiments, the targeting sequence of the gRNA for use in the systems of the disclosure is complementary to an exon of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA for use in the systems of the disclosure is complementary to a sequence of an exon selected from the group consisting of exon 1, exon 2, exon 3, and exon 4, wherein the identities of the exons correspond to those of human reference genome build hg38. In some embodiments, the targeting sequence is complementary to a sequence of exon 4, wherein the identities of the exons correspond to those of human reference genome build hg38. In some embodiments, the targeting sequence is complementary to a sequence of an APOC3 gene, and the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458-470. In some embodiments, the targeting sequence is complementary to a sequence of exon 1. In some embodiments, the targeting sequence is complementary to a sequence of exon 2. In some embodiments, the targeting sequence complementary to a sequence of exon 2 comprises a sequence of SEQ ID NO: 459. In some embodiments, the targeting sequence of the gRNA is complementary to a sequence of exon 3. In some embodiments, the targeting sequence complementary to a sequence of exon 3 comprises a sequence selected from the group consisting of SEQ ID NOS: 460 and 463. In some embodiments, the targeting sequence comprises the sequence of SEQ ID NO: 460. In some embodiments, the targeting sequence comprises the sequence of SEQ ID NO: 463. In some embodiments, the targeting sequence is complementary to a sequence of exon 4. In some embodiments, the targeting sequence complementary to exon 4 comprises a sequence selected from the group consisting of SEQ ID NOS: 461, 465, 466, 467, 468, 469, and 470. In some embodiments, the targeting sequence comprises the sequence of SEQ ID sequence comprises the sequence of SEQ ID sequence comprises the sequence of SEQ ID sequence comprises the sequence of SEQ ID sequence comprises the sequence of SEQ ID sequence comprises the sequence of SEQ ID sequence comprises the sequence of SEQ ID NO: 461. In some embodiments, the targeting NO: 465. In some embodiments, the targeting NO: 466. In some embodiments, the targeting NO: 467. In some embodiments, the targeting NO: 468. In some embodiments, the targeting NO: 469. In some embodiments, the targeting NO: 470.

[0216] In some embodiments, the targeting sequence for use in the gRNA of the disclosure is complementary to an intron of the APOC3 gene. In some embodiments, the targeting sequence is complementary to a sequence of an intron selected from the group consisting of intron 1, intron 2, and intron 3, wherein the identities of the introns correspond to those of human reference genome build hg38.

[0217] In some embodiments, the targeting sequence of the gRNA is complementary to a sequence of an APOC3 intron-exon junction. In some embodiments, the targeting sequence is complementary to an Intron 1-Exon 1 junction. In some embodiments, the targeting sequence complementary to an Intron 1-Exon 1 junction comprises the sequence of SEQ ID NO: 458. In some embodiments, the targeting sequence is complementary to an Intron 1-Exon 2 junction. In some embodiments, the targeting sequence is complementary to an Intron 2-Exon 2 junction. In some embodiments, the targeting sequence is complementary to an Intron 2-Exon 3 junction. In some embodiments, the targeting sequence is complementary to an Intron 3-Exon 3 junction. In some embodiments, the targeting sequence is complementary to an Intron 3-Exon 4 junction. In some embodiments, the targeting sequence complementary to an Intron 3-Exon 4 junction and comprises the sequence of SEQ ID NO: 464. In some embodiments, the targeting sequence of the gRNA is complementary to a sequence encoding an untranslated region (UTR) of the APOC3 gene. In some embodiments, the targeting sequences complementary to an APOC3 UTR sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458, 459, 86 462, and 470. In some embodiments, the targeting sequence of the gRNA is complementary to a sequence encoding the boundary between an UTR and the poly(A) tail of the APOC3 gene. In some embodiments, the targeting sequence complementary to a 5’ UTR comprises the sequence selected from the group consisting of SEQ ID NOS: 458 and 459. In some embodiments, the targeting sequence is complementary to a 3’ UTR boundary. In some embodiments, the targeting sequence complementary to the 3’ UTR comprises a sequence selected from the group consisting of SEQ ID NO: 462 and 470. In some embodiments, the targeting sequence comprises the sequence of SEQ ID NO: 462. In some embodiments, the targeting sequence comprises the sequence of SEQ ID NO: 470. In some embodiments, the targeting sequence is complementary to a cis-regulatory element of the APOC3 gene.

[0218] In some embodiments, the targeting sequence is complementary to a sequence located between about -5000 base pairs (bp) to about +4100 bp from the transcription start site (TSS) of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 3.2 kb of a TSS of the APOC3 gene. In some embodiments, the targeting sequence is complementary to a sequence proximal to the transcription start site of the APOC3 gene, e.g., within about 50 nucleotides, about 100 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, or about 1000 nucleotides of the transcription start site. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 1.0 kb upstream to 1.0 kb downstream of a TSS of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 500 bps upstream to 500 bps downstream of a TSS of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 100 bps upstream to 100 bps downstream of a TSS of the APOC3 gene. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid within exon 2, within exon 3, within exon 4, or is located at the intron 1-exon 1 boundary of the APOC3 gene. In some embodiments, the targeting sequence is complementary to a sequence that is known to be devoid of a single nucleotide polymorphism (SNP). In some embodiments, the targeting sequence is complementary to a sequence that harbors less than 5% SNP in the general human population.

[0219] Representative, but non-limiting examples of targeting sequences complementary to APOC3 target nucleic acids are presented as SEQ ID NOS: 458-713. The targeting sequences for targeting an APOC3 target nucleic acid may be linked to any of the gRNA scaffolds of the disclosure; e.g., including but not limited to gRNA 174, 235, or 316, or chemically-modified versions thereof. In some embodiments, the targeting sequence of the gRNA comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, or is identical to a sequence selected from the group consisting of SEQ ID NOS: 458-713. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOS: 458-713 with 1, 2, 3, 4, or 5 nucleotide(s) removed from the 3’ end of the sequence. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458-470, or a sequence that is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least 95% identical to the full-length sequence, or comprises a sequence having 1, 2, 3, 4, or 5 mismatches thereto. In some embodiments, the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 458-470. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOS: 458-470 with 1, 2, 3, 4, or 5 nucleotide(s) removed from the 3’ end of the sequence. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458-464, or a sequence that is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least 95% identical to the full-length sequence, or comprises a sequence having 1, 2, 3, 4, or 5 mismatches thereto. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458-464. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOS: 458-464 with 1, 2, 3, 4, or 5 nucleotide(s) removed from the 3’ end of the sequence. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458, 459, 460, 461, 462, and 463, or a sequence that is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least 95% identical to the full-length sequence, or a sequence having 1, 2, 3, 4, or 5 mismatches thereto. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458, 459, 460, 461, 462, and 463. In some embodiments, the targeting sequence consists of a sequence selected from the group consisting of SEQ ID NOS: 458, 459, 460, 461, 462, and 463. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458, 459, 88 and 460, or a sequence that is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least 95% identical to the full-length sequence, or a sequence having 1, 2, 3, 4, or 5 mismatches thereto. In some embodiments, the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458, 459 and 460. In some embodiments, the targeting sequence of the gRNA is selected from the group consisting of SEQ ID NOS: 458, 459, and 460 with 1, 2, 3, 4, or 5 nucleotide(s) removed from the 3’ end of the sequence.

[0220] In some embodiments, the targeting sequence of the gRNA is chemically-modified by the addition of a 2’O-methyl group to the last 1, 2, or 3 nucleosides of the 3’ end of the targeting sequence and each of the modified nucleosides is linked to the adjoining nucleotide by a phosphorothioate bond. In some embodiments, the targeting sequence of the gRNA comprises a sequence of SEQ ID NOS: 458, 459, 460, 461, 462, or 463 that is chemically-modified by the addition of a 2’O-methyl group to the last 1, 2, or 3 nucleosides of the 3’ end of the targeting sequence and each of the modified nucleosides is linked to the adjoining nucleoside by a phosphorothioate bond. c. gRNA Modifications

[0221] In another aspect, the disclosure relates to gRNA for use in the APOC3 gene-editing systems of the disclosure, which comprise one or more modifications relative to a gRNA scaffold from which it was derived. In some embodiments, a gRNA variant for use in the systems of the disclosure comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced domains relative to a gRNA sequence of the disclosure that improve a characteristic relative to the reference gRNA. Exemplary regions for modifications and swapped regions or domains include the RNA triplex, the pseudoknot, the scaffold stem loop, and the extended stem loop. In some embodiments, the gRNA variant of the disclosure comprises at least a first swapped region from a different gRNA, resulting in a chimeric gRNA. A representative example of such a chimeric gRNA is guide 316 (SEQ ID NO: 359), in which the extended stem loop of gRNA scaffold 235 is replaced with the extended stem loop of gRNA scaffold 174, wherein the resulting 316 variant retains the ability to form an RNP with an engineered X-editor protein and exhibits an improved functional characteristic compared to the parent 235, when assessed in an in vitro or in vivo assay under comparable conditions.

[0222] All gRNAs that have one or more improved functions, characteristics, or add one or more new functions when the gRNA variant is compared to a gRNA from which it was derived, while retaining the functional properties of being able to complex with the engineered X-editor and guide the engineered X-editor ribonucleoprotein holo complex to the target nucleic acid are envisaged as within the scope of the disclosure. In some embodiments, the gRNA has an improved characteristic selected from the group consisting of increased editing activity, increased pseudoknot stem stability, increased triplex region stability, increased scaffold stem stability, extended stem stability, reduced off-target folding intermediates, and increased binding affinity to an engineered X-editor protein, or any combination thereof. In some cases of the foregoing, the improved characteristic is assessed in an in vitro assay, including the assays of the Examples. In other cases of the foregoing, the improved characteristic is assessed in vivo.

[0223] In other embodiments, the gRNA variant has improved manufacturability compared to the gRNA scaffold from which it was derived. In a particular embodiment, the 316 gRNA scaffold has a shorter sequence compared to the 235 scaffold from which it was derived. In some embodiments, the shorter sequence length of the 316 scaffold confers the improvements of a higher fidelity in the ability to create the guide synthetically with the correct and complete sequence, as well as an enhanced ability to be successfully incorporated into an LNP.

[0224] Table 9 provides exemplary gRNA variant scaffold sequences of the disclosure that are utilized as gRNA scaffolds or for the generation of the gRNAs (with linked targeting sequences) for use in the engineered X-editor:gRNA systems of the disclosure. In some embodiments, the gRNA variant scaffold for use in the systems comprises any one of the sequences listed in Table 9, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto, wherein the gRNA variant retains the ability to form an RNP with an engineered X-editor of the disclosure. In other embodiments, the gRNA variant scaffold for use in the engineered X-editor:gRNA systems comprises any one of the sequences listed in Table 9, wherein the gRNA variant retains the ability to form an RNP with an engineered X-editor of the disclosure. It is understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gRNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gRNA sequence embodiments described herein. Table 9: Exemplary gRNA Scaffold Sequences SEQ ID NO: Scaffold variant ID Nucleotide sequence 357 174 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCC AUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 358 235 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGAC UAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 359 316 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUA UGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG

[0225] Additional gRNA scaffold variants contemplated for use in the systems of the disclosure include those selected from the group consisting of SEQ ID NOS: 360-434. Further gRNA scaffold variants for use in the systems of the disclosure are described e.g., in International Publication Nos. WO2020 / 247882 and WO2022 / 120095.

[0226] Guide scaffolds can be made by any suitable methods known in the art, including recombinantly or by solid-phase RNA synthesis. However, the length of the scaffold can affect the manufacturability when using solid-phase RNA synthesis, with longer lengths resulting in increased manufacturing costs, decreased purity and yield, and higher rates of synthesis failures. For use in lipid nanoparticle (LNP) formulations, solid-phase RNA synthesis of the scaffold is preferred to generate the quantities needed for commercial development. While previous experiments had identified gRNA scaffold 235 as having enhanced properties relative to gRNA scaffold 174, its increased length (in nucleotides) rendered its use for LNP formulations problematic due to synthetic manufacturing constraints. Accordingly, alternative sequences were sought. In some embodiments, the disclosure provides a gRNA wherein the gRNA scaffold and linked targeting sequence has a sequence less than about 115 nucleotides, less than about 110 nucleotides, or less than about 100 nucleotides.

[0227] In one embodiment, a gRNA scaffold was designed wherein the scaffold 235 sequence (SEQ ID NO: 358) was modified by a domain swap in which the extended stem loop of scaffold 174 replaced the extended stem loop of the 235 scaffold, resulting in the chimeric gRNA scaffold 316, having the sequence ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGU G GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 359), having 89 nucleotides, compared with the 99 nucleotides of gRNA scaffold 235. The resulting 316 scaffold had the further advantage in that the extended stem loop does not contain CpG motifs; an enhanced property conferring reduced potential to elicit an immune response. In some embodiments, the shorter sequence length of the 316 scaffold confers the improvements of a higher fidelity in the ability to create the guide synthetically with the correct and complete sequence, as well as an enhanced ability to be successfully incorporated into an LNP. In some embodiments, the disclosure provides gRNA 316 variants that are chemically-modified, described below. d. Chemically-modified gRNAs

[0228] As described further herein, the disclosure provides gRNA variants having one or more chemical modifications. In some embodiments, the present disclosure provides gRNA variants having one or more chemical modifications in order to enhance the chemical stability and manufacturability of the gRNA. In some embodiments, the chemical modification results in an enhanced property compared to a gRNA that is not modified. In some embodiments, the chemically modified gRNA are utilized in LNP formations, wherein the gRNA of the LNP folds and maintains its structural conformation in order to complex with the CasX variant protein to form an RNP. In some embodiments, the chemically modified gRNA resists nuclease degradation and / or elicits a reduced immune response when introduced into a target cell environment. Chemical modification of RNAs has been shown to improve stability, increase nuclease resistance by cellular RNase, increase duplex bond formation, and reduce immune responses by the selective modification of the nucleotides, resulting in enhanced editing in CRISPR systems (Basila, M., et al. Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity. PLoS ONE 12(11): e0188593 (2017)).

[0229] In some embodiments, the chemical modification is the addition of a 2’O-methyl group to the sugar group of one or more nucleosides of the sequence of the gRNA and linked targeting sequence, resulting in a 2' O-methyl ribonucleoside modification. In some embodiments, the chemical modification is the addition of one or more 2’O-methyl groups on each terminal end, 5' and 3', of the gRNA. In some embodiments, the chemical modification is substitution of a phosphorothioate bond between two or more nucleosides of the sequence. In some embodiments, the first 1, 2, or 3 nucleosides of the 5’ end of the scaffold (i.e., A, C, and U in the case of scaffolds 174, 235, and 316) are modified by the addition of a 2’O-methyl group and each of the modified nucleosides is linked to the adjoining nucleoside by a phosphorothioate bond. Similarly, in some embodiments, the last 1, 2, or 3 nucleosides of the 3’ end of the targeting sequence linked to the 3’ end of the scaffold are similarly modified to produce an end-protected variant (collectively, the construct with the foregoing modifications termed "vl").

[0230] In some embodiments, the gRNA with chemical modifications comprises a scaffold of SEQ ID NOS: 255-263; 265-273; or 275-283, i.e., a sequence of SEQ ID NOS: 255-263; 265273; or 275-283 without the 20 nucleotides of the 3' spacer represented in the foregoing sequences as undefined nucleotides. In the foregoing, the 20 nucleotides on the 3' end of the sequence are replaced with a targeting sequence complementary to a APOC3 target nucleic acid sequence (e.g., a sequence selected from the group consisting of SEQ ID NOS: 458-464) wherein the last 1, 2, or 3 nucleosides of the 3’ end of the targeting sequence are chemically modified by the addition of a 2’O-methyl group and each of the modified nucleosides is linked to the adjoining nucleotide by a phosphorothioate bond.

[0231] In some embodiments, the disclosure provides gRNA with chemical modifications selected from the group consisting of the sequences of SEQ ID NOS: 727-741, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides gRNA with chemical modifications selected from the group consisting of SEQ ID NOS: 727-729, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the disclosure provides a gRNA sequence with chemical modifications selected from the group consisting of SEQ ID NOS: 727-729.

[0232] A schematic of the structure of unmodified and modified gRNA variants 174, 235, and 316 are shown in FIGS. 1-8 and 22. In some embodiments, a gRNA with chemical modifications exhibits enhanced properties compared to gRNA without chemical modifications, as described herein.

[0233] It has been discovered that the incorporation of 2’O-methyl groups to modify one or more nucleotides at select locations in the interior of the gRNA sequence leads to improved manufacturability, increased recovery of full-length gRNA (i.e., a reduction in truncations during synthesis) compared to gRNA that are not modified, in addition to the enhanced properties of increased stability, increased nuclease resistance by cellular RNase, increased duplex bond formation, and a reduced ability to elicit an immune response upon administration to a subject. As used herein "interior of the gRNA sequence" means more than 20 nucleotides from the 5' or 3' end of the gRNA.

[0234] In some embodiments, 2’O-methyl groups are incorporated to modify 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, or 30 or more nucleotides of the gRNA. In some embodiments, 2’O-methyl groups are incorporated to modify 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 or 30 nucleotides of the gRNA.

[0235] In some embodiments, the incorporation of at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 2’O-methyl groups to modify one or more nucleotides at select locations in the interior of the gRNA sequence leads to improved manufacturability and recovery of gRNA with reduced truncations compared to gRNA that has not been modified or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA.

[0236] In some embodiments, 2’O-methyl groups are incorporated to modify at least about 1, at least about 2 at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 nucleotides of the scaffold stem loop of the gRNA. In some embodiments, 2’O-methyl groups are incorporated to modify at least about 1, at least about 2 at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12 nucleotides of the scaffold extended stem loop of the gRNA. In some embodiments, 2’O-methyl groups are incorporated to modify one or more nucleotides at positions selected from the group consisting of nucleotide positions 1, 2, 3, 37, 42, 43, 44, 45, 48, 49, 50, 51, 64, 65, 66, 67, 68, 69, 76, 77, 78, 79, 80, 81, 107, 108, and 109 as compared to SEQ ID NO: 274. In a particular embodiment, 2’O-methyl groups are incorporated to modify nucleotides at positions 1, 2, 3, 37, 42, 43, 44, 45, 48, 49, 50, 51, 64, 65, 66, 67, 68, 69, 76, 77, 78, 79, 80, 81, 107, 108, and 109 as compared to SEQ ID NO: 274 and each of the modified nucleosides at position 1, 2, 3, 107, 108, and 109 as compared to SEQ ID NO: 274 is linked to the adjoining nucleoside by a phosphorothioate bond (the construct termed "vl05"; see FIG. 22.

[0237] In some embodiments, the incorporation of the 2’0-methyl groups to modify the nucleotides at the foregoing locations results in recovery of at least at least about 80%, of at least at least about 85%, of at least at least about 90%, of at least at least about 91%, of at least at least about 92%, of at least at least about 93%, of at least at least about 94%, or of at least at least about 95% full-length gRNA in the manufacture of an gRNA. In some embodiments, the incorporation of the 2’O-methyl groups to modify the nucleotides at the foregoing locations results in recovery of at least about 10%, of at least about 15%, of at least about 20%, or at least about 25% greater amounts of full-length gRNA in the manufacture of the gRNA compared to a gRNA that has not been modified or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA, but not internally. In some embodiments, the incorporation of the 2’O-methyl groups to modify the nucleotides at the foregoing locations results in an increase in stability of at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% in an in vitro stability assay compared to a gRNA that has not been modified or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA. In some embodiments, the incorporation of the 2’O-methyl groups to modify the nucleotides at the foregoing locations results in an increase in nuclease resistance by cellular RNase as compared to a gRNA that has not been modified. In some embodiments, the incorporation of the 2’O-methyl groups to modify the nucleotides at the foregoing locations results in an increase in nuclease resistance by cellular RNase by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% in an in vitro or in vivo assay compared to a gRNA that has not been modified. In some embodiments, the incorporation of the 2’O-methyl groups to modify the nucleotides at the foregoing locations results in an increase in duplex bond formation as compared to a gRNA that has not been modified. In some embodiments, the incorporation of the 2’O-methyl groups to modify the nucleotides at the foregoing locations results in an increase in duplex bond by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% in an in vitro or in vivo assay as compared to a gRNA that has not been modified. In some embodiments, the incorporation of the 2’O-methyl groups to modify the nucleotides at the foregoing locations results in a reduced ability to elicit an immune response upon administration to a subject as compared to a gRNA that has not been modified. In some embodiments, the incorporation of the 2’0-methyl groups to modify the nucleotides at the foregoing locations results in a reduced ability to elicit an immune response by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% in an in vitro or in vivo assay as compared to a gRNA that has not been modified.

[0238] In some embodiments, the incorporation of the 2’O-methyl groups to modify the nucleotides at the foregoing locations results in a chemically modified gRNA that is capable of forming a RNP with the engineered X-editor protein and binding and modifying the APOC3 target nucleic acid with an editing rate in an in vitro assay of at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more of the editing rate a gRNA that has not been modified or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA, but not internally. In some embodiments, the chemically modified gRNA is capable of forming a RNP with the engineered X-editor protein and binding and modifying the APOC3 target nucleic acid in an in vitro assay with off-target effects no greater than or are lower than the off-target effects of a gRNA that has not been modified or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA, but not internally.

[0239] In some embodiments, the gRNA with chemical modifications produces off-target effects of less than about 5%, less than about 4%, less than 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than 0.1% in cells.

[0240] In some embodiments, the gRNA comprises a sequence of SEQ ID NO: 1115. In some embodiments, the gRNA with chemical modifications comprises a scaffold of SEQ ID NO: 1115 with the 20 wildcard nucleotides of the 3' spacer are replaced with a targeting sequence complementary to an APOC3 target nucleic acid sequence (e.g., a sequence selected from the group consisting of SEQ ID NOS: 458-464). In some embodiments, the gRNA comprises a sequence selected from the group consisting of SEQ ID NO: 1116-1130. In some embodiments, a gRNA consists of a sequence selected from the group consisting of SEQ ID NO: 1116-1130.

[0241] In some embodiments, the gRNA modified by the incorporation of the 2’O-methyl groups in the scaffold and the linked targeting sequence comprises a sequence selected from the group consisting of SEQ ID NO: 1116-1128, or a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches 96 thereto. In some embodiments, the gRNA modified by the incorporation of the 2’0-methyl groups in the scaffold and the linked targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 1116-1128. In some embodiments, the gRNA modified by the incorporation of the 2’0-methyl groups in the scaffold and the linked targeting sequence consists of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

[0242] In another aspect, the disclosure provides a composition comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the composition comprises a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 1116-1128. In some embodiments, the composition comprises a gRNA consisting of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128. In some embodiments, the composition comprises a gRNA consisting of the sequence of SEQ ID NO: 1115.

[0243] In another aspect, the disclosure provides a method of enhancing the manufacture and recovery of full-length gRNA comprising the chemical modification of nucleotides by the incorporation of 2’O-methyl groups in one or more select locations in the interior sequence of the gRNA. In some embodiments, the method comprises incorporation of at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 2’O-methyl groups to nucleotides at select locations in the interior sequence of the gRNA. In some embodiments of the method, the incorporation of the one or more 2’O-methyl groups is at least about 10, at least about 15, or at least about 20 nucleotides distal to the 5' and 3' ends of the gRNA. In some embodiments of the method, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 nucleotides of the scaffold stem loop of the gRNA are modified by incorporation of a 2’O-methyl group. In some embodiments of the method, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12 nucleotides of the scaffold extended stem loop of the gRNA are modified by incorporation of a 2’O-methyl group. In some embodiments of the method, the 2’O-methyl groups are incorporated into a gRNA selected from the group consisting of SEQ ID NOS: 742, 743, 748, 749, 756, and 757. In some embodiments of the method, 2’0-methyl groups are incorporated to modify nucleotides at positions 1, 2, 3, 37, 42, 43, 44, 45, 48, 49, 50, 51, 64, 65, 66, 67, 68, 69, 76, 77, 78, 79, 80, 81, 107, 108, and 109 as compared to SEQ ID NO: 274 and each of the modified nucleosides at position 1, 2, 3, 107, 108, and 109 as compared to SEQ ID NO: 274 is linked to the adjoining nucleoside by a phosphorothioate bond.

[0244] In some embodiments of the method, the incorporation of the one or more 2’O-methyl groups in the foregoing locations results in an enhanced recovery of at least about 10%, of at least about 15%, of at least about 20%, or at least about 25% greater amounts of full-length gRNA in the manufacture of the gRNA compared to a gRNA that has not been modified or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA. In some embodiments of the method, the incorporation of the one or more 2’O-methyl groups in the foregoing locations results in an increase in stability of the modified gRNA of at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% in an in vitro stability assay compared to a gRNA that has not been modified or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA. e. Complex Formation with Engineered X-editor Proteins

[0245] Upon delivery or expression of the components of the system in a target cell, the gRNA is capable of complexing as an RNP with an engineered X-editor protein and binding to the target nucleic acid of the APOC3 gene. In some embodiments, a gRNA variant has an improved ability to form an RNP complex with an engineered X-editor protein when compared to a reference gRNA or a gRNA variant from which it was derived. Improving ribonucleoprotein complex formation may, in some embodiments, improve the efficiency with which functional RNPs are assembled. In some embodiments, greater than 90%, greater than 93%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% of RNPs comprising a gRNA variant and its targeting sequence are competent for gene editing or modification of a target nucleic acid. VII. Nucleic Acid Sequences and Vectors

[0246] In another aspect, the present disclosure relates to nucleic acids encoding the engineered X-editor nucleases and gRNA disclosed herein. The disclosure provides nucleic 98 acids encoding mRNA encoding the engineered X-editor nucleases, e.g. DNA nucleic acids encoding the corresponding mRNA. Additionally, the disclosure provides vectors comprising gRNA and / or nucleic acids encoding the engineered X-editor proteins described herein. In some cases, the vectors are utilized for the expression and recovery of the engineered X-editor and gRNA components of the gene editing pair. In other cases, the vectors are utilized for the delivery of the gRNA and encoding mRNA to target cells for the editing of the target nucleic acid, as described more fully, below. In some embodiments, sequences encoding the engineered X-editor and a gRNA are templated on the same vector. In some embodiments, sequences encoding the engineered X-editor and a gRNA are templated on different vectors. Suitable vectors are described, for example, in WO2022 / 120095 and WO2020 / 247882. As described in WO2022 / 120095 and WO2020 / 247882, depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[0247] In some embodiments, the disclosure provides polynucleotide sequences encoding the engineered X-editor of any of the embodiments described herein, including the engineered X-editor proteins of SEQ ID NOS: 22-25, or sequences having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In a particular embodiment, the disclosure provides polynucleotide sequences encoding the engineered X-editor of SEQ ID NO: 23.

[0248] An mRNA encoding an engineered X-editor of the disclosure may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring nucleotides may be substituted with unnatural nucleotides. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. A gRNA can also be produced synthetically; for example, by use of a T7 RNA polymerase system known in the art. In some embodiments, the mRNA is generated by in vitro transcription (IVT) using a PCR product or linearized plasmid DNA template and a T7 RNA polymerase, wherein the plasmid contains a T7 promoter and a poly(A) tail. If using a PCR product, DNA sequences encoding candidate mRNAs will be cloned into a plasmid containing a T7 promoter, wherein the plasmid DNA template will be linearized and then used to perform IVT reactions for expression of the mRNA. Exemplary methods for the production and recovery of the mRNA are provided in the Examples.

[0249] The gRNA and / or the mRNA encoding the engineered X-editor may also be prepared by recombinantly producing a polynucleotide sequence coding for the engineered X-editor or gRNA of any of the embodiments described herein using standard recombinant techniques known in the art and incorporating the encoding gene into an expression vector appropriate for a host cell, see e.g., Sambrook, supra, and incorporating the encoding gene into an expression vector appropriate for a host cell. Methods for the recombinant production and recovery of polynucleotide sequences coding for the fusion proteins or gRNA are described, for example, in WO2023235818A2, WO2022120095A1 and WO2020247882A1, incorporated by reference herein. As described in WO2023235818A2, WO2022120095A1 and WO2020247882A1, depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector. VIII. Particles for Delivery of Engineered X-editor:gRNA systems

[0250] In another aspect, the present disclosure provides particle compositions for delivery of the engineered X-editor:gRNA systems to cells or to subjects for the modification of the APOC3 gene. In some embodiments, the disclosure provides synthetic nanoparticles that encapsulate gRNA variants and mRNA encoding engineered X-editor of any of the embodiments described herein. In some embodiments, materials for the creation of biodegradable polymeric nanoparticles (PNP) include polylactide, poly (lactic-co-glycolic acid) (PLGA), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), and poly(isohexyl cyanoacrylate), polyglutamic acid (PGA), poly (8-caprolactone) (PCL), cyclodextrin, and natural polymers for instance chitosan, albumin, gelatin, and alginate are the most utilized polymers for the synthesis of PNP (Production and clinical development of nanoparticles for gene delivery. Molecular Therapy-Methods & Clinical Development 3:16023; doi:10.1038 (2016)). In some embodiments, the disclosure provides virus-like particles for delivery of the engineered X-editor and gRNA variants (see, e.g., WO / 2021 / 113772A1). In other embodiments, the disclosure provides lipid nanoparticles that encapsulate gRNA variants and mRNA encoding engineered X- editors of any of the embodiments described herein, described more fully, below. In other embodiments, the particles are LNP that separately encapsulate a gRNA variant and a mRNA encoding an engineered X-editor of any of the embodiments described herein in different particles, that may be co-formulated as a mixture for administration, described more fully, below. In other embodiments, the particles are LNP that separately encapsulate a gRNA variant and a mRNA encoding an engineered X-editor of any of the embodiments, and the two types of particles may be administered separately. a. Lipid Nanoparticles (LNP)

[0251] In another aspect, the present disclosure provides lipid nanoparticles (LNP) for delivery of engineered X-editor:gRNA systems to cells or to subjects for the modification of the APOC3 gene. In some embodiments, the LNPs of the disclosure are organ or tissue-specific, have excellent biocompatibility, and can deliver the engineered X-editor:gRNA systems with high efficiency, and thus can be used for the modification (e.g., editing) of the target nucleic acid.

[0252] In their native forms, nucleic acid polymers are unstable in biological fluids and cannot penetrate into the cytoplasm of target cells, thus requiring delivery systems. Lipid nanoparticles (LNP) have proven useful for both the protection and delivery of nucleic acids to tissues and cells. Furthermore, the use of mRNA in LNPs to encode a CRISPR nuclease eliminates the possibility of undesirable genome integration compared to DNA vectors. Moreover, mRNA efficiently transfects both mitotic and non-mitotic cells, as it does not require to enter into the nucleus since it exerts its function in the cytoplasmic compartment. LNPs as a delivery platform offers the additional advantage of being able to co-formulate both the mRNA encoding the nuclease and the gRNA into single LNP particles.

[0253] Accordingly, in various embodiments, the disclosure encompasses lipid nanoparticles and compositions that may be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells, both in vitro and in vivo. In certain embodiments, the disclosure encompasses methods of treating or preventing diseases or disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle that encapsulates or is associated with a suitable therapeutic agent.

[0254] In certain embodiments, the lipid nanoparticles are useful for the delivery of nucleic acids, including, e.g., the mRNA encoding the engineered X-editor of the disclosure, including the sequences of SEQ ID NOS: 22-25 and the gRNA embodiments of the disclosure, including the sequences of SEQ ID NOS: 714-726. In some embodiments, the present disclosure provides LNP in which the gRNA and mRNA encoding the engineered X-editor are incorporated into single LNP particles. In other embodiments, the present disclosure provides LNP in which the gRNA and mRNA encoding the engineered X-editor are incorporated into separate LNP particles, which can be formulated together in varying ratios for administration. In some embodiments, the mRNA for incorporation singly into the LNP of the disclosure encodes any of the engineered X-editor described herein, including the sequences of SEQ ID NOS: 22-25. In some embodiments, the gRNA for incorporation singly into the LNP of the disclosure encodes any of the engineered gRNA described herein, including the sequences of SEQ ID NOS: 714726.

[0255] In some embodiments, the mRNA encoding the engineered X-editor for incorporation into the LNP of the disclosure comprises the sequences selected from the group consisting of SEQ ID NOS: 51-54, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the mRNA further comprises a 5' cap sequence linked 5’ to the 5’ UTR of the mRNA sequence wherein the 5’ cap has the nucleic acid sequence selected from the group consisting of m7G(5’)ppp(5’)mAG, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. In some embodiments, the gRNA scaffold for use in the LNP is the sequence of SEQ ID NO: 359 or a chemically modified version thereof. In some embodiments, the chemically-modified gRNA targeting APOC3 for use in the LNP comprises a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto. In some embodiments, the chemically-modified gRNA targeting APOC3 for use in the LNP comprises a sequence selected from the group consisting of SEQ ID NOS: 727-729 and 1116-1118, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

[0256] The lipid nanoparticles and compositions of certain embodiments of the disclosure may be used to induce expression of an engineered X-editor of the disclosure both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel ionizable lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA encoding the engineered X-editor protein). In some embodiments, the lipid nanoparticles and compositions may be used to decrease or eliminate the expression of the APOC3 target gene both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with one or more nucleic acids of the engineered X-editor:gRNA systems of the disclosure that modify the target nucleic acid. The lipid nanoparticles and compositions of embodiments of the disclosure may also be used for co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, such as may be useful to provide an effect requiring colocalization of different nucleic acids (e.g., mRNA encoding for a suitable gene modifying enzyme and gRNA for targeting of the target nucleic acid).

[0257] In some embodiments, LNPs and LNP compositions described herein include at least one cationic lipid, at least one conjugated lipid, at least one steroid or derivative thereof, at least one additional lipid, or any combination thereof. Alternatively, the lipid compositions of the disclosure can include an ionizable lipid, such as an ionizable cationic lipid, a helper lipid (usually a phospholipid), cholesterol, and a polyethylene glycol-lipid conjugate (PEG-lipid) to improve the colloidal stability in biological environments by, for example, reducing a specific absorption of plasma proteins and forming a hydration layer over the nanoparticles. Such lipid compositions can be formulated at typical mole ratios of 50:10:37-39:1.5-2.5 or 20-50:8-65:2540:1-2.5, with variations made to adjust individual properties.

[0258] The LNPs and LNP compositions of the present disclosure are configured to protect and deliver an encapsulated payload of the systems of the disclosure to tissues and cells, both in vitro and in vivo. Various embodiments of the LNPs and LNP compositions of the present disclosure are described in further detail herein. Cationic lipid

[0259] In some embodiments, the LNPs and LNP compositions of the present disclosure include at least one cationic lipid. The term “cationic lipid,” refers to a lipid species that has a net positive charge. In some embodiments, the cationic lipid is an ionizable cationic lipid that has a net positive charge at a selected pH, such as physiological pH. In some embodiments, the ionizable cationic lipid has a pKa less than 7 such that the LNPs and LNP compositions achieve efficient encapsulation of the payload at a relatively low pH. In some embodiments, the cationic lipid has a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. In some embodiments, the cationic lipid may be protonated at a pH below the pKa of the cationic lipid, and it may be substantially neutral at a pH over the pKa. The LNPs and LNP compositions may be safely delivered to a target organ (for example, the liver,) and / or cell (hepatocyte) in vivo, and after endocytosis, exhibit a positive charge to release the encapsulated payload through electrostatic interaction with an anionic protein of the endosome membrane.

[0260] Early formulations of LNP utilizing permanently cationic lipids resulted in LNPs with positive surface charge that proved toxic in vivo, plus were rapidly cleared by phagocytic cells. By changing to ionizable cationic lipids bearing tertiary amines, especially those with pKa < 7, results in LNP achieving efficient encapsulation of nucleic acid polymers at low pH by interacting electrostatically with the negative charges of the phosphate backbone of mRNA, that also result in largely neutral systems at physiological pH values, thus alleviating problems associated with permanently-charged cationic lipids.

[0261] As used herein, “ionizable lipid” means an amine-containing lipid which can be easily protonated, and, for example, it may be a lipid of which charge state changes depending on the surrounding pH. The ionizable lipid may be protonated (positively charged) at a pH below the pKa of a cationic lipid, and it may be substantially neutral at a pH over the pKa. In one example, the LNP may comprise a protonated ionizable lipid and / or an ionizable lipid showing neutrality. In some embodiments, the LNP has a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. The pKa of the LNP is important for in vivo stability and release of the nucleic acid payload of the LNP in the target cell or organ. In some embodiments, the LNP having the foregoing pKa ranges may be safely delivered to a target organ (for example, the liver) and / or target cell (hepatocyte) in vivo, and after endocytosis, exhibit a positive charge to release the encapsulated payload through electrostatic interaction with an anionic protein of the endosome membrane.

[0262] The ionizable lipid is an ionizable compound having characteristics similar to lipids generally, and through electrostatic interaction with a nucleic acid (for example, an mRNA of the disclosure), may play a role of encapsulating the nucleic acid within the LNP with high efficiency.

[0263] According to the type of the amine and the tail group comprised in the ionizable lipid, (i) the nucleic acid encapsulation efficiency, (ii) PDI (polydispersity index) and / or (iii) the nucleic acid delivery efficiency to tissue and / or cells constituting an organ (for example, hepatocytes or liver sinusoidal endothelial cells in the liver) of the LNP may be different. In certain embodiments, the ionizable lipid cationic lipid comprises from about 46 mol % to about 66 mol % of the total lipid present in the particle.

[0264] The LNP comprising an ionizable lipid comprising an amine may have one or more kinds of the following characteristics: (1) encapsulating a nucleic acid with high efficiency; (2) uniform size of prepared particles (or having a low PDI value); and / or (3) excellent nucleic acid delivery efficiency to organs such as the liver, and / or cells constituting such organs (for example, hepatocytes).

[0265] In some embodiments, the cationic lipid form plays a role both in nucleic acid encapsulation through electrostatic interactions and intracellular release by disrupting endosomal membranes. The nucleic acid payloads are encapsulated within the LNP by the ionic interactions they form with the positively charged cationic lipid. Non-limiting examples of cationic lipid components utilized in the LNP of the disclosure are selected from DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), DLin- KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane), and TNT (1,3,5-triazinane-2,4,6-trione) and TT (Nl,N3,N5-tris(2-aminoethyl)benzene-l,3,5-tricarboxamide). Non-limiting examples of helper lipids utilized in the LNP of the disclosure are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (2-Oleoyl-l- palmitoyl-sn-glycero-3-phosphocholine) and DOPE (l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) DOPG, l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), sphingolipid, and ceramide. Cholesterol and PEG-DMG ((R)-2,3- bis(octadecyloxy)propyl-l-(methoxy polyethylene glycol 2000) carbamate), PEG-DSG (l,2-Distearoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000), or DSPE-PEG2k (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]), are components utilized in the LNP of the disclosure for the stability, circulation, and size of the LNP.

[0266] In some embodiments, the cationic lipid in the LNP of the disclosure comprises a tertiary amine. In some embodiments, the tertiary amine includes alkyl chains connected to N of the tertiary amine with ether linkages. In some embodiments, the alkyl chains comprise C12-C30 105 alkyl chains having 0 to 3 double bonds. In some embodiments, the alkyl chains comprise C16-C22 alkyl chains. In some embodiments, the alkyl chains comprise Cl8 alkyl chains. A number of cationic lipids and related analogs have been described in U.S. Patent Publication Nos. 20060083780, 20060240554, 20110117125, 20190336608, 20190381180 and 20200121809; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; 5,785,992; 9,738,593; 10,106,490; 10,166,298; 10,221,127; and 11,219,634; and PCT Publication No. WO 96 / 10390, the disclosures of which are herein incorporated by reference in their entirety.

[0267] In some embodiments, the cationic lipid in the LNP of the disclosure may comprise, for example, one or more ionizable cationic lipids wherein the ionizable cationic lipid is a dialkyl lipid. In other embodiments, the ionizable cationic lipid is a trialkyl lipid.

[0268] In some embodiments, the cationic lipid in the LNP of the disclosure is selected from 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[l,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[l,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[l,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpe...

Claims

1. A system for modifying an apolipoprotein C3 (APOC3) gene target nucleic acid sequence, comprising:a. an mRNA comprising a sequence encoding an engineered X-editor protein; andb. a guide ribonucleic acid (gRNA), wherein the gRNA comprises a scaffold and a linked targeting sequence complementary to the APOC3 gene target nucleic acid sequence,wherein the gRNA is capable of forming a ribonucleoprotein (RNP) with the engineered X-editor protein and binding and modifying the target nucleic acid, and wherein the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the APOC3 gene.

2. The system of claim 1, wherein the gRNA is a single-molecule gRNA (sgRNA).

3. The system of claim 1 or claim 2, wherein the targeting sequence is complementary to anAPOC3 gene target nucleic acid sequence located between about -5000 base pairs (bp) to about +4100 bp from the transcription start site (TSS) of the APOC3 gene.

4. The system of any one of claims 1-3, wherein the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 3.2 kb of a TSS of the APOC3 gene.

5. The system of any one of claims 1-4, wherein the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 1.0 kb upstream to 1.0 kb downstream of a TSS of the APOC3 gene.

6. The system of any one of claims 1-5, wherein the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 500 bps upstream to 500 bps downstream of a TSS of the APOC3 gene.

7. The system of any one of claims 1-6, wherein the targeting sequence of the gRNA is complementary to a target nucleic acid sequence within 100 bps upstream to 100 bps downstream of a TSS of the APOC3 gene.

8. The system of claim 1 or claim 2, wherein the targeting sequence of the gRNA is complementary to a target nucleic acid within exon 2, within exon 3, within exon 4, or is located at the intron 1-exon 1 boundary of the APOC3 gene.

9. The system of any one of claims 1-8, wherein the targeting sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 458-713, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the full-length sequence, or a sequence having 1, 2, 3, 4, or 5 mismatches thereto.

10. The system of any one of claims 1-9, wherein the targeting sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 458-713.

11. The system of claim 10, wherein the targeting sequence comprises a sequence of SEQ ID NOS: 458-713 with 1, 2, 3, 4, or 5 nucleotide(s) removed from the 3’ end of the sequence.

12. The system of any one of claims 1-11, wherein the targeting sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 458-470, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the full-length sequence, or a sequence having 1, 2, 3, 4, or 5 mismatches thereto.

13. The system of any one of claims 1-12, wherein the targeting sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 458-470.

14. The system of claim 13, wherein the targeting sequence comprises a sequence of SEQ ID NOs: 458-470 with 1, 2, 3, 4, or 5 nucleotide(s) removed from the 3’ end of the sequence.

15. The system of any one of claims 1-13, wherein the targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 458-464.

16. The system of claim 15, wherein the targeting sequence consists of a sequence selected from the group consisting of SEQ ID NO: 458, 459, 460, 461, 462 and 463.

17. The system of any one of claims 1-16, wherein the gRNA scaffold comprises a scaffold stem loop comprising the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 435), or a sequence having 1, 2, 3, 4, or 5 mismatches thereto.

18. The system of any one of claims 1-17, wherein the gRNA scaffold comprises a sequence selected from the group consisting of SEQ ID NOS: 357-434 or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

19. The system of claim 18, wherein the gRNA scaffold comprises a sequence selected from the group consisting of SEQ ID NOS: 357-434.

20. The system of claim 18, wherein the gRNA scaffold consists of a sequence selected from the group consisting of SEQ ID NOS: 357-359.

21. The system of claim 18, wherein the gRNA scaffold comprises a sequence of SEQ ID NO: 359, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

22. The system of claim 21, wherein the gRNA scaffold comprises a sequence of SEQ ID NO: 359.

23. The system of claim 21, wherein the gRNA scaffold consists of the sequence of SEQ ID NO: 359.

24. The system of any one of claims 1-23, wherein the gRNA is chemically modified.

25. The system of claim 24, wherein the chemical modification results in an enhancedproperty compared to a gRNA that is not modified.

26. The system of claim 24 or claim 25, wherein the chemical modification to the gRNA comprises an addition of a 2’0-methyl group to one or more nucleotides of the gRNA.

27. The system of claim 26, wherein one or more nucleotides located 1, 2, 3, or 4 nucleotides from the 5’ terminal end, the 3’ terminal end, or both terminal ends of the gRNA are modified by an addition of a 2’0-methyl group.

28. The system of claim 26, wherein the chemical modification to the gRNA comprises an addition of a 2’0-methyl group to 5 or more, 10 or more, 15 or more, 20 or more, or 25 or more nucleotides of the gRNA.

29. The system of any one of claims 26, wherein the chemical modification to the gRNA comprises an addition of a 2’O-methyl group 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 or 30 nucleotides of the gRNA.

30. The system of claim 29, wherein the chemical modification to the gRNA comprises an addition of a 2’O-methyl group to 27 nucleotides of the gRNA.

31. The system of any one of claims 24-30, wherein one or more nucleotides located in the scaffold stem loop of the gRNA, the extended stem loop of the gRNA, or both are modified by an addition of a 2’O-methyl group.

32. The system of claim 31, wherein the chemical modification to the gRNA comprises an addition of a 2’O-methyl group to at least 1, at least 2 at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nucleotides of the scaffold stem loop.

33. The system of claim 31 or claim 32, wherein the chemical modification to the gRNA comprises an addition of a 2’O-methyl group to at least about 1, at least about 2 at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12 nucleotides of the scaffold extended stem loop.

34. The system of any one of claims 24-33, wherein one or more nucleotides selected from nucleotide positions 1, 2, 3, 37, 42, 43, 44, 45, 48, 49, 50, 51, 64, 65, 66, 67, 68, 69, 76, 77, 78,79, 80, 81, 107, 108, and 109 as compared to SEQ ID NO: 274 are modified by an addition of a 2’O-methyl group.

35. The system of any one of claims 24-34, wherein nucleotide positions 1, 2, 3, 37, 42, 43, 44, 45, 48, 49, 50, 51, 64, 65, 66, 67, 68, 69, 76, 77, 78, 79, 80, 81, 107, 108, and 109, as compared to SEQ ID NO:

274. are modified by an addition of a 2’O-methyl group.

36. The system of any one of claims 24-35, wherein the chemical modification to the gRNA is a substitution of a phosphorothioate bond between two or more nucleosides of the gRNA, optionally wherein the chemical modification comprises a substitution of phosphorothioate bonds between two or more nucleosides located 1, 2, 3 or 4 nucleosides from the 5’ terminal end, the 3’ terminal end, or both terminal ends of the gRNA.

37. The system of claim 36, wherein the chemical modification comprises a substitution of phosphorothioate bonds between two or more nucleosides located 1, 2, 3 or 4 nucleosides the from the 5’ terminal end, the 3’ terminal end, or both terminal ends of the gRNA.

38. The system of any one of claims 24-37, wherein the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 11161128, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

39. The system of any one of claims 24-38, wherein the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

40. The system of any one of claims 24-39, wherein the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

41. The system of any one of claims 24-39, wherein the chemically modified gRNA consists of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

42. The system of any one of claims 25-41, wherein the enhanced property of the chemically modified gRNA is one or more of improved manufacturability, increased ability to recover fulllength gRNA, a reduction in truncations during or after synthesis, increased stability, reduced off-target effects, increased nuclease resistance by cellular RNase, increased duplex bond formation, and a reduced ability to elicit an immune response as compared to a gRNA that is not chemically modified.

43. The system of claim 42, wherein the enhanced property comprises recovery of at least about 10%, of at least about 15%, of at least about 20%, or at least about 25% greater amounts of full-length gRNA in the manufacture of the gRNA compared to a gRNA that has not been modified.

44. The system of claim 42, wherein the enhanced property comprises an increase in stability of the chemically modified gRNA of at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% in an in vitro stability assay compared to a gRNA that has not been modified.

45. The system of any one of claims 24-44, wherein the chemically modified gRNA is capable of forming a RNP with the engineered X-editor protein and binding and modifying the APOC3 target nucleic acid with an editing rate in an in vitro assay of at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more the editing rate a gRNA that has not been modified.

46. The system of any one of claims 24-45, wherein the chemically modified gRNA is capable of forming a RNP with the engineered X-editor protein and binding and modifying the APOC3 target nucleic acid in an in vitro assay with off-target effects equal to or lower than the off-target effects of a gRNA that has not been modified.

47. The system of any one of claims 1-46, wherein the engineered X-editor protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 22-25, or a sequence having at least about 80%, at least about 90%, or at least about 95%, or at least about95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

48. The system of claim 47, wherein the engineered X-editor protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 22-25.

49. The system of claim 47, wherein the engineered X-editor protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 22-25.

50. The system of claim 47, wherein the engineered X-editor protein comprises the amino acid sequence of SEQ ID NO: 23, or a sequence having at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96% , or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

51. The system of claim 50, wherein the engineered X-editor protein comprises the amino acid sequence of SEQ ID NO: 23.

52. The system of claim 50, wherein the engineered X-editor protein consists of the amino acid sequence of SEQ ID NO: 23.

53. The system of any one of claims 1-52, wherein the engineered X-editor protein comprises one or more nuclear localization signals (NLS).

54. The system of claim 53, wherein the one or more NLS comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 67-136.

55. The system of claim 53 or claim 54, wherein the one or more NLS is an SV40 NLS or a c-MYC NLS.

56. The system of claim 54 or claim 55, wherein the one or more NLS comprises an amino acid sequence selected from SEQ ID NO: 67 or SEQ ID NO: 69.

57. A nucleic acid comprising:a. a sequence that encodes the engineered X-editor of any one of claims 47-56;b. a sequence that encodes or comprises a gRNA of any one of claims 1-44; or c. a sequence that encodes the engineered X-editor of (a) and the gRNA of (b).

58. The nucleic acid of claim 57, wherein the sequence encoding the engineered X-editor is codon-optimized.

59. The nucleic acid of claim 58, wherein the sequence encoding the engineered X-editor is codon-optimized for expression in a eukaryotic cell.

60. The nucleic acid of any one of claims 57-59, wherein the sequence encoding the engineered X-editor and / or the gRNA is a DNA sequence.

61. The nucleic acid of any one of claims 57-59, wherein the sequence encoding the engineered X-editor is a messenger RNA (mRNA).

62. The nucleic acid of claim 61, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 47-50, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96% , or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the fulllength sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

63. The nucleic acid system of claim 61 or claim 62, wherein the mRNA is chemically modified.

64. The nucleic acid system of claim 63, wherein the sequence encoding the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 51-54, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

65. The nucleic acid system of claim 64, wherein the mRNA comprises a sequence of SEQ ID NO: 52, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at leastabout 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

66. The nucleic acid system of claim 65, wherein the sequence encoding the mRNA comprises a sequence of SEQ ID NO: 52.

67. A lipid nanoparticle (LNP) comprising:a. the gRNA of any one of claims 1-56;b. the mRNA of any one of claims 61-66; orc. the gRNA of any one of claims 1-56 and the mRNA of any one of claims 61-66.

68. The LNP of claim 67, wherein the LNP comprises one or more components selected from the group consisting of one or more ionizable lipids, one or more helper phospholipids, one or more polyethylene glycol (PEG)-modified lipids, and cholesterol or a derivative thereof.

69. The LNP of claim 67 or claim 68, wherein the LNP comprises an ionizable lipid, a helper phospholipid, a polyethylene glycol (PEG)-modified lipid, and cholesterol or a derivative thereof.

70. A vector comprising the nucleic acid system of any one of claims 57-60.

71. The vector of claim 70, wherein the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex virus (HSV) vector, a plasmid, a minicircle, a nanoplasmid, a DNA vector, and an RNA vector.

72. A host cell comprising the vector of claim 70 or claim 71.

73. The host cell of claim 72, wherein the host cell is selected from the group consisting of a Baby Hamster Kidney fibroblast (BHK), a human embryonic kidney 293 (HEK293) cell, a human embryonic kidney 293T (HEK293T) cell, a NS0 cell, a SP2 / 0 cell, a YO myeloma cell, a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell, a hybridoma cell, a NIH3T3 cell, a CV-1 (simian) in Origin with SV40 genetic material (COS) cell, a HeLa cell, a Chinese hamster ovary (CHO) cell, and a yeast cell.

74. A pharmaceutical composition comprising:a. the system of any one of claims 1-56;b. the nucleic acid of any one of claims 57-66;c. the LNP of any one of claims 67-69; ord. the vector of claim 70 or claim 71, and one or more pharmaceutically suitable excipients.

75. The pharmaceutical composition of claim 74, wherein the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intraarterial, intraportal vein injection, intraperitoneal, intramuscular, intraocular, subcutaneous, and oral routes.

76. A method of enhancing the manufacture and recovery of full-length gRNA, comprising incorporating one or more 2’0-methyl groups at least about 10, at least about 15, or at least about 20 nucleotides distal to the 5' and 3' ends of the gRNA.

77. The method of claim 76, comprising incorporating at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 2’O-methyl groups into scaffold stem loop nucleotides of the gRNA.

78. The method of claim 76 or claim 77, comprising incorporating at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12 2’O-methyl groups into extended stem loop nucleotides of the gRNA.

79. The method of any one of claims 76-78, comprising an enhanced recovery of at least about 10%, of at least about 15%, of at least about 20%, or at least about 25% greater amounts of full-length gRNA compared to a gRNA that has not been modified, or has only been modified at 1, 2, 3 or 4 nucleotides at the 5’ terminal end, at the 3’ terminal end, or at both terminal ends of the gRNA.

80. The method of any one of claims 76-79, wherein the 2’O-methyl groups are incorporated into a gRNA selected from the group consisting of SEQ ID NOS: 742, 743, 748, 749, 756, and 757.

81. A method of modifying an APOC3 target nucleic acid sequence in a population of cells, the method comprising introducing into cells of the population:a. the system of any one of claims 1-56;b. the nucleic acid system of any one of claims 57-66;c. the LNP of any one of claims 67-69;d. the vector of claim 70 or claim 71;e. the pharmaceutical composition of claim 74 or claim 75; orf. combinations of two or more of (a)-(e), wherein the APOC3 target nucleic acid sequence of the cells targeted by the gRNA is modified by the engineered X-editor protein.

82. The method of claim 81, wherein the modifying comprises introducing a single-stranded break in the APOC3 target nucleic acid sequence of the cells of the population.

83. The method of claim 81, wherein the modifying comprises introducing a double-stranded break in the APOC3 target nucleic acid sequence of the cells of the population.

84. The method of any one of claims 81-83, wherein the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the APOC3 target nucleic acid of the cells of the population85. The method of any one of claims 81-84, wherein the modifying produces off-target effects of less than about 5%, less than about 4%, less than 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than 0.1% in the cells.

86. The method of any one of claims 81-85, wherein the APOC3 target nucleic acid is modified in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% or more of the cells of the population.

87. The method of any one of claims 81-86, wherein the modifying results in a knocking down expression of the APOC3 gene in the cells of the population such that expression of apolipoprotein C3 is decreased by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in comparison to a cell where the APOC3 gene has not been modified.

88. The method of any one of claims 81-87, wherein the APOC3 gene of the cells of the population is modified such that at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the modified cells do not express a detectable level of apolipoprotein C3.

89. The method of any one of claims 81-88, wherein the cells are eukaryotic cells.

90. The method of claim 89, wherein the eukaryotic cells are selected from the groupconsisting of rodent cells, mouse cells, rat cells, and non-human primate cells.

91. The method of claim 89, wherein the eukaryotic cells are human cells.

92. The method of any one of claims 89-91, wherein the eukaryotic cells are selected from the group consisting of hepatocytes, intestine cells, kidney cells, and cells of arterial endothelial cells.

93. The method of any one of claim 81-92, wherein the modifying of the APOC3 gene target nucleic acid sequence of the population of cells occurs in vitro or ex vivo.

94. The method of any one of claims 81-92, wherein the modifying of the APOC3 gene target nucleic acid sequence of the population of cells occurs in vivo in a subject.

95. The method of claim 94, wherein the subject is selected from the group consisting of a rodent, a mouse, a rat, and a non-human primate.

96. The method of claim 94, wherein the subject is a human.

97. A method of treating an ApoC3-related disease in a subject in need thereof, comprising administering a therapeutically effective dose ofa. the system of any one of claims 1-56;b. the nucleic acid system of any one of claims 57-66;c. the LNP of any one of claims 67-69;d. the vector of claim 70 or claim 71;e. the pharmaceutical composition of claim 74 or claim 75; or f. a combination of two or more of (a)-(e),wherein the APOC3 target nucleic acid sequence of cells of the subject targeted by the gRNA are modified by the engineered X-editor protein.

98. The method of claim 97, wherein the modifying comprises introducing a single-stranded break in the APOC3 gene of the cells.

99. The method of claim 97, wherein the modifying comprises introducing a double-stranded break in the APOC3 gene of the cells.

100. The method of any one of claims 97-99, wherein the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the APOC3 gene of the cells.

101. The method of any one of claims 97-100, wherein the modifying results in a knocking down of the expression of the APOC3 gene in the modified cells of the subject such that expression of apolipoprotein C3 in the subject is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in comparison to a subject where the APOC3 gene has not been modified.

102. The method of any one of claims 97-101, wherein the modifying results in a knocking down expression of the APOC3 gene in the modified cells of the subject such that expression of apolipoprotein C3 in the subject is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in comparison to a subject where the APOC3 gene has not been modified.

103. The method of any one of claims 97-102, wherein the subject is selected from the group consisting of a rodent, a mouse, a rat, and a non-human primate.

104. The method of any one of claims 97-102, wherein the subject is a human.

105. The method of any one of claims 97-104, wherein the cells that are modified are selected from the group consisting of hepatocytes, intestine cells, kidney cells, and arterial endothelial cells.

106. The method of any one of claims 97-105, wherein the subject is treated with a therapeutically effective dose of the LNP.

107. The method of claim 106, wherein the LNP is administered by a route of administration selected from the group consisting of intravenous, intraarterial, intraportal vein injection, intraperitoneal, intramuscular, subcutaneous, and oral routes.

108. The method of any one of claims 97-107, wherein the ApoC3-related disease is selected from the group consisting of: hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or abnormal cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia induced pancreatitis, metabolic syndrome, type II diabetes mellitus, familial chylomicronemia syndrome, and familial partial lipodystrophy or combinations thereof.

109. The method of any one of claims 97-108, wherein the method results in improvement in at least one clinically-relevant endpoint associated with ApoC3-related disease.

110. The method of any one of claims 97-109, wherein the method results in improvement in at least two clinically-relevant endpoints associated with ApoC3-related disease.

111. The method of claim 109 or claim 110, wherein the clinically-relevant endpoint is selected from the group consisting of: percent change in fasting triglyceride levels from baseline, percent change from baseline in apoC-III, percent change from baseline in total cholesterol, percent change from baseline in LDL-C, percent change from baseline in HDL-C, percent change from baseline in non-HDL-C, percent change from baseline in VLDL-C, percent change from baseline in apoB, percent change from baseline in apoA-I, percent change from baseline in lipoprotein(a) and percent change from baseline in angiopoietin-like 3 (ANGPTL3).

112. A gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least 221about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

113. A gRNA consisting of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

114. A composition comprising a gRNA consisting of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

115. A composition comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

116. A composition comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

117. A composition comprising a gRNA consisting of a sequence selected from the group consisting of SEQ ID NOS: 1116-1128.

118. A composition consisting of the sequence of SEQ ID NO: 1115.

119. A composition comprising an mRNA encoding an engineered X-editor protein, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 33-54, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto, and comprising a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 727-741 and 1116-1128, or a sequence having at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least 222about 99% sequence identity to the full-length sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatches thereto.

120. The composition of any one of claims 114-119 for use in the manufacture of a medicament for the treatment of a subject with an ApoC3-related disease.

121. The system of any one of claims 1-56 for use in the treatment of a subject with an ApoC3-related disease.

122. A kit comprising the system of any one of claims 1-56, the nucleic acid system of any one of claims 57-66, the LNP of any one of claims 67-69, the vector of claim 70 or claim 71, the composition of any one of claims 112-120, or combinations thereof, and a suitable container.

123. The kit of claim 121, comprising a buffer, an excipient, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

124. A method of reducing a level of apolipoprotein C3 in a subject, comprising administering a therapeutically effective dose of:a. the system of any one of claims 1-56;b. the nucleic acid system of any one of claims 57-66;c. the LNP of any one of claims 67-69;d. the vector of claim 70 or claim 71;e. the pharmaceutical composition of claim 74 or claim 75; orf. a combination of two or more of (a)-(e),wherein the APOC3 target nucleic acid sequence of cells of the subject targeted by the gRNA are modified by the engineered X-editor protein.

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