Engineered cells having reduced cancer-associated mutation risk and methods of making the same

Abstract

The present technology comprises engineered cells having a reduced mutation risk and methods of making the same. In some embodiments, the engineered cells disrupt a cytosine-guanine (CG) dinucleotide that drives disease when mutated, including cancer. The CG dinucleotide is synonymously edited such that the nucleotide sequence of the codons comprising the CG dinucleotide changes, but the respective amino acids do not.

Description

ENGINEERED CELLS HAVING REDUCED CANCER-ASSOCIATED MUTATION RISK AND METHODS OF MAKING THE SAMECROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 653,71 1 , filed May 30, 2024, the contents of which are incorporated herein by reference in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

[0002] This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on May 23, 2025, is named “155336.8001 .US00 PCT. xml” and is 25,301 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.BACKGROUND

[0003] Over 35% of disease-driving mutations in coding regions comprise a single nucleotide polymorphism (SNP) at a cytosine-guanine (CG) dinucleotide, 90% of which involve a C to thymine mutation. CG dinucleotides are error prone, having a 42-fold higher mutation rate than predicted, relative to random mutations at other dinucleotides. For instance, Tumor protein p53 (TP53), a gene encoding a tumor suppressor protein, is the most frequently mutated gene across all cancers, where more than 20% of all cancers may be attributable to 4 mutations in TP53, each comprising a cytosine-to- thymine variant at a CG dinucleotide.

[0004] CG dinucleotides may be prone to mutation, including cancer-associated mutations. Cytosine-to-thymine mutations may occur through deamination of 5- methylcytosine, forming thymine. Unlike deamination of cytosine, which generates Uracil that may be readily repaired by a uracil DNA glycosylase, deamination of 5- methylcytosine results in thymine, a native DNA base not readily recognized by repair machinery. Cytosine methylation may be complete and tissue-independent across CG dinucleotides in the TP53 gene and occur in most CG dinucleotides genome-wide.

[0005] Current therapies either do not consider this genetic component of cancer, are met with limited success, or are not preventative therapies. As such, novel therapies which address disease-driving mutations and that have increased efficacy in cancer treatment and prevention are needed.SUMMARY

[0006] The present technology comprises engineered cells and methods of making the same.

[0007] In some embodiments, the present technology comprises an engineered cell having one or more edited codons encoding an amino acid at one or more genomic loci, the engineered cell generated by the steps of (i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a cytosine- guanine (CG) dinucleotide at the genomic loci encoding the amino acid; and (ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein the one or more edited codons comprises at least one nucleotide that is different from nucleotides in the codon encoding the amino acid.

[0008] In some embodiments, the present technology comprises an engineered cell having one or more edited codons each encoding a first amino acid and a second amino acid at a genomic loci generated by the steps of (i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits one or more codons comprising a CG dinucleotide at a codon junction; and (ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein at least one of the edited codons comprises a nucleotide sequence that is different from at least one codon at the codon junction encoding the first amino acid and the second amino acid.

[0009] In some embodiments, the present technology comprises an engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence encoding an amino acid at one or more genomic loci selected from the group consisting of chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , Chr17:7,674,943-7,674,945, ch r 17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776, chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803,chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40,064,352- 40,064,354, the engineered cell generated by the steps of (i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at the genomic loci; and (ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein the one or more edited codons is different from the codon encoding the amino acid.

[0010] In some embodiments, the present technology comprises a method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising (i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at a genomic loci; and (ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate one or more edited codons in the engineered cell, wherein the one or more edited codons is different from the codon comprising the CG dinucleotide.

[0011] In some embodiments, the present technology comprises a method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising (i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at a genomic loci; and (ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate one or more edited codons in the engineered cell, the engineered cell comprising or consisting of an AGA or an AGG nucleotide sequence at chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945, Chr17:7,674,892-7,674,894, ch r 17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776, chr17:7,674,219-7,674,221 , chr 17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354, wherein the one or more edited codons is different from the codon comprising the CG dinucleotide.

[0012] In some embodiments, the present technology comprises a method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising (i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits one or more codons comprising a CG dinucleotide at a codon junction; and (ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein at least oneof the edited codons comprises a nucleotide sequence that is different from at least one codon at the codon junction encoding a first amino acid and a second amino acid.

[0013] In some embodiments, the present technology comprises an engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence at one or more genomic loci selected from the group consisting of chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945, chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776, chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354.

[0014] In some embodiments, the present technology comprises an engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence at chr17:7, 675, 087-7, 675, 089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945, chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776, chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40,064,352- 40,064,354.

[0015] In some embodiments, the one or more edited codons comprise two or more codons.

[0016] In some embodiments, the one or more edited codons comprise an edited codon in Table 3, Table 6, Table 7, or Table 8.

[0017] In some embodiments, the codon comprises a nucleotide sequence endogenous to the isolated cell.

[0018] In some embodiments, the isolated cell comprises a cell selected from the group consisting of a cancer cell, a cell susceptible to becoming a cancer cell, a tumor cell, a cyst cell, and a cell that promotes cancer cell growth.

[0019] In some embodiments, the one or more edited codons is on a sense strand of a deoxyribonucleic acid molecule.

[0020] In some embodiments, the one or more edited codons comprises a codon in a tumor protein 53 (TP53) gene.

[0021] In some embodiments, the one or more edited codons is a codon in a BCL6 corepressor (BCOR) gene.

[0022] In some embodiments, the nucleotide editing apparatus comprises a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system, a transcription activator-like effector nuclease (TALENs) system, a zinc finger nuclease system, a base editing system, or a prime editing system.

[0023] In some embodiments, the CRISPR-Cas system further comprises a guide ribonucleic acid (gRNA) that selectively binds to the codon comprising the CG dinucleotide.

[0024] In some embodiments, the engineered cell is a mammalian cell.

[0025] In some embodiments, the mammalian cell is human.

[0026] In some embodiments, the engineered cell is present in a population of engineered cells.

[0027] In some embodiments, the population of engineered cells is transplanted into a subject.

[0028] In some embodiments, the steps of generating the engineered cell do not introduce a CG dinucleotide at a codon junction in the genome of the isolated cell.

[0029] In some embodiments, the steps of generating the engineered cell do not introduce a splice site in the genome of the isolated cell.

[0030] In some embodiments, the steps of generating the engineered cell do not introduce a start codon in the genome of the isolated cell.

[0031] In some embodiments, the steps of generating the engineered cell do not introduce a repeated DNA element in the genome of the isolated cell.

[0032] In some embodiments, the amino acid, the first amino acid, or the second amino acid is selected from the group consisting of arginine, serine, proline, threonine, and alanine.

[0033] In some embodiments, the first amino acid is selected from the group consisting of phenylalanine, leucine, isoleucine, serine, proline, threonine, tyrosine, histidine, asparagine, cysteine, arginine, valine, alanine, aspartic acid, and glycine, and the second amino acid is selected from the group consisting of valine, alanine, aspartic acid, glycine, and glutamic acid.

[0034] In some embodiments, the phenylalanine is encoded by a phenylalanine codon comprising a TTC nucleotide acid sequence.

[0035] In some embodiments, the phenylalanine codon is 5’ to a codon comprising an amino acid sequence selected from the group consisting of GTT, GTC, GTA, GTG, GCT, GCC, GCA, GCG, GAT, GAC, GAA, GAG, GGC, GGC, and GGA, GGG.

[0036] In some embodiments, the one or more edited codons comprises a TTT nucleotide sequence.

[0037] In some embodiments, the one or more edited codons comprises a nucleotide sequence selected from the group consisting of GGAGGC, GGGGGC, and GGTGGC.

[0038] In some embodiments, the nucleotide sequence is at chr17:7,674,228- 7,674,233.

[0039] In some embodiments, the arginine is encoded by an arginine codon selected from the group consisting of CGT, CGA, CGG, and CGC.

[0040] In some embodiments, the one or more edited codons comprises an AGA or an AGG nucleotide sequence.

[0041] In some embodiments, the serine is encoded by a serine codon comprising the nucleotide sequence TCG.

[0042] In some embodiments, the one or more edited codons comprises a TCT, a TCA, a TCC, an AGT, or an AGC nucleotide sequence.

[0043] In some embodiments, the threonine is encoded by a threonine codon comprising the nucleotide sequence ACG.

[0044] In some embodiments, the one or more edited codons comprises an ACT, an ACA, or an ACC nucleotide sequence.

[0045] In some embodiments, the proline is encoded by a proline codon comprising the nucleotide sequence CCG.

[0046] In some embodiments, the one or more edited codons comprises a CCT, a CCA, or a CCC nucleotide sequence.

[0047] In some embodiments, the alanine is encoded by an alanine codon comprising the nucleotide sequence GCG.

[0048] In some embodiments, the one or more edited codons comprises a GOT, a GCA, or a GCC nucleotide sequence.

[0049] In some embodiments, the period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell is about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 minutes, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, or about 4 weeks.

[0050] In some embodiments, the mutation is selected from the group consisting of rs28934578, rs138729528, rs28934575, rs1057519989, rs11540652, rs121912651 , rs121913343, and rs28934576.

[0051] In some embodiments, the engineered cell is a somatic cell.

[0052] In some embodiments, the somatic cell is a somatic stem cell.

[0053] In some embodiments, the somatic cell expresses a chimeric antigen receptor (CAR).

[0054] In some embodiments, the somatic cell is a cell engineered with T-cell receptor (TCR) engineering.

[0055] In some embodiments, the somatic cell is a cell selected from the group consisting of a T-cell, a natural killer cell, a monocyte, a macrophage, a blood cell, a neural cell, a skin cell, a myocyte, an epithelial cell, an islet cell, and a donor derived beta cell.

[0056] In some embodiments, the engineered cell is a non-somatic cell.

[0057] In some embodiments, the non-somatic cell is a cell selected from the group consisting of a stem cell, a germ cell, and a stem cell-derived cell.

[0058] In some embodiments, the stem cell is selected from the group consisting of a totipotent stem cell, a pluripotent stem cell, and a multipotent stem cell.

[0059] In some embodiments, the stem cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell (iPSC), and a hematopoietic stem cell.

[0060] In some embodiments, the stem cell-derived cell is selected from the group consisting of a totipotent-derived stem cell, a pluripotent-derived stem cell, and a multipotent-derived stem cell.

[0061] In some embodiments, the stem cell-derived cell is selected from the group consisting of an embryonic stem cell-derived cell, an iPSC-derived cell, and a hematopoietic stem cell-derived cell.

[0062] In some embodiments, the iPSC or the iPSC-derived cell is derived from a cell selected from the group consisting of a skin cell, a blood cell, and a urine cell.

[0063] In some embodiments, the engineered cell is a cell selected from the group consisting of a progenitor cell, neural progenitor cell, a progenitor-derived cell, a neural progenitor-derived cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIGS. 1 A and 1 B illustrate exemplary codon edits at codon junctions for generating the engineered cells of the present technology. FIG. 1 A illustrates potential deoxyribonucleic acid (DNA) sequence edits on the forward strand. FIG. 1 B illustrates potential DNA sequence edits on the reverse strand. Codons annotated with “+” indicate alternative codons that do not have capacity to form cytosine (C) or guanine (G) at codon junctions. Codons annotated with indicate codons where C or G are at the ends of codons that have potential to form CG at codon junctions.

[0065] FIG. 2 shows an example of codon editing in the Tumor protein p53 (TP53) gene, in accordance with the methods of the present technology. CG-containing codons are underlined, cancer-associated C to thymine (T) mutation positions are indicated with a star, and the alternative synonymous codons that do not contain a CG dinucleotide are shown in the far-right column.

[0066] FIGS. 3A-3D illustrate commonly observed amino acid substitutions in the TP53 gene in cancer cells. FIG. 3A shows the incidence of amino acid substitutions across the full TP53 protein. FIGS. 3B-3D show the amino acid frequencies observedafter pathogenic mutation occurs at the codons encoding the reference amino acids R175, R248, and R273, respectively. R: Arginine.

[0067] FIG. 4 shows a TP53 complementary DNA (cDNA) sequence (line 1 ; SEQ ID NO: 1 ) with a corresponding amino acid sequence line 2; (SEQ ID NO: 2) and synonymous edits (bolded) that are in accordance with the methods of the present technology.

[0068] FIGS. 5A-5F illustrate CG dinucleotide edits observed in cells before extended culturing. FIGS. 5A-5D illustrates CG dinucleotide variants in the TP53 gene of non-edited control embryonic stem cells (ESCs), non-edited control induced pluripotent stem cells (IPSCs), edited ESCs, and edited iPSCs, respectively, before extended culturing. FIGS. 5E and 5F illustrate CG dinucleotide variants in the BCL6 corepressor (BCOR) gene in non-edited control PSCs and edited PSCs, respectively, before extended culturing.

[0069] FIGS. 6A-6J illustrate CG dinucleotide variants observed in cells after extended culturing. FIGS. 6A-6D illustrates CG dinucleotide variants in the TP53 gene of edited ESCs (FIG. 6A), edited iPSCs (FIG. 6B), and non-edited control ESCs (FIGS. 6C-6G) after extended culturing. FIGS. 6H-6J illustrate CG dinucleotide variants in the BCOR gene in edited PSCs (FIG. 6H), and non-edited control iPSCs (FIGS. 6I and 6J) after extended culturing.

[0070] FIGS. 7A and 7B illustrate a meta-analysis of CG single nucleotide variants (SNVs) in human pluripotent stem cells (hPSCs). FIG. 7A shows the number of hPSC samples assayed in each study and the sum of all samples in this analysis. FIG. 7B shows the total number of SNVs and the number of SNVs occurring at CG dinucleotides, stratified as C to (>) T / (G > A) mutations or the sum of all other possible mutations: C > A / (G > T), C > G / (G > C) in CG dinucleotides. C: Cytosine; T : Thymine; A: Adenine; G: Guanine.

[0071] FIG. 8 illustrates a proposed mechanism of CG mutagenesis in hPSCs. Methyl-cytosine undergoes deamination when present as single-stranded DNA. The resulting C to T mutated strand is replicated, resulting in one daughter cell stably harboring the C to T (G to A) mutation.

[0072] FIGS. 9A and 9B illustrate generation of CG-disrupted induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC) lines. FIG. 9A shows sequencing data demonstrating 12 homozygous synonymous edits disrupt CG dinucleotides known to be mutated in cancers and hPSCs. FIG. 9B shows edited hPSC lines harboring the 12 homozygous edits have normal morphology.DETAILED DESCRIPTION

[0073] The present technology comprises engineered cells having a reduced risk of undesired mutations, and methods of making the same. The undesired mutations may occur at a cytosine-guanine (CG) dinucleotide, which may be present in a single codon or at a codon junction. The engineered cells and associated methods may be useful in preventing clonal dominance in cell populations and for treating or preventing diseases associated with undesired mutations, including cancer.

[0074] The following description is merely exemplary in nature and is not intended to limit the present technology, its applications, or its uses. It should be understood that throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features. The description of specific examples indicated in various embodiments of the present technology are intended for purposes of illustration only and are not intended to limit the scope of the present technology disclosed herein. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features or other embodiments incorporating different combinations of the stated features.

[0075] Furthermore, the detailed description of various embodiments herein makes reference to the accompanying drawing / FIGS, which show various embodiments by way of illustration. While the embodiments are described in sufficient detail to enable those skilled in the art to practice the present technology, it should be understood that other embodiments may be realized, and that logical and mechanical changes may be made without departing from the spirit and scope of the present technology. Thus, the detailed description herein is presented for purposes of illustration only and not of limitation. For example, steps or functions recited in any description, method, system, or process, may be executed in any order and are not limited to the order presented. Moreover, any of the steps or functions thereof may be outsourced to or performed by one or more third parties.Definitions

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present technology belongs. For the purposes of the present technology, the following terms are defined below.

[0077] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0078] The term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by acceptable levels in the art. Typically, such variation may be as much 10% above and below a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length and such variation may be influenced by standard applicable measurement practices. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.

[0079] “Amino acids” are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen and have the generic formula H2N — CHR — COOH, wherein R represents a side chain group. The various a-amino acids differ in the side-chain moiety that is attached to the o-carbon. The “amino acids” of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User’s Guide, G. A. Grant, editor, W.H. Freeman & Co., New York (1992), the teachings of which are incorporated herein by reference, including the text and table set forth at pages 11 through 24. As set forth above, the term “amino acid” also includes stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. Modified and unusual amino acids are described generally in Synthetic Peptides: A User’s Guide, supra; Hruby et al., Biochem. J. 268:249-262 (1990); and Toniolo, Int. J. Peptide ProteinRes. 35:287-300 (1990); the teachings of all of which are incorporated herein by reference.

[0080] As used herein, “conservative amino acid substitution” means a given amino acid may be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Polypeptides comprising conservative amino acid substitutions may be tested in any one of the assays described herein to confirm that a desired activity, e.g., antigen-binding activity and specificity of a native or reference polypeptide is retained. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1 ) nonpolar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into H is; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0081] As used herein, “control” may comprise a cell at baseline (i.e., before editing) or a cell comprising a sequence that is different from the engineered cell at one or more loci. A “control” may comprise a method lacking or changing one or more steps of the methods of the present technology.

[0082] All nucleotide sequences of the present technology, including genomic coordinates, are referenced with respect to the Genome reference Consortium Human Build 38 (i.e., “GRCh38” or “hg38”).Engineered CellsCG Dinucleotide Edits

[0083] Cytosine-guanine (CG) dinucleotides may be prone to mutation, including cancer-associated mutations. Cytosine-to-thymine mutations may occur through cytosine deamination, generating uracil. Uracil may be readily recognized and repaired by a uracil DNA glycosylase, thereby generating thymine. Cytosine to thymine mutations may also occur through cytosine methylation, thereby generating 5- methylcytosine. Deamination of 5-methylcytosine forms thymine. Additionally, there may be an increase cytosine to thymine transition at CG dinucleotides due to CpG site methylation.

[0084] When the cytosine in the CG dinucleotide is mutated to thymine, this may create an alternate codon at the CG dinucleotide site that results missense, nonsense, loss-of-function, and gain-of-function mutations, or mutations that otherwise disrupt protein function.

[0085] The present technology comprises engineered cells that are genetically engineered to have a reduced risk of mutation by editing a CG dinucleotide. The CG dinucleotide is synonymously edited such that the nucleotide sequence of the codons comprising the CG dinucleotide changes, but the respective amino acids do not, relative to baseline (i.e., before editing). These edits may comprise CG dinucleotides and / or codons that are on a forward strand of a deoxyribonucleic acid (DNA) molecule or a reverse strand of a DNA molecule. The engineered cell may be homozygous, heterozygous, or hemizygous for any edit of the present technology. In some embodiments, the CG dinucleotide is encoded on an autosome or a sex chromosome.

[0086] Nonlimiting examples of codons comprising CG dinucleotides include TCG (Ser), CCG (Pro), ACG (Thr), GCG (Ala), CGT (Arg), CGC (Arg), CGA (Arg), and CGG (Arg). One or more of these codons may be edited at the CG dinucleotide site to generate a synonymous mutation. For example, when the CG dinucleotide is in (a) an Arginine codon of CGT, CGA, CGG, or CGC, the codon is edited to Arginine codonAGG or AGA; (b) a Serine codon of TCG, the codon is edited to Serine codon TCT, TCA, TCC, AGT, or AGC; (c) a Threonine codon ACG, the codon is edited to Threonine codon ACT, ACA, or ACC; (d) a Proline codon CCG, the codon is edited to Proline codon CCT, CCA, or CCC; (e) an Alanine codon GCG, the codon is edited to Alanine codon GCT, GCA, or GCC. Additional examples of synonymous edits useful for generating engineered cells of the present technology are outlined in Tables 1 -4.Table 1 : Potential codon edits on the forward strand*denotes codons containing a cytosine-guanine (CG) dinucleotide where cytosine (C) to thymine (T) nucleotide change may result in an undesired mutation+: denotes alternative codons that do not have a CG with capacity for a C to T nucleotide change*al I other codons represent codons where none of the codons encoding the amino acid contain a CG dinucleotideTable 2: Potential codon edits on the reverse strand*denotes codons containing a cytosine-guanine (CG) dinucleotide where cytosine (C) to thymine (T) nucleotide change may result in an undesired mutation+: denotes alternative codons that do not have a CG with capacity for a C to T nucleotide change*all other codons represent codons where none of the codons encoding the amino acid contain a CG dinucleotideTable 3: Exemplary codon edits for CG-containing codonsTable 4: Exemplary Codon EditsNCBI: National Center for Biotechnology InformationGenomic Loci

[0087] In some embodiments, the present technology comprises engineered cells having one or more edited codons encoding an amino acid at one or more genomic loci. Nonlimiting examples of the one or more genomic loci comprise a Tumor protein p53 (TP53) locus comprising at least a portion of a TP53 coding region and a BCL6 corepressor (BCOR) locus comprising at least a portion of a BCOR coding region. In some embodiments, the one or more genomic loci comprise at least a portion of a gene selected from Table 5.Table 5: Edited Genes

[0088] In some embodiments, the one or more edited codons comprise codons in the TP53 gene. Nonlimiting examples of edited codons in the TP53 gene include an AGA or an AGG nucleotide sequence at one or more genomic loci selected from the group consisting of chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945, chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776 chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40,064,352- 40,064,354.

[0089] In some embodiments, the one or more edited codons in the TP53 gene comprise an edited codon outlined in Table 6 or Table 7.Table 6: Exemplary TP53 EditsTable 7: Additional TP53 Mutations

[0090] *[]: bracketed sequences represent codons; underlined sequences represent site of observed C to T pathogenic mutation. The sense codon sequences (5’ - 3’) and reverse complement sequences (3’ - 5’) are shown for TP53, a reverse- stranded gene.

[0091] The one or more edited codons may comprise edited codons at the same gene or loci or different gene or loci. For example, the engineered cell may comprise two or more edited codons, wherein a first edited codon is at a first gene or loci and a second edited codon is present at a second gene or loci.Codon Junctions

[0092] In some embodiments, when the engineered cells comprise one or more edited codons, the one or more edited codons are edited at a codon junction, wherein a CG dinucleotide spans across the edited codons (i.e., the cytosine nucleotide is present in a first codon and the guanine dinucleotide is present in a second codon). When the CG dinucleotide is at a codon junction, a codon with a terminal cytosine must be upstream of a codon beginning with a guanine. For example, a first codon selected from the group consisting of TTC, TCC, TAC, TGC, CTC, CCC, CAC, CGC, ATC, ACC, AAC, AGC, GCC, GAC, GTC, and GGC must be upstream of a second codon selected from the group consisting of GTT, GTC, GTA, GTG, GCT, GCC, GCA, GCG, GAT, GAC, GAA, GAG, GGT, GGC, GGA, and GGG.

[0093] In some embodiments, when the CG dinucleotide is at a codon junction comprising a Phenylalanine codon TTC and another codon beginning with a guanine nucleotide (e.g., GTT, GTC, GTA, GTG, GCT, GCC, GCA, GCG, GAT, GAC, GAA, GAG, GGT, GGC, GGA, or GGG), the Phenylalanine codon TTC is synonymously edited to TTT.

[0094] In some embodiments, when the CG dinucleotide is at a codon junction comprising a Serine codon TCC and another codon beginning with guanine nucleotide (e.g., GTT, GTC, GTA, GTG, GCT, GCC, GCA, GCG, GAT, GAC, GAA, GAG, GGT, GGC, GGA, or GGG), the Serine codon TCC is synonymously edited to TCT, TCA, or TCG.

[0095] The codon junctions encode a first amino acid and a second amino acid. In some embodiments, the first amino acid and the second amino acid encoded by the codon junction comprise a first amino acid selected from the group consisting of phenylalanine, leucine, isoleucine, serine, proline, threonine, tyrosine, histidine, asparagine, cysteine, arginine, valine, alanine, aspartic acid, and glycine; and / or a second amino acid selected from the group consisting of phenylalanine, leucine,isoleucine, serine, proline, threonine, tyrosine, histidine, asparagine, cysteine, arginine, valine, alanine, aspartic acid, glycine, and glutamic acid.

[0096] Exemplary codon junction edits in accordance with the present technology are shown in Table 8.Table 8: Exemplary sense strand codon junction editsCell Types, Cell Populations, and Subjects

[0097] The engineered cells of the present technology may comprise a somatic cell or a non-somatic cells. In some embodiments, the somatic cell expresses a chimeric antigen receptor (CAR) or is engineered with T-cell receptor (TCR) engineering. In some embodiments the somatic cell comprises a cell selected from the group consisting of a T-cell, a natural killer cell, a monocyte, a macrophage, a blood cell, a neural cell, a skin cell, a myocyte, an epithelial cell, an islet cell, and a donor derived beta cell. In some embodiments, the somatic cell is a somatic stem cell (i.e., an adult stem cell). In some embodiments, the non-somatic cell comprises a stem cell (e.g., an embryonic stem cell), a germ cell, or an embryonic stem cell-derived cell.

[0098] In some embodiments the cell comprises an induced pluripotent stem cell (iPSC), an iPSC-derived cell. In some embodiments, the IPSC or the IPSC cell comprises a totipotent stem cell, a pluripotent stem cell, or a multipotent stem cell, or a cell derived thereof. In some embodiments, the IPSC or the iPSC-derived cell is derived from a skin cell, a blood cell, or a urine cell. In some embodiments, the cell comprises a progenitor cell, neural progenitor cell, a progenitor-derived cell, a neural progenitor- derived cell, an adult stem cell, or a hematopoietic stem cell.

[0099] In some embodiments, the engineered cell is a mammalian cell. In some embodiments, the mammalian cell is human.

[0100] In some embodiments, the engineered cell is a cell derived from a pet (e.g., a domesticated household animal), livestock, or working animal. Nonlimiting examples of pet, livestock, or working animals include dogs, cats, chicken, cattle, sheep, pigs, goats, horses, donkeys, and mules.

[0101] In some embodiments, the engineered cells of the present technology are present in a population of cells (i.e., a population of engineered cells). In some embodiments, about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of cells are engineered cells.

[0102] In some embodiments, at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of cells are engineered cells.

[0103] In some embodiments, at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of cells are engineered cells.

[0104] In some embodiments, the allele frequency of an edit to a CG dinucleotide is about 0.01 , 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, 0.99, or 1.00 in the population of engineered cells. In some embodiments, about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of engineered cells are homozygous for the edit to the CG dinucleotide. In some embodiments, about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of engineered cells are heterozygous for the edit to the CG dinucleotide.

[0105] In some embodiments, the allele frequency of an edit to a CG dinucleotide is at least 0.01 , 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, 0.99, or 1.00 in the population of engineered cells. In some embodiments, at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of engineered cells are homozygous for the edit to the CG dinucleotide. In some embodiments, at least 1 %, 5%, 10%, 15%, 20%, 25%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of engineered cells are heterozygous for the edit to the CG dinucleotide.

[0106] In some embodiments, the allele frequency of an edit to a CG dinucleotide at least about 0.01 , 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, 0.99, or 1.00 in the population of engineered cells. In some embodiments, at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of engineered cells are homozygous for the edit to the CG dinucleotide. In some embodiments, at least about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in the population of engineered cells are heterozygous for the edit to the CG dinucleotide.

[0107] In some embodiments, the engineered cell or the population of engineered cells are formulated for administration to or transplantation in a subject.

[0108] In some embodiments, the subject has cancer or is at risk of developing cancer, tumors, or cysts. Nonlimiting examples of cancer include a bladder cancer, a blood cancer, a brain cancer, a breast cancer, a colorectal cancer, an endocrine cancer, a gastrointestinal cancer, a kidney (renal) cancer, a lung cancer, a lymphoma, a pancreatic cancer, a prostate cancer, a skin cancer, a uterine cancer, and a teratoma.

[0109] The subject of the present technology may comprise a human subject or a non-human animal (e.g., a livestock animal).Methods

[0110] The present technology comprises methods of generating the foregoing engineered cells. In some embodiments, the methods comprise preventing or reducing a mutation risk. In some embodiments, the mutation is a disease-associated mutation (e.g., cancer-associated mutation). Nonlimiting examples of mutations include rs28934578, rs138729528, rs28934575, rs1057519989, rs1 1540652, rs121912651 , rs121913343, and rs28934576.

[0111] The methods of the present technology may be useful in preventing or reducing one or more of (a) a cell culture variant (e.g., an iPSC variant); (b) clonal dominance; (c) aberrant splice mutations; (d) aberrant intronic mutations; (e) aberrant miRNA targeting; (f) induction of a splice site; (g) induction of an alternative start codon;induction of a frameshift mutation; (h) induction of a repeated DNA element, or (i) aberrant point mutations relative to baseline or control.

[0112] In some embodiments, the methods of the present technology increase a probability, that, in the instance of a mutation, that mutation will result in an amino acid that is more biochemically similar to the amino acid encoded by the unedited codon (e.g., a conservative amino acid substitution), relative to an amino acid encoded by a mutated codon that is not subjected to the methods of the present technology.

[0113] The methods of the present technology are designed to prevent or reduce the introduction of unwanted nucleotide changes. For example, in some embodiments, the methods do not introduce a CG dinucleotide in a codon or at a codon junction in the genome of the isolated cell. In some embodiments, the methods do not introduce a splice site in the genome of the isolated cell. In some embodiments, the methods do not introduce a start codon in the genome of the isolated cell. In some embodiments, the methods do not introduce a repeated DNA element in the genome of the isolated cell. In some embodiments, the methods do not introduce a polyadenylation signal in the genome of the isolated cell.

[0114] In some embodiments, the methods of the present technology comprise: (i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a cytosine-guanine (CG) dinucleotide at the genomic loci encoding the amino acid; and (ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein the one or more edited codons comprises at least one nucleotide that is different from nucleotides in the codon encoding the amino acid.

[0115] In some embodiments, the methods comprise: (i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits one or more codons comprising a CG dinucleotide at a codon junction; and (ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein at least one of the edited codons comprises a nucleotide sequence that is different from at least one codon at the codon junction encoding the first amino acid and the second amino acid.

[0116] In some embodiments, the one or more edited codons, the at least one nucleotide, or the at least one codon comprises a nucleotide sequence that is endogenous to the isolated cell.

[0117] In some embodiments, the isolated cell comprises a cancer cell, or a cell susceptible to becoming a cancer cell, a tumor cell, a cyst cell, or a cell that promotes cancer cell growth. Nonlimiting examples of cancer cells include a cell causing or derived from a bladder cancer, a blood cancer, a brain cancer, a breast cancer, a colorectal cancer, an endocrine cancer, a gastrointestinal cancer, a kidney (renal) cancer, a lung cancer, a lymphoma, a pancreatic cancer, a prostate cancer, a skin cancer, a uterine cancer, and a teratoma.

[0118] The nucleotide editing apparatus of the methods of the present technology may comprise a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system, a transcription activator- 1 ike effector nuclease (TALENs) system, a zinc finger nuclease system, a base editing system, or a prime editing system.

[0119] In some embodiments, the CRISPR-Cas system comprises a guide ribonucleic acid (gRNA) that selectively binds to the codon comprising the CG dinucleotide or distal to the codon comprising the CG dinucleotide.

[0120] In some embodiments, the gRNA comprises a sequence in Table 9.

[0121] In some embodiments, the gRNA comprises a sequence about 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequence of any one of SEQ ID NOs: 3-6, 8, or 9.

[0122] In some embodiments, the gRNA comprises a sequence at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequence of any one of SEQ ID NOs: 3-6, 8, or 9.

[0123] In some embodiments, the gRNA comprises a sequence at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequence of any one of SEQ ID NOs: 3-6, 8, or 9.Table 9: gRNA sequences

[0124] In some embodiments, the period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell is about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 minutes, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, or about 4 weeks.

[0125] In some embodiments, the period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell is at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 minutes, at least 2 hours, at least 4 hours, at least 6 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, or at least 4 weeks.

[0126] In some embodiments, the period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell is at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 minutes, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 72 hours, at least about 1 week, at least about 2 weeks, or at least about 4 weeks.

[0127] The methods of the present technology may disrupt less than about 1%, 5%, 10%, 20%, 50%, or 75% of CG dinucleotides in a locus (e.g., a single exon, a gene coding region) comprising more than one exon or all exons in a gene, a gene locus, a regulatory element (e.g., a promoter, an intron, an untranslated region).EXAMPLESExample 1 : Exemplary Codon Edits

[0128] Exemplary forward and reverse strand codon edits for generating an engineered cell of the present technology are shown in Tables 1 and 2, respectively. Exemplary sense strand codon edits for generating an engineered cell of the present technology are shown in Table 3 and Table 4. Where an undesired mutation (e.g., missense, nonsense, loss-of-function, or gain-of-function mutations) may occur in a CG-containing codon, an alternative codon may be used by editing the codon according to Table 1 or Table 2. This strategy may work for codons encoding serine, proline, threonine, alanine, or arginine amino acid residues.

[0129] When an undesired mutation occurs in a CG dinucleotide-containing codon-junction (i.e., a CG dinucleotide spanning across two codons), alternative codon(s) may be used. Exemplary codon edits at codon junctions on the forward strand and reverse strand are shown in FIGS. 1 A and 1 B, respectively. Exemplary codon edits at codon junctions on the sense strand are shown in Table 8. CG dinucleotide junctioncontaining codons should only be edited when a diagonal-striped or a dotted checkered codon (codon ending with C) is upstream of bolded-outlined or dotted codon (codon starting with G) (FIGS. 1 A and 1 B).Example 2: Editing the TP53 Locus

[0130] The TP53 gene comprises CG dinucleotides that are commonly mutated in cancer cells, thereby altering the encoded amino acid sequence (FIG. 2 and FIGS. 3A- 3D). Exemplary TP53 mutations are shown in Table 7 and FIG. 2. Exemplary editing sites of TP53 according to the methods of the present technology is shown in Table 6 and FIG. 4.Example 3: Assessing CG dinucleotides after extended culturing

[0131] Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) lines were transfected with Cas9, guide RNA (gRNAs), and donortemplates to generate gene-edited lines with 12 codons synonymously edited to ablate commonly mutated CG dinucleotides in TP53 (R175, R181 , R196, R213, G244-G245, R248, R267, R273, R282) and BCOR (R1162, R 1163, R1164). The gene-edits ablated CG dinucleotides without changing the encoded amino acid (FIGS. 5C, 5D, 5F, and 9A). gRNAs and donor templates used are shown in Table 10.Table 10: gRNAs and Donor Templates

[0132] Non-edited and gene-edited hESC and hiPSC samples were extracted for DNA, TP53 and BCOR gene sequences were amplified by polymerase chain reaction (PCR), and the resulting amplicons were sequenced using next generation sequencing (NGS). Sequencing reads were aligned to the TP53 and BCOR reference sequences (hg38) using the Geneious DNA sequencing alignment software and reads with a mapping quality of 40 or greater were mapped. All variants in TP53 (R175, R181 , R196, R213, G244-G245, R248, R267, R273, R282) and BCOR (R1162, R1 163, R1164) with an allele fraction of at least 2% and p-value of less than 0.0001 were called. No proteinchanging variants were called in any of the cell lines prior to extended culture (FIGS. 5A-5F).

[0133] Non-edited hESC (48 samples), non-edited hiPSC (24 samples), gene- edited hESCs (48 samples), and gene-edited hiPSCs (24 samples) were subjected to 4 months of culture and the samples were extracted for DNA, analyzed by NGS, and assessed for variants (FIGS. 6A-6J). Protein changing variants in at least one of the amino acids of interest:TP53 R175, R181 , R196, R213, G244-G245, R248, R267, R273, or R282; and BOOR R1 162, R1 163, or R1 164, were identified in 20 of 72 nonedited hPSCs (allele frequencies ranging from 2.3% - 20.6%) and 0 of 72 edited- hPSCs. Chi squared analysis demonstrated a p-value of 0.00000144 when comparing samples with protein sequence changing variants in at least one of these amino acids in non-edited compared to edited hPSCs.

[0134] Sequencing read pile up views were generated to show examples of protein sequence changing variants occurring at CG dinucleotides in TP53 (R175, R181 , R196, R213, G244-G245, R248, R267, R273, R282) and BCOR (R1162, R1 163, R1 164) in the non-edited hPSCs after extended culture (FIGS. 6C-6G, 6I, and 6J). No protein sequence changing CG dinucleotide mutations at these amino acids were observed in the edited group (FIGS. 6A, 6B, and 6H).Example 4: Assessing mechanisms of commonly occurring oncogenic mutations in hPSCs. mC substrates and deaminases may promote transition from cytosine to thymine in hPSCs.

[0135] Despite representing less than 1 % of the genome, mutations in CG dinucleotides underly 35% of human acquired genetic diseases, a 42-fold higher rate than would be expected if mutations occurred randomly. While thousands of hPSC samples have been sequenced and specific mutations have been identified, no analysis has been performed to assess the frequency of mutations at CG dinucleotides. Therefore, a meta-analysis of published sequencing data from 3167 hPSC samples was performed, finding that CG dinucleotides are hot-spots for oncogenic mutations.Meta-Analysis Results

[0136] Merkle: The first large scale study of acquired oncogenes in hPSCs, Merkle et. al., examined 391 hPSC samples (FIG. 7A) derived from 135 unique humanembryonic (hESC) lines and 1 12 unique human induced pluripotent (hiPSC) lines (see Merkle, F. et al. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature 545, (2017), the entire contents of which are incorporated herein by reference). In total, 31 1 acquired single nucleotide variants (SNVs) were reported (FIG. 7B). The genomic coordinates of the SNVs were examined and neighboring bases were assessed to determine whether the reported mutations occurred at CG dinucleotides. It was found that 33.1% (103 / 311 ) of the SNVs reported were C to T mutations at CG dinucleotides (FIG. 7B), consistent with a mechanism of mC deamination to thymine. Additionally, the analysis revealed that TP53 was the most commonly mutated gene, and that specific SNVs were recurrent, arising independently in multiple cell lines. Furthermore, all of the 9 unique TP53 SNVs reported in hPSCs have also been found in human cancers and are annotated in the COSMIC database as pathogenic mutations. This analysis found that 74.6% (44 / 59) of the mutated TP53 samples were the result of C to T mutations at CG dinucleotides.

[0137] Rouhani: In another large-scale study, Rouhani et. al., mutations in hiPSCs were examined genome-wide (see Rouhani, F. J. et al. Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells. Nat Genet 54, 1406-1416 (2022), the entire contents of which are incorporated herein by reference). A total of 263 SNVs were detected in 555 hiPSC samples (FIG. 7A and 7B) that were distinct from hPSC samples studied by Merkle et al. Notably, specific recurrent TP53 mutations that were reported in hESCs by Merkle et. al. were also detected in hiPSCs in this study, though mutations in BCOR were observed more frequently. An analysis of the SNV neighboring bases was performed, finding that 33.5% (88 / 263) of the SNVs reported were C to T mutations at CG dinucleotides (FIG. 7B).

[0138] Lezmi: In the largest and most recent study, Lezmi et. al. assessed 2,221 hPSC samples (FIG. 7A) derived from 146 unique hESC and hiPSC lines and found cancer-related mutations in 25% of hPSC cell lines, with 64% of mutations occurring in TP53 (see Lezmi, E., Jung, J. & Benvenisty, N. High prevalence of acquired cancer- related mutations in 146 human pluripotent stem cell lines and their differentiated derivatives. Nat Biotechnol 1-5 (2024), the entire contents of which are incorporated herein by reference). This analysis demonstrated that 54.3% (294 / 541 ) of the SNVs detected in hPSC genomes were C to T mutations at CG dinucleotides (FIG. 7B).

[0139] In total, this meta-analysis revealed that 43.5% (485 / 1 115) of cancer- related mutations detected in hPSCs were C to T SNVs that occur at CG dinucleotides (FIG. 7B). CG dinucleotides may represent 0.88% of the human genome, suggesting a 49-fold enrichment of mutations at CG dinucleotides in hPSCs. These findings support that CG dinucleotides may be frequent sites of mutations in hPSCs. Because hPSCs and human cancers may both exhibit mutations at a much higher frequency at CG dinucleotides, this suggests that a common mechanism may be responsible. In human cancers, the mechanism for the high rate of mutations at CG dinucleotides may occur via deamination of mC. Deamination of non-methylated cytosine, adenine, or guanine results in uracil, hypoxanthine, or xanthine, respectively, which may be corrected by high efficiency base excision repair pathways. In contrast, deamination of mC results in thymine, which may not be recognized by base excision repair pathways, and may only be corrected by error-prone mismatch repair pathways. Because repair of the resulting T:G mismatch may be a low efficiency process and mC deamination may occur on single-stranded DNA in a helicase-dependent manner, the C to T mutations may be stably inherited to daughter cells (FIG. 8). Consistent with this mechanism, in hPSCs, it was found that 94.2% (485 / 515) of the detected SNVs at CG dinucleotides were specifically C to T (G to A) mutations, in respect to the hg38 forward strand reference genome, with C to T mutations on the forward strand called as CG to TG, and C to T mutations on the reverse stand called as CG to CA. CG to AG(TC) / GG(CC) mutations in sum accounted for the other 5.8% (30 / 515) of SNVs at CG dinucleotides (FIG. 7B). Considering the frequency of CG dinucleotides in the genome alongside C to T representing 1 / 3 of possible CG SNVs, less than 4 of the 11 15 total SNVs analyzed would be expected to be C to T if mutations arose randomly, though 485 were detected. Thus, this demonstrates that deamination of mC may be responsible for nearly half of all SNVs in hPSCs. mC Substrates

[0140] To directly test whether mC substrates are required for frequently occurring SNVs in hPSCs, CG dinucleotides, the substrates for mC, will be ablated, by knocking in synonymous codons that do not contain CGs.

[0141] Using a CRISPR-Cas9 approach, engineered hESC and hiPSC lines were engineered with homozygous synonymous edits at 12 CG dinucleotides that arefrequently mutated in hPSCs and human cancers, with 9 in the TP53 gene and 3 in the BCOR gene (FIG. 9A). hESC and hiPSC lines generated with disrupted CG dinucleotides appear normal (FIG 9B). Synonymous mutations do not change the protein coding sequence of a gene and may have a minimal role in human diseases, though, synonymous mutations may have aberrant effects. Therefore, expression and splicing of the edited genes will be examined.

[0142] To demonstrate the role of CG dinucleotide mC substrates for recurrent oncogenic mutations found in hPSCs, four distinct hPSC parental lines will be edited such that CG dinucleotides are ablated with synonymous mutations that maintain the wildtype protein sequence. Parental hPSC lines derived from diverse donors will be used to strengthen assessments of the universality and reproducibility of the mechanism. One hiPSC and one hESC line have been engineered (FIGS. 9A and 9B), and the same reagents and methods will be used to engineer two additional hiPSC lines. hPSC lines will be cultured using standard methods and passaging techniques. hPSCs will be electroporated with Cas9 and guide RNA ribonucleoprotein complexes and donor DNA. For TP53 edits, two gRNAs, one targeting intron 4 and the other targeting intron 9, and a synthesized 2.6kb double-stranded DNA donor are used to knock-in a 1 .4kb sequence that contains twelve synonymous SNVs. For BCOR edits, one gRNA targeting intron 7 and a 0.2 kb single-stranded DNA donor will be used to knock-in four synonymous SNVs. Bulk edited hPSC populations will be subjected to single-cell microfluidic dispensing in 96-well format and confirmed to be clonal using standard techniques or adaptations thereof. Clones will be screened by amplicon sequencing to confirm whether the intended edits are generated. For each of the four hPSC parental lines, a non-edited control clone and an edited clone that is homozygous for all 16 SNV edits will be selected for further experiments. Maintenance of pluripotency will be confirmed with flow cytometry and only cell lines displaying >90% SOX2+ / OCT4+ will be utilized. SOX2 NCBI Accession: NP_003097.1 ; OCT4 NCBI Accession(s): NP 002692.2; NP_001 167002.1 ; NP_001272916.1 ; NP_001272915.1 ).

[0143] Initial low-passage, banked samples will be assayed to survey 447 cancer- associated genes with targeted sequencing. Their end-to-end service includes isolation of DNA from cell samples, DNA fragmentation and size selection, library preparation, PCR enrichment, hybridization-based capture, amplicon sequencing, and reporting certified by a clinical geneticist for any variants with >3% VAF. Additionally, clones willbe subjected to amplicon NGS sequencing (see FIGS. 5A-5F) to confirm no acquired TP53 or BCOR mutations are present at >2% VAF prior to downstream experiments. PCR primers that capture the coding regions of TP53 and BCOR have been designed. Amplicon libraries are generated using Illumina Nextera Flex and sequenced on an Illumina NovaSeq X sequencer, resulting in at least four million 150bp paired-end reads per amplicon and 5000x coverage. Raw data will be processed with Geneious Prime to trim adapter sequences and filter out bases with phred quality scores less than 30. Sequencing reads will be aligned to the TP53 and BCOR reference sequences (hg38) using the Geneious DNA sequencing alignment software. Reads with a mapping quality of 40 or greater will be mapped. All variants in TP53 (R175, R181 , R196, R213, G244- G245, R248, R267, R273, R282) and BCOR (R1162, R1163, R1 164) with an allele fraction of at least 2% and p-value of less than 0.0001 will be called. Any clones that display oncogenic mutations in the low-passage cell banks will be discarded and replaced with alternate clones free of oncogenic mutations.

[0144] Based on the meta-analysis in FIG. 7A and 7B, 10.1% (321 / 3167) of all hPSC samples acquired one of the TP53 or BCOR C to T mutations in CG nucleotides at the 12 codons synonymously edited via 16 SNVs. Many samples will be assessed to prevent lack of detecting a sufficient number of mutations in the control group to enable robust statistical comparisons with the CG-ablated edited group. Clones will be expanded and subjected to 4 months of extended culture with 35 bi-weekly passages to maximize the occurrence and clonal dominance of mutations to detectable levels. Based on routine culture experiments, it is anticipated that >19 of the 192 control samples will display mutations in the CG dinucleotides ablated in the edited hPSCs. Because of the 49-fold enrichment of mutations at CG dinucleotides in hPSCs, it is anticipated that <1 of the synonymously edited codons will acquire mutations in this sample size. Four independent experimental runs will be assessed with 96 samples in each run (12 control and 12 edited subclones for each of the four cell lines). Two-way ANOVA with Tukey posthoc testing will determine the impact of mC substrate ablation and parental cell line on the number of samples having acquired mutation with VAF>1 % in the target codons in TP53 (R175, R181 , R196, R213 G244-G245, R248, R267, R273, R282) or BCOR (R1 162, R1163, R1 164). A 10-fold mean difference, standard deviation of 2x the mean, and 3 replicates per group is 99.28% powered to achieve a p-value of >0.05. Thus, the number of samples and independent experimental runs may besufficient to detect differences between CG-ablated and control hPSCs that are even less robust than anticipated. An alternative approach would be to expand the number of ablated CG dinucleotides in the edited hPSCs, increasing the pool of CG dinucleotides in the analysis. Synonymous edits have been designed for additional CG dinucleotides with known C to T mutations in hPSCs and cancers.

[0145] To identify potential impacts on gene expression and splicing, quantitative reverse transcriptase polymerase chain reaction (qRTPCR), cDNA sequencing, and protein analysis will be performed on the edited lines. qRTPCR primers will be designed on constitutively utilized exons upstream of any of the edits to capture total transcript expression. For splicing detection, primers will be designed on the 5' and 3’ UTRs to amplify full length cDNA, and the resulting TP53 and BCOR amplicons will be subjected to targeted NGS and analyzed using Cuffdiff (Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28, 511-515 (2010), the contents of which are incorporated herein by reference in their entirety). Notably, many CG mutations may occur in arginine codons. All six arginine codons (CGA, CGC, CGG, CGT, AGA, AGG) may be utilized at similar frequencies in human protein-coding sequences, though the two arginine codons that lack a CG dinucleotide contain the canonical splice acceptor sequence AG. While 99% of splice sites may utilize AG / GT sequences, AG / GT dinucleotides comprise 12% of the genome, and less than 1 % of AG / GT dinucleotides may be utilized as splice donors / acceptor sites, with the context sequences surrounding AG and GT sites that may have major role in splice site recognition. If altered splicing is found in the edited cells, the cryptic splice site will be mapped to identify the causal synonymous edit and re-engineer the hPSC line such that the responsible synonymous edit and flanking sequences lack the aberrant AG / GT splice site. Western blotting will be performed using standard methods and antibodies to TP53 and BCOR. Notably, none of the proposed synonymous edits are catalogued in the ClinVar database (see Landrum MJ et al., ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014 Jan 1 ;42(1 ):D980-5; the contents of which are incorporated herein by reference in their entirety) as pathogenic or likely pathogenic mutations and genome wide analysis supports the expectation that synonymous variants will not impact normal gene regulation.Additional Embodiments

[0146] Various embodiments of the present technology are set forth below in paragraphs

[0147] to

[0208] :

[0147] 1. An engineered cell having one or more edited codons encoding an amino acid at one or more genomic loci, the engineered cell generated by the steps of(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a cytosine-guanine (CG) dinucleotide at the genomic loci encoding the amino acid; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein the one or more edited codons comprises at least one nucleotide that is different from nucleotides in the codon encoding the amino acid.

[0148] 2. An engineered cell having one or more edited codons each encoding a first amino acid and a second amino acid at a genomic loci generated by the steps of(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits one or more codons comprising a CG dinucleotide at a codon junction; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein at least one of the edited codons comprises a nucleotide sequence that is different from at least one codon at the codon junction encoding the first amino acid and the second amino acid.

[0149] 3. An engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence encoding an amino acid at one or more genomic loci selected from the group consisting of chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945,chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776 chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354, the engineered cell generated by the steps of(I) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at the genomic loci; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein the one or more edited codons is different from the codon encoding the amino acid.

[0150] 4. A method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at a genomic loci; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate one or more edited codons in the engineered cell, wherein the one or more edited codons is different from the codon comprising the CG dinucleotide.

[0151] 5. A method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at a genomic loci; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate one or more edited codons in the engineered cell, the engineered cell comprising or consisting of an AGA or an AGG nucleotide sequence atChr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945, chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776 chr17:7,674,219-7,674,221 ,Chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354, wherein the one or more edited codons is different from the codon comprising the CG dinucleotide.

[0152] 6. A method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits one or more codons comprising a CG dinucleotide at a codon junction; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein at least one of the edited codons comprises a nucleotide sequence that is different from at least one codon at the codon junction encoding a first amino acid and a second amino acid.

[0153] 7. An engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence at one or more genomic loci selected from the group consisting of chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945, chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776 chr17:7,674,219-7,674,221 ,Chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354.

[0154] 8. An engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence at chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945, chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 ,Chr17:7,673,774-7,673,776Chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354.

[0155] 9. The engineered cell of any one of embodiments 1 -6, wherein the one or more edited codons comprise two or more codons.

[0156] 10. The engineered cell of any one of embodiments 1 , 2, or 9, wherein the one or more edited codons comprise an edited codon in Table 3, Table 4, Table 6, Table 7, or Table 8.

[0157] 11. The method of any one of embodiments 4-6, wherein the one or more edited codons comprise two or more codons.

[0158] 12. The method of any one of embodiments 4-6 or 11 , wherein the one or more edited codons comprise an edited codon in Table 3, Table 4, Table 6, Table 7, or Table 8.

[0159] 13. The engineered cell or the method of any one of embodiments 1 -12, wherein the codon comprises a nucleotide sequence endogenous to the isolated cell.

[0160] 14. The engineered cell or the method of embodiment 13, wherein the isolated cell comprises a cell selected from the group consisting of a cancer cell, a cell susceptible to becoming a cancer cell, a tumor cell, a cyst cell, and a cell that promotes cancer cell growth.

[0161] 15. The engineered cell or the method of any one of embodiments 1 -14, wherein the one or more edited codons is on a sense strand of a deoxyribonucleic acid molecule.

[0162] 16. The engineered cell or the method of any one of embodiments 1 -15, wherein the one or more edited codons comprises a codon in a tumor protein 53 (TP53) gene.

[0163] 17. The engineered cell or the method of any one of embodiments 1 -15, wherein the one or more edited codons is a codon in a BCL6 corepressor (BCOR) gene.

[0164] 18. The engineered cell or the method of any one of embodiments 1 -6 and 9-17, wherein the nucleotide editing apparatus comprises a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system, a transcription activatorlike effector nuclease (TALENs) system, a zinc finger nuclease system, a base editing system, or a prime editing system.

[0165] 19. The engineered cell or the method of embodiment 18, wherein theCRISPR-Cas system further comprises a guide ribonucleic acid (gRNA) that selectively binds to the codon comprising the CG dinucleotide.

[0166] 20. The engineered cell of any one of embodiments 1 -3 or 7-19, wherein the engineered cell is a mammalian cell.

[0167] 21. The engineered cell of embodiment 20, wherein the mammalian cell is human.

[0168] 22. The engineered cell of any one of embodiments 1 -3 or 7-21 , wherein the engineered cell is present in a population of engineered cells.

[0169] 23. The engineered cell of embodiment 22, wherein the population of engineered cells is transplanted into a subject.

[0170] 24. The engineered cell of any one of embodiments 1 -3, wherein the steps of generating the engineered cell do not introduce a CG dinucleotide at a codon junction in the genome of the isolated cell.

[0171] 25. The engineered cell of any one of embodiments 1 -3 or 24, wherein the steps of generating the engineered cell do not introduce a splice site in the genome of the isolated cell.

[0172] 26. The engineered cell of any one of embodiments 1 -3, 24, or 25, wherein the steps of generating the engineered cell do not introduce a start codon in the genome of the isolated cell.

[0173] 27. The engineered cell of any one of embodiments 1 -3 or 24-26, wherein the steps of generating the engineered cell do not introduce a repeated DNA element in the genome of the isolated cell.

[0174] 28. The engineered cell or the method of any one of embodiments 1 ,3, 4, or 5, wherein the amino acid, the first amino acid, or the second amino acid is selected from the group consisting of arginine, serine, proline, threonine, and alanine.

[0175] 29. The engineered cell of embodiment 5, wherein the first amino acid is selected from the group consisting of phenylalanine, leucine, isoleucine, serine, proline, threonine, tyrosine, histidine, asparagine, cysteine, arginine, valine, alanine, aspartic acid, and glycine, and

[0176] the second amino acid is selected from the group consisting of valine, alanine, aspartic acid, glycine, and glutamic acid.

[0177] 30. The engineered cell of embodiment 29, wherein the phenylalanine is encoded by a phenylalanine codon comprising a TTC nucleotide acid sequence.

[0178] 31 . The engineered cell of embodiment 30, wherein the phenylalanine codon is 5’ to a codon comprising an amino acid sequence selected from the group consisting of GTT, GTC, GTA, GTG, GOT, GCC, GCA, GCG, GAT, GAC, GAA, GAG, GGC, GGC, and GGA, GGG.

[0179] 32. The engineered cell of any one of embodiments 29-31 , wherein the one or more edited codons comprises a TTT nucleotide sequence.

[0180] 33. The engineered cell of any one or embodiments 29-31 , wherein the one or more edited codons comprises a nucleotide sequence selected from the group consisting of GGAGGC, GGGGGC, and GGTGGC.

[0181] 34. The engineered cell of embodiment 33, wherein the nucleotide sequence is at chr17:7,674,228-7,674,233.

[0182] 35. The engineered cell or the method of embodiment 28, wherein the arginine is encoded by an arginine codon selected from the group consisting of CGT, CGA, CGG, and CGC.

[0183] 36. The engineered cell or the method of embodiment 35, wherein the one or more edited codons comprises an AGA or an AGG nucleotide sequence.

[0184] 37. The engineered cell or the method of embodiment 28, wherein the serine is encoded by a serine codon comprising the nucleotide sequence TOG.

[0185] 38. The engineered cell or the method of embodiment 37, wherein the one or more edited codons comprises a TCT, a TCA, a TCC, an AGT, or an AGO nucleotide sequence.

[0186] 39. The engineered cell or the method of embodiment 28, wherein the threonine is encoded by a threonine codon comprising the nucleotide sequence ACG.

[0187] 40. The engineered cell or the method of embodiment 39, wherein the one or more edited codons comprises an ACT, an ACA, or an ACC nucleotide sequence.

[0188] 41 . The engineered cell or the method of embodiment 28, wherein the proline is encoded by a proline codon comprising the nucleotide sequence CCG.

[0189] 42. The engineered cell or the method of embodiment 41 , wherein the one or more edited codons comprises a CCT, a CCA, or a CCC nucleotide sequence.

[0190] 43. The engineered cell or the method of embodiment 28 or 29, wherein the alanine is encoded by an alanine codon comprising the nucleotide sequence GCG.

[0191] 44. The engineered cell or the method of embodiment 43, wherein the one or more edited codons comprises a GCT, a GCA, or a GCC nucleotide sequence.

[0192] 45. The engineered cell or the method of any one of embodiments 1 -6 or 28-44, wherein the CG dinucleotide is encoded on an autosome.

[0193] 46. The engineered cell or the method of any one of embodiments 1 -6 or 28-44, wherein the CG dinucleotide is encoded on a sex chromosome.

[0194] 47. The engineered cell or the method of any one of embodiments 1 -6 or 28-46, wherein the period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell is about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 minutes, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, or about 4 weeks.

[0195] 48. The method of any one of embodiments 4-6, 1 1 -19, or 28-47, wherein the mutation is selected from the group consisting of rs28934578, rs138729528, rs28934575, rs1057519989, rs11540652, rs121912651 , rs121913343, and rs28934576.

[0196] 49. The engineered cell or the method of any one of embodiments 1 -48, wherein the engineered cell is a somatic cell.

[0197] 50. The engineered cell or the method of embodiment 49, wherein the somatic cell is a somatic stem cell.

[0198] 51 . The engineered cell or the method of embodiment 49, wherein the somatic cell expresses a chimeric antigen receptor (CAR).

[0199] 52. The engineered cell or the method of embodiment 49, wherein the somatic cell is a cell engineered with T-cell receptor (TCR) engineering.

[0200] 53. The engineered cell or the method of any one of embodiments 49-52, wherein the somatic cell is a cell selected from the group consisting of a T-cell, a natural killer cell, a monocyte, a macrophage, a blood cell, a neural cell, a skin cell, a myocyte, an epithelial cell, an islet cell, and a donor derived beta cell.

[0201] 54. The engineered cell or the method of any one of embodiments 1 -48, wherein the engineered cell is a non-somatic cell.

[0202] 55. The engineered cell or the method of embodiment 54, wherein the non-somatic cell is a cell selected from the group consisting of a stem cell, a germ cell, and a stem cell-derived cell.

[0203] 56. The engineered cell or the method of embodiment 55, wherein the stem cell is selected from the group consisting of a totipotent stem cell, a pluripotent stem cell, and a multipotent stem cell.

[0204] 57. The engineered cell or the method of embodiment 50, 55 or 56, wherein the stem cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell (iPSC), and a hematopoietic stem cell.

[0205] 58. The engineered cell or the method of embodiment 55, wherein the stem cell-derived cell is selected from the group consisting of a totipotent-derived stem cell, a pluripotent-derived stem cell, and a multipotent-derived stem cell.

[0206] 59. The engineered cell or the method of embodiment 50, 55 or 58, wherein the stem cell-derived cell is selected from the group consisting of an embryonic stem cell-derived cell, an iPSC-derived cell, and a hematopoietic stem cell-derived cell.

[0207] 60. The engineered cell or the method of embodiment 57 or 59, wherein the iPSC or the iPSC-derived cell is derived from a cell selected from the group consisting of a skin cell, a blood cell, and a urine cell.

[0208] 61 . The engineered cell or the method of any one of embodiments 1 -60, wherein the engineered cell is a cell selected from the group consisting of a progenitor cell, neural progenitor cell, a progenitor-derived cell, a neural progenitor- derived cell.

[0209] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the present technology. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMS l / We claim:1 . An engineered cell having one or more edited codons encoding an amino acid at one or more genomic loci, the engineered cell generated by the steps of(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a cytosine-guanine (CG) dinucleotide at the genomic loci encoding the amino acid; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein the one or more edited codons comprises at least one nucleotide that is different from nucleotides in the codon encoding the amino acid.

2. An engineered cell having one or more edited codons each encoding a first amino acid and a second amino acid at a genomic loci generated by the steps of(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits one or more codons comprising a CG dinucleotide at a codon junction; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein at least one of the edited codons comprises a nucleotide sequence that is different from at least one codon at the codon junction encoding the first amino acid and the second amino acid.

3. An engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence encoding an amino acid at one or more genomic loci selected from the group consisting ofChr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945,chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776 chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354, the engineered cell generated by the steps of(I) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at the genomic loci; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein the one or more edited codons is different from the codon encoding the amino acid.

4. A method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at a genomic loci; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate one or more edited codons in the engineered cell, wherein the one or more edited codons is different from the codon comprising the CG dinucleotide.

5. A method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits a codon comprising a CG dinucleotide at a genomic loci; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate one or more edited codons in the engineered cell, the engineered cell comprising or consisting of an AGA or an AGG nucleotide sequence at chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , Chr17:7,674,943-7,674,945, chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776 Chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354, wherein the one or more edited codons is different from the codon comprising the CG dinucleotide.

6. A method of engineering a cell having a reduced mutation risk, relative to a control, the method comprising(i) introducing to an isolated cell, a nucleotide editing apparatus that selectively edits one or more codons comprising a CG dinucleotide at a codon junction; and(ii) culturing the isolated cell for a period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell, wherein at least one of the edited codons comprises a nucleotide sequence that is different from at least one codon at the codon junction encoding a first amino acid and a second amino acid.

7. An engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence at one or more genomic loci selected from the group consisting of chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 , chr17:7,674,943-7,674,945,Chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776 chr17:7,674,219-7,674,221 ,Chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354.

8. An engineered cell having one or more edited codons comprising or consisting of an AGA or an AGG nucleotide sequence at chr17:7,675,087-7,675,089, chr17:7,675,069-7,675,071 ,Chr17:7,674,943-7,674,945, chr17:7,674,892-7,674,894, chr17:7,673,819-7,673,821 , chr17:7,673,774-7,673,776Chr17:7,674,219-7,674,221 , chr17:7,673,801 -7,673,803, chrX:40, 064, 346-40, 064, 348, chrX:40, 064, 349-40, 064, 351 , and chrX:40, 064, 352-40, 064, 354.

9. The engineered cell of any one of claims 1-6, wherein the one or more edited codons comprise two or more codons.

10. The engineered cell of any one of claims 1 , 2, or 9, wherein the one or more edited codons comprise an edited codon in Table 3, Table 4, Table 6, Table 7, or Table 8.11 . The method of any one of claims 4-6, wherein the one or more edited codons comprise two or more codons.

12. The method of any one of claims 4-6 or 11 , wherein the one or more edited codons comprise an edited codon in Table 3, Table 4, Table 6, Table 7, or Table 8.

13. The engineered cell or the method of any one of claims 1 -12, wherein the codon comprises a nucleotide sequence endogenous to the isolated cell.

14. The engineered cell or the method of claim 13, wherein the isolated cell comprises a cell selected from the group consisting of a cancer cell, a cell susceptible to becoming a cancer cell, a tumor cell, a cyst cell, and a cell that promotes cancer cell growth.

15. The engineered cell or the method of any one of claims 1 -14, wherein the one or more edited codons is on a sense strand of a deoxyribonucleic acid molecule.

16. The engineered cell or the method of any one of claims 1 -15, wherein the one or more edited codons comprises a codon in a tumor protein 53 (TP53) gene.

17. The engineered cell or the method of any one of claims 1 -15, wherein the one or more edited codons is a codon in a BCL6 corepressor (BCOR) gene.

18. The engineered cell or the method of any one of claims 1-6 and 9-17, wherein the nucleotide editing apparatus comprises a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system, a transcription activator-like effector nuclease (TALENs) system, a zinc finger nuclease system, a base editing system, or a prime editing system.

19. The engineered cell or the method of claim 18, wherein the CRISPR-Cas system further comprises a guide ribonucleic acid (gRNA) that selectively binds to the codon comprising the CG dinucleotide.

20. The engineered cell of any one of claims 1-3 or 7-19, wherein the engineered cell is a mammalian cell.21 . The engineered cell of claim 20, wherein the mammalian cell is human.

22. The engineered cell of any one of claims 1-3 or 7-21 , wherein the engineered cell is present in a population of engineered cells.

23. The engineered cell of claim 22, wherein the population of engineered cells is transplanted into a subject.

24. The engineered cell of any one of claims 1 -3, wherein the steps of generating the engineered cell do not introduce a CG dinucleotide at a codon junction in the genome of the isolated cell.

25. The engineered cell of any one of claims 1 -3 or 24, wherein the steps of generating the engineered cell do not introduce a splice site in the genome of the isolated cell.

26. The engineered cell of any one of claims 1 -3, 24, or 25, wherein the steps of generating the engineered cell do not introduce a start codon in the genome of the isolated cell.

27. The engineered cell of any one of claims 1 -3 or 24-26, wherein the steps of generating the engineered cell do not introduce a repeated DNA element in the genome of the isolated cell.

28. The engineered cell or the method of any one of claims 1 , 3, 4, or 5, wherein the amino acid, the first amino acid, or the second amino acid is selected from the group consisting of arginine, serine, proline, threonine, and alanine.

29. The engineered cell of claim 5, wherein the first amino acid is selected from the group consisting of phenylalanine, leucine, isoleucine, serine, proline, threonine, tyrosine, histidine, asparagine, cysteine, arginine, valine, alanine, aspartic acid, and glycine, and the second amino acid is selected from the group consisting of valine, alanine, aspartic acid, glycine, and glutamic acid.

30. The engineered cell of claim 29, wherein the phenylalanine is encoded by a phenylalanine codon comprising a TTC nucleotide acid sequence.31 . The engineered cell of claim 30, wherein the phenylalanine codon is 5’ to a codon comprising an amino acid sequence selected from the group consisting of GTT, GTC, GTA, GTG, GOT, GCC, GCA, GCG, GAT, GAC, GAA, GAG, GGC, GGC, and GGA, GGG.

32. The engineered cell of any one of claims 29-31 , wherein the one or more edited codons comprises a TTT nucleotide sequence.

33. The engineered cell of any one or claims 29-31 , wherein the one or more edited codons comprises a nucleotide sequence selected from the group consisting of GGAGGC, GGGGGC, and GGTGGC.

34. The engineered cell of claim 33, wherein the nucleotide sequence is at chr17:7,674,228-7,674,233.

35. The engineered cell or the method of claim 28, wherein the arginine is encoded by an arginine codon selected from the group consisting of CGT, CGA, CGG, and CGC.

36. The engineered cell or the method of claim 35, wherein the one or more edited codons comprises an AGA or an AGG nucleotide sequence.

37. The engineered cell or the method of claim 28, wherein the serine is encoded by a serine codon comprising the nucleotide sequence TCG.

38. The engineered cell or the method of claim 37, wherein the one or more edited codons comprises a TOT, a TCA, a TCC, an AGT, or an AGC nucleotide sequence.

39. The engineered cell or the method of claim 28, wherein the threonine is encoded by a threonine codon comprising the nucleotide sequence ACG.

40. The engineered cell or the method of claim 39, wherein the one or more edited codons comprises an ACT, an ACA, or an ACC nucleotide sequence.

41. The engineered cell or the method of claim 28, wherein the proline is encoded by a proline codon comprising the nucleotide sequence CCG.

42. The engineered cell or the method of claim 41 , wherein the one or more edited codons comprises a CCT, a CCA, or a CCC nucleotide sequence.

43. The engineered cell or the method of claim 28 or 29, wherein the alanine is encoded by an alanine codon comprising the nucleotide sequence GCG.

44. The engineered cell or the method of claim 43, wherein the one or more edited codons comprises a GCT, a GCA, or a GCC nucleotide sequence.

45. The engineered cell or the method of any one of claims 1 -6 or 28-44, wherein the CG dinucleotide is encoded on an autosome.

46. The engineered cell or the method of any one of claims 1 -6 or 28-44, wherein the CG dinucleotide is encoded on a sex chromosome.

47. The engineered cell or the method of any one of claims 1 -6 or 28-46, wherein the period of time sufficient to edit the CG dinucleotide to generate the one or more edited codons in the engineered cell is about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 minutes, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, or about 4 weeks.

48. The method of any one of claims 4-6, 11 -19, or 28-47, wherein the mutation is selected from the group consisting of rs28934578, rs138729528, rs28934575, rs1057519989, rs11540652, rs121912651 , rs121913343, and rs28934576.

49. The engineered cell or the method of any one of claims 1 -48, wherein the engineered cell is a somatic cell.

50. The engineered cell or the method of claim 49, wherein the somatic cell is a somatic stem cell.

51. The engineered cell or the method of claim 49, wherein the somatic cell expresses a chimeric antigen receptor (CAR).

52. The engineered cell or the method of claim 49, wherein the somatic cell is a cell engineered with T-cell receptor (TCR) engineering.

53. The engineered cell or the method of any one of claims 49-52, wherein the somatic cell is a cell selected from the group consisting of a T-cell, a natural killer cell, a monocyte, a macrophage, a blood cell, a neural cell, a skin cell, a myocyte, an epithelial cell, an islet cell, and a donor derived beta cell.

54. The engineered cell or the method of any one of claims 1 -48, wherein the engineered cell is a non-somatic cell.

55. The engineered cell or the method of claim 54, wherein the non-somatic cell is a cell selected from the group consisting of a stem cell, a germ cell, and a stem cell-derived cell.

56. The engineered cell or the method of claim 55, wherein the stem cell is selected from the group consisting of a totipotent stem cell, a pluripotent stem cell, and a multipotent stem cell.

57. The engineered cell or the method of claim 50, 55 or 56, wherein the stem cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell (iPSC), and a hematopoietic stem cell.

58. The engineered cell or the method of claim 55, wherein the stem cell- derived cell is selected from the group consisting of a totipotent-derived stem cell, a pluripotent-derived stem cell, and a multipotent-derived stem cell.

59. The engineered cell or the method of claim 50, 55 or 58, wherein the stem cell-derived cell is selected from the group consisting of an embryonic stem cell-derived cell, an iPSC-derived cell, and a hematopoietic stem cell-derived cell.

60. The engineered cell or the method of claim 57 or 59, wherein the iPSC or the iPSC-derived cell is derived from a cell selected from the group consisting of a skin cell, a blood cell, and a urine cell.61 . The engineered cell or the method of any one of claims 1 -60, wherein the engineered cell is a cell selected from the group consisting of a progenitor cell, neural progenitor cell, a progenitor-derived cell, a neural progenitor-derived cell.

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