Compositions and Methods for Genome Editing

Abstract

Compositions and methods for ex vivo genome editing using the Neisseria meningitidis (Nme) CRISPR / Cas9 system are disclosed. The present disclosure provides engineered cells, including for example, gene modifications for use in adoptive cell transfer therapy.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,990, filed on June 16, 2022, under 35 U.S.C. § 119(e), and the entire content thereof is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in XML file format, and the entire content thereof is incorporated herein by reference. The XML file was created on June 13, 2023, named "01155 - 0055 - 00PCT_SL.xml", and has a size of 20,586,099 bytes.

Summary of the Invention

[0003] The present disclosure relates to genome editing using the Neisseria meningitidis CRISPR / Cas9 system.

[0004] The ability to down - regulate endogenous T - cell receptor (TCR) alpha and beta subunits, MHC class I, and MHC class II loci is important for many in vivo and ex vivo applications, for example, for transplantation or for creating, in vitro, cell populations that do not activate T cells using allogeneic cells (derived from a donor). In particular, the transfer of allogeneic cells into a subject has received great interest in the field of cell therapy. The use of allogeneic cells is limited due to the problem of rejection by the recipient's immune cells that recognize the transplanted cells as foreign and mount an attack. To avoid the problem of immune rejection, cell - based therapies have focused on an autologous approach that uses the subject's own cells as the cell source for therapy, which is time - consuming and costly.

[0005] Typically, immune rejection of allogeneic cells is due to a mismatch of major histocompatibility complex (MHC) molecules between the donor and the recipient. Within the human population, MHC molecules exist in various forms, including, for example, multiple genetic variants, i.e., alleles, of any given MHC gene that encode different forms of MHC proteins. The primary classes of MHC molecules are referred to as MHC class I and MHC class II. MHC class I molecules (e.g., HLA-A, HLA-B, and HLA-C in humans) are expressed on all nucleated cells, present antigens, and activate cytotoxic T cells (CD8+ T cells or CTLs). MHC class II molecules (e.g., HLA-DP, HLA-DQ, and HLA-DR in humans) are expressed only on certain cell types (e.g., B cells, dendritic cells, and macrophages), present antigens, and activate helper T cells (CD4+ T cells or Th cells), thereby providing signals to B cells to produce antibodies.

[0006] For example, minor differences in MHC alleles between individuals can activate T cells within the recipient. During T cell development, an individual's T cell repertoire is tolerant to its own MHC molecules, but T cells that recognize MHC molecules of another individual may persist in circulation and are referred to as alloreactive T cells. Alloreactive T cells can be activated, for example, by the presence of cells of another individual expressing MHC molecules in the body, causing, for example, graft-versus-host disease and transplant rejection.

[0007] For example, methods and compositions for reducing the susceptibility to allogeneic cell rejection reactions, including reducing the expression of the cell's MHC proteins to avoid recipient T cell responses, are of interest. In fact, the ability to genetically modify allogeneic cells for transplantation into a subject has been hampered by the requirements for multiple genome edits to reduce all MHC protein expression while simultaneously avoiding other harmful recipient immune responses. For example, strategies to deplete MHC class I proteins can reduce CTL activation, while cells lacking MHC class I on their surface are susceptible to lysis by natural killer (NK) cells of the immune system. This is because NK cell activation is regulated by MHC class I-specific inhibitory receptors. Thus, it has proven difficult to safely reduce or eliminate the expression of MHC class I. Genome editing strategies for depleting MHC class II molecules have also proven difficult, particularly in certain cell types, due to reasons including low editing efficiency and low cell viability, which has hindered their practical application as cell therapies.

[0008] Accordingly, there is a need for improved methods and compositions for modifying cells to overcome the problems of recipient immune rejection reactions and the technical difficulties associated with the multiple gene modifications required to produce safer cells for transplantation. The present disclosure provides genome editing using the Neisseria meningitidis CRISPR / Cas9 system. NmeCas9 is smaller than Streptococcus pyogenes Cas9 (SpyCas9), enabling NmeCas9 to be suitable for messenger RNA (mRNA)-based delivery methods. NmeCas9 has advantageous specificity and a low off-target cleavage rate.

[0009] The engineered cells include genetic modifications in HLA-A, TRAC, TRBC, or CIITA (class II major histocompatibility complex transactivator) that may be useful for cell therapy. The present disclosure further provides compositions and methods for reducing or eliminating surface expression of endogenous T cell receptors, MHC class I or II proteins in cells by genetically modifying the HLA-A, TRAC, TRBC, or CIITA gene.

[0010] In some embodiments, provided is a method for reducing surface expression of HLA-A protein in engineered cells as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments shown herein. In some embodiments, provided is a method for reducing surface expression of TRAC protein in engineered cells as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments shown herein. In some embodiments, provided is a method for reducing surface expression of TRBC protein in engineered cells as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments shown herein. In some embodiments, provided is a method for reducing surface expression of MHC class II protein in engineered cells as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments shown herein.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0012] The present disclosure provides engineered cells that are useful, for example, in adoptive cell transfer (ACT) therapy, as well as methods and compositions for genetically modifying cells to produce engineered cells and populations of engineered cells that are useful, for example, in adoptive cell transfer (ACT) therapy. The disclosure provided herein overcomes certain hurdles of conventional methods by providing methods and compositions for genetically modifying the HLA-A, TRAC, TRBC, CIITA, or AAVS1 locus to reduce the expression of HLA-A, TRAC, TRBC, or MHC class II proteins on the surface of cells. In some embodiments, the present disclosure provides engineered cells in which the surface expression of HLA-A, TRAC, TRBC, or MHC class II is reduced or eliminated as a result of genetic modification in the HLA-A, TRAC, TRBC, or CIITA gene. In some embodiments, the present disclosure provides compositions and methods for reducing or eliminating the expression of HLA-A, TRAC, TRBC, or MHC class II proteins, as well as compositions and methods for further reducing the susceptibility of cells to immune rejection reactions. For example, in some embodiments, the methods and compositions include reducing or eliminating the surface expression of HLA-A protein by genetically modifying the HLA-A gene. In some embodiments, the methods and compositions include reducing or eliminating the surface expression of TRAC protein by genetically modifying the TRAC gene. In some embodiments, the methods and compositions include reducing or eliminating the surface expression of TRBC1 protein by genetically modifying the TRBC1 gene. In some embodiments, the methods and compositions include reducing or eliminating the surface expression of TRBC2 protein by genetically modifying the TRBC2 gene. In some embodiments, the methods and compositions include reducing or eliminating the surface expression of MHC class II protein by genetically modifying CIITA. Engineered cell compositions produced by the methods disclosed herein have desirable properties including, for example, reduced expression of MHC molecules, reduced immunogenicity in vitro and in vivo, increased survival time, and increased genetic compatibility with a larger subject population for transplantation.

[0013] The terms "about" or "approximately" mean an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, or the degree of variation that does not materially affect the characteristics of the subject matter being described, or the acceptable ranges accepted in the art (e.g., within 10%, 5%, 2%, or 1%). Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0014] The following numbered embodiments are provided herein.

[0015] Embodiment 1 is an engineered cell comprising a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to an unmodified cell, wherein the genetic modification comprises at least 1 nucleotide within genomic coordinates chr6:29942540-29945459.

[0016] Embodiment 2 is the engineered cell according to Embodiment 1, comprising a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to an unmodified cell, wherein the genetic modification comprises at least 1 nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 1, or the genetic modification comprises at least 1 nucleotide within a genomic coordinate targeted by a guide RNA comprising any one of the guide sequences of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80.

[0017] Embodiment 3 is an engineered cell comprising a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to an unmodified cell, wherein the genetic modification comprises at least 1 nucleotide within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494.

[0018] Embodiment 4 is an engineered human cell comprising a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to an unmodified cell, wherein the genetic modification comprises at least 1 nucleotide within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809.

[0019] Embodiment 5 is an engineered human cell comprising a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to an unmodified cell, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494.

[0020] Embodiment 6 is an engineered human cell comprising a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to unmodified cells, wherein the genetic modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within a genomic locus selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809.

[0021] Embodiment 7 is an engineered cell according to any one of Embodiments 1-6, wherein the expression of HLA-A is reduced or eliminated by a genome editing system that binds to an HLA-A genomic target sequence comprising at least 5 consecutive nucleotides within a genomic locus selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494, or within a genomic locus selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809.

[0022] Embodiment 8 is an engineered cell according to any one of Embodiments 1-7, wherein the genetic modification comprises at least 1 nucleotide within a genomic locus selected from any one of the genomic loci listed in Table 1.

[0023] Embodiment 9 is an engineered cell according to any one of Embodiments 1-8, wherein the cell is homozygous for HLA-C.

[0024] Embodiment 10 is the engineered cell according to any one of Embodiments 1 to 9, wherein the cell is homozygous for HLA-B and homozygous for HLA-C.

[0025] Embodiment 11 is a composition comprising an HLA-A guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the HLA-A guide RNA comprises: i. a guide sequence selected from SEQ ID NOs: 66, 61, 2 to 60, 62 to 65, 67 to 80; ii. at least 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 66, 61, 2 to 60, 62 to 65, 67 to 80; or iii. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 66, 61, 2 to 60, 62 to 65, 67 to 80; iv. a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides; v. at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence from (iv); or vi. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0026] Embodiment 12 is a method of producing engineered human cells with reduced or eliminated surface expression of HLA-A protein compared to unmodified cells, comprising contacting the cells with a composition comprising an HLA-A guide RNA and optionally an RNA-guided DNA binder, or a nucleic acid encoding an RNA-guided DNA binder, wherein the HLA-A guide RNA comprises: i. a guide sequence selected from SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; ii. at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; or iii. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; iv. a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides; v. at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence from (iv); or vi. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0027] Embodiment 13 is a method for reducing the surface expression of HLA-A protein in human cells as compared to unmodified cells, comprising contacting the cells with a composition comprising an HLA-A guide RNA and optionally an RNA-guided DNA binder, or a nucleic acid encoding an RNA-guided DNA binder, wherein the HLA-A guide RNA is: i. a guide sequence selected from SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; ii. at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; or iii. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; iv. a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides; v. at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence from (iv); or vi. a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0028] Embodiment 14 is the composition or method according to any one of Embodiments 11-13, wherein the HLA-A guide RNA comprises a guide sequence of any one of SEQ ID NOs: 66, 61, 13, 55, 70, and 71.

[0029] Embodiment 15 is the composition or method according to any one of Embodiments 11-13, wherein the HLA-A guide RNA comprises a guide sequence of any one of SEQ ID NOs: 61, 66, 13, 17, 55, and 70.

[0030] Embodiment 16 is the composition or method according to any one of Embodiments 11-13, wherein the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 61.

[0031] Embodiment 17 is the composition or method according to any one of Embodiments 11-13, wherein the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 66.

[0032] Embodiment 18 is a cell population comprising engineered cells produced by the engineered cells described in any one of Embodiments 1 to 10, or by the method described in any one of Embodiments 12 to 17, or by the use of the composition described in Embodiment 11.

[0033] Embodiment 19 is a pharmaceutical composition comprising (a) engineered cells produced by the engineered cells described in any one of Embodiments 1 to 10, by the method described in any one of Embodiments 12 to 17, or by the use of the composition described in Embodiment 11, or (b) the cell population described in Embodiment 18.

[0034] Embodiment 20 is an engineered human cell comprising a gene modification in the TRAC gene that reduces or eliminates surface expression of TRAC as compared to unmodified cells, wherein the gene modification comprises at least 1 nucleotide within genomic coordinates chr14:22547505-22551621, or chr14:22547462-22551621.

[0035] Embodiment 21 is the engineered cell according to Embodiment 20, wherein the gene modification comprises at least 1 nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 2, or the gene modification comprises at least 1 nucleotide within a genomic coordinate targeted by a guide RNA comprising any one of the guide sequences of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120.

[0036] Embodiment 22 is the engineered cell according to Embodiment 20 or 21, wherein the gene modification comprises at least 1 nucleotide within a genomic coordinate selected from chr14:22550574-22550598, chr14:22550544-22550568, chr14:22547505-22547529, or chr14:22547525-22547549, chr14:22547674-22547698.

[0037] Embodiment 23 is the engineered cell according to Embodiment 20 or 21, wherein the gene modification comprises at least 1 nucleotide within genomic coordinates chr14:22547481-22547505.

[0038] Embodiment 24 is the engineered cell according to Embodiment 20 or 21, wherein the gene modification comprises at least 1 nucleotide within genomic coordinates chr14:22547471-22547495.

[0039] Embodiment 25 is the engineered cell according to Embodiment 20 or 21, wherein the gene modification comprises at least 1 nucleotide within genomic coordinates chr14:22547470-22547494.

[0040] Embodiment 26 is the engineered cell according to Embodiment 20 or 21, wherein the gene modification comprises at least 1 nucleotide within genomic coordinates chr14:22547462-22547486.

[0041] Embodiment 27 is an engineered human cell comprising a gene modification in the TRAC gene that reduces or eliminates the expression of TRAC as compared to an unmodified cell, wherein the gene modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr14:22550574-22550598, chr14:22550544-22550568, chr14:22547505-22547529, chr14:22547525-22547549, or chr14:22547674-22547698.

[0042] Embodiment 28 is the engineered cell according to any one of Embodiments 20 to 27, wherein the expression of TRAC is reduced or eliminated by a genome editing system that binds to a TRAC target sequence containing at least 5 consecutive nucleotides within a genomic locus selected from chr14:22550574-22550598, chr14:22550544-22550568, chr14:22547505-22547529, chr14:22547525-22547549, or chr14:22547674-22547698.

[0043] Embodiment 29 is a composition comprising a TRAC guide RNA comprising a guide sequence, which a) directs an RNA-guided DNA binding agent to i) target a TRAC genomic target sequence or ii) induce a double-strand break (DSB) or single-strand break (SSB) within a TRAC genomic target sequence containing at least 10 consecutive nucleotides within a genomic locus selected from chr14:22550574-22550598, chr14:22550544-22550568, chr14:22547505-22547529, chr14:22547525-22547549, or chr14:22547674-22547698.

[0044] Embodiment 30 is a composition comprising (a) a TRAC guide RNA and optionally (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the TRAC guide RNA comprises: i) a guide sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 2 ± 10 nucleotides; v) at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0045] Embodiment 31 is the composition according to Embodiment 30 for use in altering a DNA sequence within the TRAC locus in a cell.

[0046] Embodiment 32 is the composition according to Embodiment 30 for use in reducing or eliminating the expression of the TRAC protein in a cell.

[0047] Embodiment 33 is a method of generating engineered human cells with reduced or eliminated surface expression of the TRAC protein as compared to unmodified cells, the method comprising contacting the cells with a composition comprising (a) a TRAC guide RNA and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the TRAC guide RNA comprises: i) a guide sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 2 ± 10 nucleotides; v) at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0048] Embodiment 34 is a method for reducing the surface expression of TRAC protein in human cells as compared to unmodified cells, the method comprising contacting the cells with a composition comprising (a) a TRAC guide RNA and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the TRAC guide RNA comprises: i) a guide sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 2 ± 10 nucleotides; v) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0049] Embodiment 35 is the composition or method according to any one of Embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence of any one of SEQ ID NOs: 111, 107, 101, 102, and 103.

[0050] Embodiment 36 is the composition or method according to any one of Embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 107.

[0051] Embodiment 37 is the composition or method according to any one of Embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 111.

[0052] Embodiment 38 is the composition or method according to any one of Embodiments 29 to 34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 117.

[0053] Embodiment 39 is the composition or method according to any one of Embodiments 29 to 34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 118.

[0054] Embodiment 40 is the composition or method according to any one of Embodiments 29 to 34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 119.

[0055] Embodiment 41 is the composition or method according to any one of Embodiments 29 to 34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 120.

[0056] Embodiment 42 is an engineered cell according to any one of Embodiments 20 to 28, or the use of a composition according to Claim 29 or 30, or an engineered cell produced by the method according to any one of Embodiments 33 to 41, wherein more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% of the cell population is CD3− cells.

[0057] Embodiment 43 is an engineered cell according to any one of Embodiments 20 to 28, or the use of a composition according to Embodiment 29 or 30, or an engineered cell produced by the method according to any one of Embodiments 33 to 41, or the cell population according to Embodiment 42, wherein more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% of the population lacks an endogenous T cell receptor.

[0058] Embodiment 44 is the use of an engineered cell according to any one of Embodiments 20-28, or a composition according to Embodiment 29 or 30, or an engineered cell produced by the method according to any one of Embodiments 33-41, wherein the expression of the TRAC gene in the population is reduced by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%, or at least about 99% compared to an unaltered population of the same cells, or a cell population comprising the engineered cell, or a cell population according to Embodiment 42 or 43.

[0059] Embodiment 45 is a pharmaceutical composition comprising an engineered cell according to any one of Embodiments 20-28, or a composition according to Embodiment 29 or 30, or an engineered cell produced by the method according to any one of Embodiments 33-41, or a cell population according to any one of Embodiments 42-44.

[0060] Embodiment 46 is an engineered cell comprising a genetic modification at the TRBC locus that reduces or eliminates surface expression of TRBC compared to an unmodified cell, wherein the genetic modification comprises at least 1 nucleotide within genomic coordinates chr7:142791756-142802543.

[0061] Embodiment 47 is an engineered cell comprising a genetic modification at the TRBC locus that reduces or eliminates surface expression of TRBC compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within genomic coordinates: (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543, or the genetic modification comprises at least one nucleotide within genomic coordinates targeted by a guide RNA comprising any one of the guide sequences of SEQ ID NOs: 215, 201-214, and 216-265.

[0062] Embodiment 48 is the engineered cell according to Embodiment 46 or 47, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from any one of the genomic coordinates listed in Table 3.

[0063] Embodiment 49 is the engineered cell according to Embodiment 46 or 47, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from: (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130.

[0064] Embodiment 50 is the engineered cell according to Embodiment 46 or 47, wherein the gene modification contains at least one nucleotide within the genomic coordinates selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213.

[0065] Embodiment 51 is an engineered cell comprising a gene modification in the human TRBC gene that reduces or eliminates the expression of TRBC as compared to an unmodified cell, wherein the gene modification contains an indel, a substitution from C to T, or a substitution from A to G within the genomic coordinates (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543.

[0066] Embodiment 52 is an engineered human cell comprising a gene modification in the TRBC gene that reduces or eliminates the expression of TRBC as compared to an unmodified cell, wherein the gene modification contains an indel, a substitution from C to T, or a substitution from A to G within the genomic coordinates selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130.

[0067] Embodiment 53 is an engineered human cell comprising a genetic modification in the TRBC gene that reduces or eliminates the expression of TRBC as compared to an unmodified cell, wherein the genetic modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within a genomic locus selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213.

[0068] Embodiment 54 is the engineered cell according to any one of Embodiments 46 to 53, wherein the TRBC expression is reduced or eliminated by a genome editing system that binds to a TRBC target sequence comprising at least 5 consecutive nucleotides within a genomic locus selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130.

[0069] Embodiment 55 is the engineered cell according to any one of Embodiments 46 to 54, wherein the expression of TRBC is reduced or eliminated by a genome editing system that binds to a TRBC target sequence comprising at least 5 consecutive nucleotides within a genomic locus selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213.

[0070] Embodiment 56 is a composition comprising (a) a TRBC guide RNA and optionally (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the TRBC guide RNA comprises: i) a guide sequence selected from SEQ ID NOs: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NOs: 215, 201-214, and 216-265; iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 215, 201-214, and 216-265; iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 3 plus or minus 10 nucleotides; v) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0071] Embodiment 57 is the composition according to Embodiment 56 for use in altering a DNA sequence within the TRBC locus in a cell.

[0072] Embodiment 58 is the composition according to Embodiment 56 for use in reducing or eliminating the expression of a TRBC protein in a cell.

[0073] Embodiment 59 is a method of producing engineered human cells with reduced or eliminated surface expression of the TRBC protein compared to unmodified cells, the method comprising contacting the cells with (a) a TRBC guide RNA and optionally (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the TRBC guide RNA comprises: i) a guide sequence selected from SEQ ID NO: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NO: 215, 201-214, and 216-265; iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NO: 215, 201-214, and 216-265; iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 3 ± 10 nucleotides; v) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0074] Embodiment 60 is a method for reducing the surface expression of TRBC protein in human cells as compared to unmodified cells, the method comprising contacting the cells with (a) a TRBC guide RNA and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the TRBC guide RNA comprises: i) a guide sequence selected from SEQ ID NOs: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NOs: 215, 201-214, and 216-265; iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 215, 201-214, and 216-265; iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 3 ± 10 nucleotides; v) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0075] Embodiment 61 is the method or composition according to any one of Embodiments 56-60, wherein the TRBC guide RNA comprises any one of the guide sequences of SEQ ID NOs: 215, 216, 223, 224, 229, 230, 246, 259, and 260.

[0076] Embodiment 62 is the method or composition according to any one of Embodiments 56-61, wherein the TRBC guide RNA comprises any one of the guide sequences of SEQ ID NOs: 215, 216, 224, 229, 246, 259, and 260.

[0077] Embodiment 63 is the method or composition according to any one of Embodiments 56-62, wherein the TRBC guide RNA comprises any one of the guide sequences of SEQ ID NOs: 215, 259, and 260.

[0078] Embodiment 64 is a cell population in which more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% of the cell population is CD3− cells, the engineered cells described in any one of Embodiments 46 to 55, or the use of the composition described in Embodiment 56, or the engineered cells produced by the method described in any one of Embodiments 59 to 63.

[0079] Embodiment 65 is a cell population in which more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% of the population lacks an endogenous T cell receptor, the engineered cells described in any one of Embodiments 46 to 55, or the use of the composition described in Embodiment 56, or the engineered cells produced by the method described in any one of Embodiments 59 to 63.

[0080] Embodiment 66 is a cell population in which the expression of the TRBC gene in the population is reduced by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% compared to an unmodified population of the same cells, the engineered cells described in any one of Embodiments 46 to 55, or the use of the composition described in Embodiment 56, or the engineered cells produced by the method described in any one of Embodiments 59 to 63.

[0081] Embodiment 67 is a pharmaceutical composition comprising (a) the engineered cells described in any one of Embodiments 46 to 55, or the use of the composition described in Embodiment 56, or the engineered cells produced by the method described in any one of Embodiments 59 to 63, or (b) the cell population described in any one of Embodiments 64 to 66.

[0082] Embodiment 68 is an engineered cell comprising a genetic modification in the CIITA gene that reduces or eliminates surface expression of MHC class II compared to unmodified cells, wherein the genetic modification comprises at least one nucleotide within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136.

[0083] Embodiment 69 is the engineered cell according to Embodiment 68, wherein the genetic modification comprises at least one nucleotide within a genomic locus selected from any one of the genomic loci listed in Table 4.

[0084] Embodiment 70 is the engineered cell according to Embodiment 68, wherein the gene modification contains at least 1 nucleotide within the genomic coordinates selected from (a) chr16:10907504-10907528, chr16:10906643-10906667, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or (b) chr16:10906889-10906913, or chr16:10907504-10907528.

[0085] Embodiment 71 is the engineered cell according to Embodiment 68, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from chr16:10907504-10907528, chr16:10906643-10906667, chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10907477-10907501, chr16:10907497-10907521, or chr16:10907508-10907532.

[0086] Embodiment 72 is an engineered cell comprising a genetic modification at the CIITA locus that reduces or eliminates surface expression of MHC class II compared to unmodified cells, wherein the genetic modification comprises (a) indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr16:10907504-10907528, chr16:10906643-10906667, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or (b) indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from chr16:10907504-10907528.

[0087] Embodiment 73 is an engineered cell comprising a genetic modification at the CIITA locus that reduces or eliminates surface expression of MHC class II compared to an unmodified cell, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr16:10907504-10907528, chr16:10906643-10906667, chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, chr16:10907508-10907532.

[0088] Embodiment 74 is the engineered cell according to any one of Embodiments 68 to 73, wherein the expression of MHC class II is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence containing at least 5 consecutive nucleotides within a genomic locus selected from (a) chr16:10907504-10907528, chr16:10906643-10906667, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or chr16:10907504-10907528.

[0089] Embodiment 75 is the engineered cell according to any one of Embodiments 68 to 74, wherein the expression of MHC class II is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence containing at least 5 consecutive nucleotides within genomic coordinates selected from chr16:10907504-10907528, chr16:10906643-10906667, chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10907477-10907501, chr16:10907497-10907521, or chr16:10907508-10907532.

[0090] Embodiment 76 is a composition comprising (a) a CIITA guide RNA and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the CIITA guide RNA is i) a guide sequence selected from SEQ ID NOs: 301, 422, 302 to 421, and 423 to 576, or ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301, 422, 302 to 421, and 423 to 576, or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301, 422, 302 to 421, and 423 to 576, or iv) a sequence comprising 10 consecutive nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 4, or v) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence from (iv), or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0091] Embodiment 77 is the composition according to Embodiment 76 for use in altering a DNA sequence within the CIITA gene in a cell.

[0092] Embodiment 78 is the composition according to Embodiment 76 for use in reducing or eliminating the expression of CIITA in cells.

[0093] Embodiment 79 is a method of generating engineered human cells having reduced or eliminated surface expression of MHC class II proteins as compared to unmodified cells, comprising contacting the cells with (a) a CIITA guide RNA and optionally (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the CIITA guide RNA comprises i) a guide sequence selected from SEQ ID NOs: 301, 422, 302-421, and 423-576, or ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301, 422, 302-421, and 423-576, or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301, 422, 302-421, and 423-576, or iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 4 ± 10 nucleotides, or v) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence from (iv), or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0094] Embodiment 80 is a method for reducing the surface expression of MHC class II proteins in human cells as compared to unmodified cells, the method comprising contacting the cells with (a) a CIITA guide RNA and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, wherein the CIITA guide RNA comprises: i) a guide sequence selected from SEQ ID NOs: 301, 422, 302-421, and 423-576; or ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NOs: 301, 422, 302-421, and 423-576; or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301, 422, 302-421, and 423-576; or iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 4 ± 10 nucleotides; or v) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence from (iv); or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0095] Embodiment 81 is the method or composition according to any one of Embodiments 76-80, wherein the CIITA guide RNA comprises any one of the guide sequences of SEQ ID NOs: 301, 422, 302, 320, 321, 324, 326, 327, 332, 354, 361, 372, 400, 408, 414, 415, 419, 420, 428, 431, 432, 434, 451, 455, 458, 462, 463, 464, 468.

[0096] Embodiment 82 is the method or composition according to any one of Embodiments 76-81, wherein the CIITA guide RNA comprises any one of the guide sequences of SEQ ID NO: 538.

[0097] Embodiment 83 is the method or composition according to any one of Embodiments 76 to 81, wherein the CIITA guide RNA contains any one of the guide sequences of SEQ ID NOs: 301, 422, 302, 320, 372, 414, 419, 462, and 463.

[0098] Embodiment 84 is the use of the engineered cell according to any one of Embodiments 68 to 75, or the composition according to Embodiment 76, or the engineered cell produced by the method according to any one of Embodiments 79 to 83, wherein more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% of the cell population is negative MHC class II molecules when measured by flow cytometry, or a cell population comprising the engineered cell.

[0099] Embodiment 85 is the use of the engineered cell according to any one of Embodiments 68 to 75, or the composition according to Embodiment 76, or the engineered cell produced by the method according to any one of Embodiments 79 to 83, wherein more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 98%, or more than about 99% of the cell population is negative for MHC class II molecules when measured by next-generation sequencing (NGS), or a cell population comprising the engineered cell.

[0100] Embodiment 86 is the use of the engineered cell according to any one of Embodiments 68 to 75, or the composition according to Embodiment 76, or the engineered cell produced by the method according to any one of Embodiments 79 to 83, wherein the expression of MHC class II molecules in the population is reduced by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% compared to the unmodified population of the same cells, or a cell population comprising the engineered cell.

[0101] Embodiment 87 is a pharmaceutical composition comprising (a) an engineered cell according to any one of Embodiments 68 to 75, or the use of the composition according to Embodiment 76, or an engineered cell produced by the method according to any one of Embodiments 79 to 83, or (b) a cell population according to any one of Embodiments 84 to 86.

[0102] Embodiment 88 is an engineered cell comprising a gene modification at the AAVS1 locus, wherein the gene modification comprises at least one nucleotide within a genomic coordinate selected from chr19:55115151-55116209.

[0103] Embodiment 89 is the engineered cell according to Embodiment 88, wherein the gene modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 5.

[0104] Embodiment 90 is the engineered cell according to Embodiment 88, wherein the gene modification comprises at least 1 nucleotide within genomic coordinates selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030.

[0105] Embodiment 91 is an engineered cell comprising a gene modification in the AAVS1 gene, wherein the gene modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within genomic coordinates selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030.

[0106] Embodiment 92 is an engineered cell according to any one of Embodiments 88 to 91, which is induced by a genome editing system that binds to an AAVS1 genomic target sequence containing at least 5 consecutive nucleotides within a genomic locus selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030.

[0107] Embodiment 93 is a composition comprising an AAVS1 guide RNA comprising a guide sequence that directs an RNA-guided DNA binding agent to induce a double-strand break (DSB) or a single-strand break (SSB) within an AAVS1 genomic target sequence that either a) i) targets the AAVS1 genomic target sequence or ii) comprises at least 10 contiguous nucleotides within a genomic locus selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030.

[0108] Embodiment 94 is a composition comprising (a) an AAVS1 guide RNA (gRNA), and optionally, (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the AAVS1 guide RNA comprises: (i) a guide sequence selected from SEQ ID NOs: 601-774; (ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NOs: 601-774; (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 601-774; (iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 5 ± 10 nucleotides; (v) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the sequence from (iv); or (vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0109] Embodiment 95 is a method of generating an engineered human cell, comprising contacting the cell with (a) an AAVS1 guide RNA, and optionally, (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the AAVS1 guide RNA comprises: (i) a guide sequence selected from SEQ ID NOs: 601-774; (ii) at least 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a sequence selected from SEQ ID NOs: 601-774; (iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 601-774; (iv) a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 5 ± 10 nucleotides; (v) at least 20, 21, 22, 23, or 24 consecutive nucleotides of the sequence from (iv); or (vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0110] Embodiment 96 is the method or composition according to any one of Embodiments 93 to 95, wherein the AAVS1 guide RNA contains any one of the guide sequences of SEQ ID NOs: 611, 620, 622, 626, 627, 628, 629, 632, 633, 634, 656, 659, 660, 661, 673, 691, 692, 730, 734, and 746.

[0111] Embodiment 97 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 96, wherein the gene modification contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides within the genomic coordinates.

[0112] Embodiment 98 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 97, wherein the gene modification contains at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates.

[0113] Embodiment 99 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 98, wherein the genomic target sequence contains at least 10 consecutive nucleotides within the genomic coordinates.

[0114] Embodiment 100 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 99, wherein the genomic target sequence contains at least 15 consecutive nucleotides within the genomic coordinates.

[0115] Embodiment 101 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 100, wherein the genomic target sequence contains at least 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides within the genomic coordinates.

[0116] Embodiment 102 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 101, wherein the gene modification includes an indel.

[0117] Embodiment 103 is an engineered cell according to any one of Embodiments 1 to 102, wherein the gene modification includes the insertion of a heterologous coding sequence.

[0118] Embodiment 104 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 103, wherein the gene modification includes at least one substitution of A to G within a genomic coordinate.

[0119] Embodiment 105 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 104, wherein the gene modification includes at least one substitution of C to T within a genomic coordinate.

[0120] Embodiment 106 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 105, wherein the cell has a gene modification within the CIITA gene.

[0121] Embodiment 107 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 106, wherein the cell has reduced expression of the TRAC protein on the surface of the cell.

[0122] Embodiment 108 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 107, wherein the cell has reduced expression of the TRBC protein on the surface of the cell.

[0123] Embodiment 109 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 108, wherein the cell has reduced expression of MHC class II molecules on the surface of the cell.

[0124] Embodiment 110 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 109, wherein the engineered cell is an immune cell.

[0125] Embodiment 111 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 110, wherein the cell is a primary cell.

[0126] Embodiment 112 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 111, wherein the engineered cell is a monocyte, macrophage, mast cell, dendritic cell, or granulocyte.

[0127] Embodiment 113 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 112, wherein the engineered cell is a lymphocyte.

[0128] Embodiment 114 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 113, wherein the cell is a T cell.

[0129] Embodiment 115 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 114, wherein the cell is a CD8+ T cell.

[0130] Embodiment 116 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 114, wherein the cell is a CD4+ T cell.

[0131] Embodiment 117 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 113, wherein the cell is a natural killer (NK) cell.

[0132] Embodiment 118 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 113, wherein the cell is a macrophage.

[0133] Embodiment 119 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 113, wherein the cell is a B cell.

[0134] Embodiment 120 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 113, wherein the cell is a plasmacytoid B cell.

[0135] Embodiment 121 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 113, wherein the cell is a memory B cell.

[0136] Embodiment 122 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 109, wherein the cell is a stem cell.

[0137] Embodiment 123 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 109, wherein the cell is a progenitor cell.

[0138] Embodiment 124 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 109, wherein the cell is a HSC.

[0139] Embodiment 125 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 109, wherein the cell is an iPSC.

[0140] Embodiment 126 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 121, wherein the cell is an activated cell.

[0141] Embodiment 127 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 121, wherein the cell is an inactivated cell.

[0142] Embodiment 128 is the population according to Embodiment 18 or the pharmaceutical composition according to Embodiment 19, wherein the cell population is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% HLA-A negative when measured by flow cytometry.

[0143] Embodiment 129 is the population according to any one of Embodiments 42-44 and 64-66, or the pharmaceutical composition according to Embodiment 45 or 67, wherein the cell population is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% endogenous TCR protein negative when measured by flow cytometry.

[0144] Embodiment 130 is the population according to any one of Embodiments 84-86, or the pharmaceutical composition according to Embodiment 87, wherein the cell population is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% HLA-DP, DQ, DR negative when measured by flow cytometry.

[0145] Embodiment 131 is the population according to Embodiment 18 or the pharmaceutical composition according to Embodiment 19, wherein at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the cell population contains gene modification in the HLA-A gene when measured by next-generation sequencing (NGS).

[0146] Embodiment 132 is the population according to any one of Embodiments 42 to 44 or the pharmaceutical composition according to Embodiment 45, wherein at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cell population contains gene modification in the TRAC gene when measured by next-generation sequencing (NGS).

[0147] Embodiment 133 is the population according to any one of Embodiments 64 to 66 or the pharmaceutical composition according to Embodiment 67, wherein at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cell population contains gene modification in the TRBC gene when measured by next-generation sequencing (NGS).

[0148] Embodiment 134 is the population according to any one of Embodiments 84 to 86, or the pharmaceutical composition according to Embodiment 87, wherein at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cell population contains gene modification in the CIITA gene when measured by next-generation sequencing (NGS).

[0149] Embodiment 135 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 134, wherein the cell is an allogeneic cell.

[0150] Embodiment 136 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 135, wherein the cell is a primary cell.

[0151] Embodiment 137 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a CD4+ T cell.

[0152] Embodiment 138 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a CD8+ T cell.

[0153] Embodiment 139 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a memory T cell.

[0154] Embodiment 140 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a B cell.

[0155] Embodiment 141 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a plasma cell.

[0156] Embodiment 142 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a memory B cell.

[0157] Embodiment 143 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a natural killer (NK) cell.

[0158] Embodiment 144 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a macrophage.

[0159] Embodiment 145 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a stem cell.

[0160] Embodiment 146 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a pluripotent stem cell (PSC).

[0161] Embodiment 147 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a hematopoietic stem cell (HSC).

[0162] Embodiment 148 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is an induced pluripotent stem cell (iPSC).

[0163] Embodiment 149 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a mesenchymal stem cell (MSC).

[0164] Embodiment 150 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a neural stem cell (NSC).

[0165] Embodiment 151 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a limbic system stem cell (LSC).

[0166] Embodiment 152 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a progenitor cell, such as an endothelial progenitor cell or a neural progenitor cell.

[0167] Embodiment 153 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a tissue-specific primary cell.

[0168] Embodiment 154 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is selected from chondrocytes, myocytes, and keratinocytes.

[0169] Embodiment 155 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is an activated cell.

[0170] Embodiment 156 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 136, wherein the cell is a non-activated cell.

[0171] Embodiment 157 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 156, wherein the cell is engineered with a genome editing system.

[0172] Embodiment 158 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 7 to 157, wherein the genome editing system comprises an RNA-guided DNA binding agent or a nucleic acid encoding the RNA-guided DNA binding agent.

[0173] Embodiment 159 is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 158, wherein the RNA-guided DNA binding agent or the RNA-guided DNA binding agent encoded by the nucleic acid is N. meningitidis Cas9 (NmeCas9).

[0174] Embodiment 160 is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 159, wherein NmeCas9 is Nme1Cas9, Nme2Cas9, or Nme3Cas9.

[0175] Embodiment 161 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158 to 160, wherein the RNA-guided DNA binding agent or the RNA-guided DNA binding agent encoded by the nucleic acid has double-strand endonuclease activity.

[0176] Embodiment 162 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158 to 161, wherein the RNA-guided DNA binding agent or the RNA-guided DNA binding agent encoded by the nucleic acid has nickase activity.

[0177] Embodiment 163 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158 to 161, wherein the RNA-guided DNA binding agent or the RNA-guided DNA binding agent encoded by the nucleic acid comprises a dCas9 DNA binding domain.

[0178] Embodiment 164 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158-160, wherein the RNA-guided DNA binder, or the nucleic acid encoding the RNA-guided DNA binder, is a base editor from A to G.

[0179] Embodiment 165 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158-160, wherein the RNA-guided DNA binder, or the nucleic acid encoding the RNA-guided DNA binder, is a base editor from C to T.

[0180] Embodiment 166 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 158-165, wherein the RNA-guided DNA binder, or the RNA-guided DNA binder encoded by the nucleic acid, comprises a deaminase region.

[0181] Embodiment 167 is an engineered cell according to Embodiment 158, wherein the RNA-guided DNA binder, or the RNA-guided DNA binder encoded by the nucleic acid, comprises APOBEC3A deaminase (A3A) and N. meningitidis Cas9 nickase.

[0182] Embodiment 168 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1-167, wherein the guide RNA is provided to the cell by a vector.

[0183] Embodiment 169 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1-168, wherein the RNA-guided DNA binder is provided to the cell by a vector, optionally the same vector as the guide RNA.

[0184] Embodiment 170 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1-169, wherein the exogenous nucleic acid is provided to the cell by a vector.

[0185] Embodiment 171 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 168 to 170, wherein the vector is a viral vector.

[0186] Embodiment 172 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 168 to 170, wherein the vector is a non-viral vector.

[0187] Embodiment 173 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 168 to 171, wherein the vector is a lentiviral vector.

[0188] Embodiment 174 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 168 to 171, wherein the vector is a retroviral vector.

[0189] Embodiment 175 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 168 to 171, wherein the vector is AAV.

[0190] Embodiment 176 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 175, wherein the guide RNA is provided to the cell in a lipid nucleic acid assembly composition, and optionally in the same lipid nucleic acid assembly composition as the RNA guide DNA binder.

[0191] Embodiment 177 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 176, wherein the exogenous nucleic acid is provided to the cell in a lipid nucleic acid assembly composition.

[0192] Embodiment 178 is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 176 or 177, wherein the lipid nucleic acid assembly composition is a lipid nanoparticle (LNP).

[0193] Embodiment 179 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 178, wherein the exogenous nucleic acid is integrated into the genome of the cell.

[0194] Embodiment 180 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 179, wherein the exogenous nucleic acid is integrated into the genome of the cell by homologous recombination (HR).

[0195] Embodiment 181 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 180, wherein the exogenous nucleic acid is integrated into a safe harbor locus within the genome of the cell.

[0196] Embodiment 182 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 181, wherein the guide RNA is a single guide RNA.

[0197] Embodiment 183 is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 182, wherein the single guide RNA comprises the nucleotide sequence 3' of SEQ ID NO: 900 for the guide sequence.

[0198] Embodiment 184 is such that the single guide RNA includes a guide region and a conserved region, and the conserved region is (a) a shortened repeat / anti-repeat region that lacks 2 to 24 nucleotides, where (i) one or more of nucleotides 37 to 48 and 53 to 64 relative to SEQ ID NO: 900 are deleted, and optionally one or more of nucleotides 37 to 64 are substituted, and (ii) nucleotide 36 is linked to nucleotide 65 by at least two nucleotides, a shortened repeat / anti-repeat region; or (b) a shortened hairpin 1 region that lacks 2 to 10, optionally 2 to 8 nucleotides, where (i) one or more of nucleotides 82 to 86 and 91 to 95 relative to SEQ ID NO: 900 are deleted, and optionally one or more of positions 82 to 96 are substituted, and (ii) nucleotide 81 is linked to nucleotide 96 by at least four nucleotides, a shortened hairpin 1 region; or (c) a shortened hairpin 2 region that lacks 2 to 18, optionally 2 to 16 nucleotides, where (i) one or more of nucleotides 113 to 121 and 126 to 134 relative to SEQ ID NO: 900 are deleted, and optionally one or more of nucleotides 113 to 134 are substituted, and (ii) nucleotide 112 is linked to nucleotide 135 by at least four nucleotides, a shortened hairpin 2 region; includes one or more of these, one or both of nucleotides 144 to 145 relative to SEQ ID NO: 900 are optionally deleted, and at least 10 nucleotides are modified nucleotides, which is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 182 or 183.

[0199] Embodiment 185 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 184, where the guide RNA includes at least one modification.

[0200] Embodiment 186 is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 185, wherein the modification comprises a modified nucleotide selected from a 2'-O-methyl (2'-OMe) modified nucleotide, a 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotide, a 2'-fluoro (2'-F) modified nucleotide, an internucleotide phosphorothioate (PS) bond, or an inverted abasic modified nucleotide.

[0201] Embodiment 187 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 186, wherein the gRNA comprises a 5'-end modification, a modification in the repeat / anti-repeat region, a modification in the hairpin 1 region, a modification in the hairpin 2 region, or a 3'-end modification.

[0202] Embodiment 188 is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 187, wherein the 5'-end modification comprises at least one PS bond, and i. one PS bond is present and the bond is between a first nucleotide and a second nucleotide, ii. there are two PS bonds between the first three nucleotides, iii. there is a PS bond between any one or more of the first four nucleotides, iv. there is a PS bond between any one or more of the first five nucleotides, and one or more of the above.

[0203] Embodiment 189 is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 187 or 188, wherein the 5'-end modification further comprises at least one 2'-OMe, 2'-O-moe, inverted abasic, or 2'-F modified nucleotide.

[0204] Embodiment 190 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 187 to 189, wherein the 5'-end modification is: i. one or more modifications of the first 1 to 4 nucleotides, where the modification is a PS bond, an inverted abasic nucleotide, 2'-OMe, 2'-O-moe, or 2'-F; ii. a modification of the first nucleotide by 2'-OMe, 2'-O-moe, or 2'-F, and an optional one or two PS bonds to the next nucleotide or the first nucleotide of the 3'-tail; iii. a modification of the first or second nucleotide by 2'-OMe, 2'-O-moe, or 2'-F, and optionally one or more PS bonds; iv. a modification of the first, second, or third nucleotide by 2'-OMe, 2'-O-moe, or 2'-F, and optionally one or more PS bonds; or v. a modification of the first, second, third, or fourth nucleotide by 2'-OMe, 2'-O-moe, or 2'-F, and optionally one or more PS bonds.

[0205] Embodiment 191 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 187 to 180, wherein the 3'-end modification comprises at least one PS bond: i. one PS bond is present and the bond is between the last nucleotide and the second last nucleotide; ii. there are two PS bonds between the last three nucleotides; iii. there is a PS bond between any one or more of the last four nucleotides.

[0206] Embodiment 192 is the engineered cell, cell population, pharmaceutical composition, or method according to Embodiment 191, wherein the 3'-end modification further comprises at least one 2'-OMe, 2'-O-moe, inverted abasic, or 2'-F modified nucleotide.

[0207] Embodiment 193 is the engineered cell, cell population, pharmaceutical composition, or method according to 192, wherein the 3'-end modification is: i. one or more modifications of one of the last 1 to 4 nucleotides, and the modification is a PS bond, an inverted abasic nucleotide, 2'-OMe, 2'-O-moe, or 2'-F; ii. a modification of the last nucleotide by 2'-OMe, 2'-O-moe, or 2'-F, and an optional one or two PS bonds to the nucleotide next to or the first nucleotide of the 3'-tail; iii. a modification of the last or second-to-last nucleotide by 2'-OMe, 2'-O-moe, or 2'-F, and optionally one or more PS bonds; iv. a modification of the last, second-to-last, or third-to-last nucleotide by 2'-OMe, 2'-O-moe, or 2'-F, and optionally one or more PS bonds; or v. a modification of the last, second-to-last, third-to-last, or fourth-to-last nucleotide by 2'-OMe, 2'-O-moe, or 2'-F, and optionally one or more PS bonds.

[0208] Embodiment 194 is the engineered cell, cell population, pharmaceutical composition, or method according to 193, further comprising a 3'-tail comprising 2'-O-Me modified nucleotides.

[0209] Embodiment 195 is the engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 194, wherein the guide RNA comprises a 5'-end modification or a 3'-end modification.

[0210] Embodiment 196 is that the guide RNA is A guide sequence comprising 2'-O-Me modified nucleotides at the first four nucleotides 1-4; PS linkages between nucleotides 1-2, 2-3, and 3-4; and 2'-O-Me modified nucleotides at nucleotides 5, 8, 9, 11, 13, 18, and 22 of the guide sequence; nucleotides 38-48 and 53-63 are deleted relative to SEQ ID NO: 900, and a shortened repeat / anti-repeat region comprising 2'-O-Me modified nucleotides at nucleotides 25, 29, 30, 31, 32, 37, 49-52, 64, 65, 69, 70, and 73; nucleotides 86 and 91 are deleted relative to SEQ ID NO: 900, and a shortened hairpin 1 region comprising 2'-O-Me modified nucleotides at nucleotides 80, 81, 83, 84, 85, 87-90, 92-94, and 99; a 2'-O-Me modified nucleotide at nucleotide 101 between the shortened hairpin region 1 and the shortened hairpin region 2; nucleotides 112-120 and 127-135 are deleted relative to SEQ ID NO: 900, and a shortened hairpin 2 region comprising 2'-O-Me modified nucleotides at nucleotides 104, 110, 111, 122-125, 142, and 143, and a PS linkage between nucleotides 141-142 and 142-143, wherein one or both of nucleotides 144-145 are optionally deleted relative to SEQ ID NO: 900, the method or composition according to any one of embodiments 1-195 comprising the guide sequence.

[0211] Embodiment 197 is the method or composition according to any one of embodiments 1-196, wherein the guide RNA comprises a modified nucleotide of any one of SEQ ID NOs: 904-909, 911, 995-997, and 1081-1089.

[0212] Embodiment 198 is the method or composition according to any one of embodiments 1-197, wherein the guide RNA comprises the modified nucleotide of SEQ ID NO: 995.

[0213] Embodiment 199 is the method or composition according to any one of embodiments 1-198, wherein the guide RNA comprises the modified nucleotide of SEQ ID NO: 1083.

[0214] Embodiment 200 is the method or composition according to any one of Embodiments 1 to 199, wherein the guide RNA is modified according to any one pattern of SEQ ID NOs: 904 to 909, 911, and 995 to 997, each N in the pattern is any natural or unnatural nucleotide, and Ns together are any one of the guide sequences in Tables 1 to 5.

[0215] Embodiment 201 is the method or composition according to any one of Embodiments 1 to 200, wherein the guide RNA is modified according to the pattern of SEQ ID NO: 995, each N in the pattern is any natural or unnatural nucleotide, and Ns together are any one of the guide sequences in Tables 1 to 5.

[0216] Embodiment 202 is a method of administering an engineered cell, cell population, or pharmaceutical composition according to any one of Embodiments 1 to 10, 18 to 28, 42 to 55, 64 to 75, 84 to 92, and 97 to 195 to a subject in need thereof.

[0217] Embodiment 203 is a method of administering an engineered cell, cell population, or pharmaceutical composition according to any one of Embodiments 1 to 10, 18 to 28, 42 to 55, 64 to 75, 84 to 92, and 97 to 195 to a subject as adoptive cell transfer (ACT) therapy.

[0218] Embodiment 204 is a method of treating a disease or disorder comprising administering an engineered cell, cell population, or pharmaceutical composition according to any one of Embodiments 1 to 10, 18 to 28, 42 to 55, 64 to 75, 84 to 92, and 97 to 195 to a subject in need thereof.

[0219] Embodiment 205 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1 to 10, 18 to 28, 42 to 55, 64 to 75, 84 to 92, and 97 to 195 for use in administering to a subject as adoptive cell transfer (ACT) therapy.

[0220] Embodiment 206 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1-10, 18-28, 42-55, 64-75, 84-92, and 97-195 for use in treating a subject having cancer.

[0221] Embodiment 207 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1-10, 18-28, 42-55, 64-75, 84-92, and 97-195 for use in treating a subject having an infectious disease.

[0222] Embodiment 208 is an engineered cell, cell population, pharmaceutical composition, or method according to any one of Embodiments 1-10, 18-28, 42-55, 64-75, 84-92, and 97-195 for use in treating a subject having an autoimmune disease.

[0223] I. Definitions Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings.

[0224] As used herein, the term "or combinations thereof" refers to all substitutions and combinations of the terms listed before this term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Following this example, combinations containing repetitions of one or more items or terms such as BB, AAA, AAB, BBC, CBBA, CABA, etc. are explicitly included. One of ordinary skill in the art will understand that, unless otherwise apparent from the context, there is typically no limit to the number of items or terms in any combination.

[0225] As used herein, the term "kit" refers to a packaged set of related components (one or more polynucleotides or compositions) and one or more related substances (such as delivery devices (e.g., syringes), solvents, solutions, buffers, instructions, or desiccants).

[0226] "Allogeneic" cells, as used herein, refer to cells derived from a donor subject of the same species as the recipient subject, where the donor subject and the recipient subject have genetic differences, e.g., have genes at one or more loci that are not identical. Thus, for example, the cells are allogeneic with respect to the subject to whom the cells are administered. As used herein, cells removed or isolated from a donor that are not reintroduced into the original donor are considered allogeneic cells.

[0227] "Autologous" cells, as used herein, refer to cells taken from the same subject and into which the cell material will later be reintroduced. Thus, for example, if cells are removed from a subject and then reintroduced into the same subject, the cells are considered autologous.

[0228] "CIITA" or "CIITA" or "C2TA", as used herein, refers to the nucleic acid sequence or protein sequence of "class II major histocompatibility complex transactivator", and the human gene has accession number NC_000016.10 (range 10866208..10941562), reference GRCh38.p13. The nuclear CIITA protein functions as a positive regulatory gene for the transcription of MHC class II genes and is required for the expression of MHC class II proteins.

[0229] As used herein, "MHC" or "MHC molecule(s)" or "MHC protein" or "MHC complex(es)" refers to major histocompatibility complex molecule(s), including, for example, MHC class I and MHC class II molecules. In humans, MHC molecules are referred to as the "human leukocyte antigen" complex or "HLA molecule" or "HLA protein". The use of the terms "MHC" and "HLA" is not meant to be limiting, and as used herein, the term "MHC" can be used to refer to human MHC molecules, i.e., HLA molecules. Thus, the terms "MHC" and "HLA" are used interchangeably herein.

[0230] The term "HLA-A", as used herein in the context of an HLA-A protein, refers to an MHC class I protein molecule that is a heterodimer consisting of a heavy chain (encoded by the HLA-A gene) and a light chain (i.e., beta-2 microglobulin). The term "HLA-A" or "HLA-A gene", as used herein in the context of a nucleic acid, refers to the gene that encodes the heavy chain of the HLA-A protein molecule. The HLA-A gene is also referred to as "HLA class I histocompatibility, A alpha chain", and the human gene has accession number NC_000006.12 (29942532..29945870). The HLA-A gene is known to have thousands of different types (also referred to as "alleles") across the population (an individual can receive two different alleles of the HLA-A gene). A public database of HLA-A alleles containing sequence information can be accessed at IPD-IMGT / HLA: https: / / www.ebi.ac.uk / ipd / imgt / hla / . All alleles of HLA-A are encompassed by the terms "HLA-A" and "HLA-A gene".

[0231] "HLA-B", as used herein in the context of nucleic acids, refers to the gene encoding the heavy chain of the HLA-B protein molecule. HLA-B is also referred to as "HLA class I histocompatibility, B alpha chain", and the human gene has accession number NC_000006.12(31353875..31357179).

[0232] "HLA-C", as used herein in the context of nucleic acids, refers to the gene encoding the heavy chain of the HLA-C protein molecule. HLA-C is also referred to as "HLA class I histocompatibility, C alpha chain", and the human gene has accession number NC_000006.12(31268749..31272092).

[0233] The term "TRAC", as used herein in the context of the TRAC protein, refers to the T cell receptor alpha chain. As used herein in the context of nucleic acids, "TRAC" refers to the gene encoding the T cell receptor alpha chain. The human wild-type TRAC sequence is available at NCBI Gene ID:28755;Ensembl:ENSG00000277734. T cell receptor alpha constant, TCRA, IMD7, TRCA, and TRA are gene synonyms for TRAC.

[0234] The term "TRBC" (or "TRBC1 / 2") is used to refer to the "T cell receptor beta chain", for example, the nucleic acid or amino acid sequences of TRBC1 and TRBC2. The terms "TRBC1" and "TRBC2", when used herein in the context of the TRBC protein, refer to two homologous proteins that include the T cell receptor beta chain. "TRBC1" and "TRBC2", when used herein in the context of nucleic acids, refer to the genes encoding the T cell receptor beta chain. The human wild-type TRBC1 sequence is NCBI Gene ID:28639; Ensembl: ENSG00000211751. T cell receptor beta constant, V_segment translation product, BV05S1J2.2, TCRBC1, and TCRB are gene synonyms for TRBC1. The human wild-type TRBC2 sequence is available at NCBI Gene ID:28638; Ensembl: ENSG00000211772. T cell receptor beta constant, V_segment translation product, and TCRBC2 are gene synonyms for TRBC2.

[0235] As used herein, the term "AAVS1" refers to the genomic location at chr19:50900000-58617616 according to hg38.

[0236] As used herein, the term "within a genomic coordinate" includes the boundaries of the given genomic coordinate range. For example, when chr6:29942854-chr6:29942913 is given, the coordinates chr6:29942854-chr6:29942913 are included. Throughout this application, the genomic coordinates referred to are based on genomic annotations in the GRCh38 (also referred to as hg38) assembly of the human genome from the Genome Reference Consortium, which is available on the website of the National Center for Biotechnology Information. Tools and methods for converting genomic coordinates between one assembly and another are known in the art and can be used to convert the genomic coordinates provided herein to the corresponding coordinates of another assembly of the human genome, including conversions to previous assemblies generated by the same institution or using the same algorithm (e.g., from GRCh38 to GRCh37), and conversions to assemblies generated by different institutions or algorithms (e.g., from GRCh38 to NCBI33 generated by the International Human Genome Sequencing Consortium). Available methods and tools known in the art include, but are not limited to, the NCBI Genome Remapping Service available on the website of the National Center for Biotechnology Information, UCSC LiftOver available on the website of the UCSC Genome Brower, and the Assembly Converter available on the website of Ensembl.org.

[0237] As used herein, the term "homozygous" refers to having two identical alleles of a particular gene.

[0238] As used herein, the term "subject" is intended to include living organisms capable of eliciting an immune response, including, for example, mammals, primates, and humans.

[0239] "Polynucleotide" and "nucleic acid" are used herein to refer to multimeric compounds that include nucleosides or nucleoside analogs (including polymers that are conventional RNA, DNA, hybrid RNA-DNA, and their analogs) having nitrogenous heterocyclic bases or base analogs linked together along a backbone. The nucleic acid "backbone" can be composed of various linkages, including one or more of a sugar phosphate diester linkage, a peptide-nucleic acid bond ("peptide nucleic acid" or PNA, PCT WO95 / 32305), a phosphorothioate linkage, a methylphosphonate linkage, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or a similar compound having a substitution, for example, a 2'-methoxy or 2'-halide substitution. The nitrogenous bases can be conventional bases (A, G, C, T, U), their analogs (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine), inosine, purine or pyrimidine derivatives (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having substituents at the 5- or 6-position (e.g., 5-methylcytosine), purine bases having substituents at the 2, 6, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4 -alkyl-pyrimidines, U.S. Patent No. 5,378,825 and PCT WO93 / 13121). For general considerations, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11 th(see, e.g., ed., 1992). Nucleic acids can contain one or more "abasic" residues where the backbone does not contain nitrogenous bases at the position(s) of the polymer (U.S. Patent No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases, and linkages, or may contain both conventional components and substitutions (e.g., a polymer containing a conventional base with a 2'-methoxy linkage, or both a conventional base and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), analogs that have bicyclic furanose units locked in an RNA-mimicking sugar structure and contain one or more LNA nucleotide monomers that enhance hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or its analogs in RNA and thymine or its analogs in DNA.

[0240] "Guide RNA", "gRNA", and simply "guide" are used interchangeably herein, for example, for the purpose of referring to a guide that directs an RNA guide DNA binder to a target DNA, and can be a single guide RNA, or a combination of a crRNA and a trRNA (also known as a tracrRNA). Exemplary gRNAs include class II Cas nuclease guide RNAs in modified or unmodified form. The crRNA and trRNA can associate as a single RNA molecule (single guide RNA, sgRNA), or can associate in two separate RNA strands (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA can be a native sequence, or a trRNA sequence that has modifications or diversity compared to the native sequence.

[0241] As used herein, the "guide sequence" refers to the sequence within the guide RNA that is complementary to the target sequence and functions to direct the guide RNA to the target sequence to be bound or modified (e.g., cleaved) by an RNA-guided DNA binding agent. The "guide sequence" may also be referred to as the "targeting sequence" or the "spacer sequence". In the case of Neisseria meningitides, the guide sequence can be 19, 20, 21, 22, 23, or 24, or 25 nucleotides in length. In some embodiments, the Nme Cas9 guide sequence comprises at least 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 2-80, 101-120, 201, 265, 301, 302, 304-576, or 601-774. In some embodiments, the target sequence is, for example, within a gene or on a chromosome and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence is at least 80%, 85%, 90%, or 95%. For example, in some embodiments, the guide sequence comprises a sequence of 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 2-80, 101-120, 201, 265, 301, 302, 304-576, or 601-774. In some embodiments, the guide sequence and the target region can be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence. For example, the guide sequence and the target sequence may contain 1 to 2, preferably 1 or fewer mismatches, and the full length of the target sequence is 19, 20, 21, 22, 23, or 24, or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1 to 2 mismatches, and the guide sequence comprises at least 24, or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1 to 2 mismatches, and the guide sequence comprises 24 nucleotides. That is, the guide sequence and the target region may form a double-stranded region or more having base pairs.In certain embodiments, the duplex region may contain 1 to 2 mismatches such that the guide strand and the target sequence are not perfectly complementary. Mismatch positions are known in the art, for example, such that PAM-distal mismatches tend to be better tolerated than PAM-proximal matches. Mismatch tolerances at other positions are known in the art (see, e.g., Edraki et al., 2019. Mol. Cell, 73:1-13).

[0242] Since the nucleic acid substrate of the RNA-guided DNA binder is a double-stranded nucleic acid, the target sequence of the RNA-guided DNA binder includes both the plus and minus strands of genomic DNA (i.e., the given sequence and the reverse complement of the sequence). Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that the guide RNA can be directed to bind to the reverse complement of the target sequence. Thus, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to a particular nucleotide of the target sequence (e.g., a target sequence without a PAM), except for the substitution of U with T in the guide sequence.

[0243] As used herein, "RNA-guided DNA binder" means a polypeptide or complex of polypeptides having RNA and DNA binding activities, or the DNA-binding subunit of such a complex, wherein the DNA-binding activity is sequence-specific and depends on the presence of a PAM and the sequence of the guide RNA. Exemplary RNA-guided DNA binders include Cas cleases / Cas nickases, and their inactivated forms ("dCas DNA binders"). As used herein, "Cas nuclease" (also referred to as "Cas protein") includes Cas cleases, Cas nickases, and dCas DNA binders. Cas cleases / Cas nickases and dCas DNA binders include the Csm complex or Cmr complex of the type III CRISPR system, their Cas10 subunit, Csm1 subunit or Cmr2 subunit, the Cascade complex of the type I CRISPR system, its Cas3 subunit, and class 2 Cas nucleases.

[0244] As used herein, "Class 2 Cas nuclease" is a single-stranded polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas endonucleases / Cas nickases (e.g., the H840A variant, D10A variant or N863A variant of Spy Cas9, and D16A and H588A of Nme2 Cas9, e.g., Nme2 Cas9), which further have RNA-guided DNA endonuclease activity or DNA nickase activity, and Class 2 dCas DNA binders in which the endonuclease activity / nickase activity is inactivated. Examples of Class 2 Cas nucleases include proteins such as Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., the N497A variant, R661A variant, Q695A variant, Q926A variant), HypaCas9 (e.g., the N692A variant, M694A variant, Q695A variant, H698A variant), eSPCas9(1.0) (e.g., the K810A variant, K1003A variant, R1060A variant), and eSPCas9(1.1) (e.g., the K848A variant, K1003A variant, R1060A variant), and modifications thereof. The Cpf1 protein, Zetsche et al., Cell, 163:1-13 (2015) is homologous to Cas9 and includes an RuvC-like nuclease domain. The Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, for example, Tables S1 and S3 of the Zetsche reference. See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015), Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0245] Some Cas9 orthologs are obtained from N. meningitidis (Esvelt et al., Nat. Methods, vol. 10, 2013, 1116 - 1121, Hou et al., PNAS, vol. 110, 2013, pages 15644 - 15649) (Nme1Cas9, Nme2Cas9, and Nme3Cas9). The Nme2Cas9 ortholog functions efficiently in mammalian cells, recognizes the N4CC PAM, and can be used for in vivo editing with cognate gRNAs (Ran et al., Nature, vol. 520, 2015, pages 186 - 191, Kim et al., Nat. Commun., vol. 8, 2017, pages 14500). Nme2Cas9 can be specific and selective, for example, enabling low off - target editing (Lee et al., Mol. Ther., vol. 24, 2016, pages 645 - 654, Kim et al., 2017). See also, e.g., WO / 2020081568, which describes Nme2Cas9 D16A nickase and is incorporated herein by reference in its entirety (e.g., pages 28 and 42). Throughout, "NmeCas9" or "Nme Cas9" is a generic term encompassing any type of NmeCas9, including Nme1Cas9, Nme2Cas9, and Nme3Cas9.

[0246] Exemplary nucleotide and polypeptide sequences of Cas9 molecules are shown in Table 7. Methods for identifying alternative nucleotide sequences encoding Cas9 polypeptide sequences, including alternative naturally - occurring variants, are known in the art. Also contemplated are Cas9 nucleic acid sequences encoding the amino acid sequences provided herein, or sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the nucleic acid sequences.

[0247] As used herein, the term "editor" refers to an agent that includes a polypeptide capable of making modifications within a DNA sequence. In some embodiments, the editor is a nuclease such as Cas9 nuclease. In some embodiments, the editor is capable of deaminating a base within a DNA molecule and may be referred to as a base editor. In some embodiments, the editor is capable of deaminating cytosine (C) in DNA. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to deaminase APOBEC3A (A3A). In some embodiments, the editor comprises a Cas9 nickase fused to deaminase APOBEC3A (A3A). In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase and UGI. In some embodiments, the editor lacks UGI.

[0248] As used herein, "cytidine deaminase" means a polypeptide or polypeptide complex capable of cytidine deaminase activity that catalyzes the hydrolytic deamination of cytidine or deoxycytidine, typically resulting in uridine or deoxyuridine. Cytidine deaminase includes enzymes within the cytidine deaminase superfamily, in particular enzymes of the APOBEC family (enzymes APOBEC1, APOBEC2, APOBEC4, and the APOBEC3 subgroups), activation-induced cytidine deaminase (AID or AICDA), and CMP deaminase (see, for example, Conticello et al., Mol. Biol. Evol. 22:367-77, 2005, Conticello, Genome Biol. 9:229, 2008, Muramatsu et al., J. Biol. Chem. 274:18470-6, 1999, Carrington et al., Cells 9:1690 (2020)). In some embodiments, variants of any known cytidine deaminase or APOBEC protein are included. Variants include proteins having a sequence that differs from the wild-type protein by one or more mutations, such as one or more point substitutions (i.e., substitutions, deletions, insertions). For example, a truncated sequence can be used by deleting amino acids at the N-terminus, C-terminus, or internally, preferably by deleting 1 to 4 amino acids at the C-terminus of the sequence. As used herein, the term "variant" refers to allelic variants, splicing variants, and natural or artificial mutants, which are homologous to the reference sequence. Variants are "functional" in that they exhibit catalytic activity for DNA editing.

[0249] As used herein, the term "APOBEC3A" refers to a cytidine deaminase, e.g., a protein expressed by the human A3A gene. APOBEC3A can have catalytic DNA editing activity. The amino acid sequence of APOBEC3A is described (UniPROT accession ID: p31941) and is included herein as SEQ ID NO: 827. In some embodiments, the APOBEC3A protein is a human APOBEC3A protein or a wild-type protein. Variants include sequences that differ from the wild-type APOBEC3A protein by one or more mutations (i.e., substitutions, deletions, insertions), e.g., proteins having one or more point substitutions. For example, a truncated APOBEC3A sequence can be used by deleting 1 to 4 amino acids, e.g., at the N-terminus, C-terminus, or internal amino acids, preferably at the C-terminus of the sequence. As used herein, the term "variant" refers to allelic variants, splicing variants, and natural or artificial mutants that are homologous to the APOBEC3A reference sequence. Variants are "functional" in that they exhibit catalytic activity for DNA editing. In some embodiments, APOBEC3A (such as human APOBEC3A) has the 57th position of the wild-type amino acids (when numbered in the wild-type sequence). In some embodiments, APOBEC3A (such as human APOBEC3A) has asparagine at the 57th position of the amino acids (when numbered in the wild-type sequence).

[0250] As used herein, "nickase" is an enzyme that creates a single-strand break (also known as a "nick") within double-stranded DNA, i.e., it cleaves one strand of the DNA double helix but not the other. As used herein, "RNA-guided DNA nickase" means a polypeptide or complex of polypeptides having DNA nickase activity, and the DNA nickase activity is sequence-specific and dependent on the sequence of an RNA. Exemplary RNA-guided DNA nickases include Cas nickases. Class 2 Cas nickases include polypeptides in which either the HNH or RuvC catalytic domain has been inactivated, e.g., Cas9 (e.g., the H840A, D10A, or N863A variants of SpyCas9, or the D16A variant of NmeCas9). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain, or the RuvC or RuvC-like domain of N. meningitidis, include Nme2Cas9 D16A (HNH nickase) and Nme2Cas9 H588A (RuvC nickase). Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., the N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., the N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., the K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., the K848A, K1003A, R1060A variants) proteins, as well as modifications thereof, are included. The Cpf1 protein, Zetsche et al., Cell, 163:1-13 (2015) is homologous to Cas9 and contains an RuvC-like protein domain. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety. See, for example, Tables S1 and S3 of the Zetsche reference. "Cas9" includes S. pyogenes (Spy) Cas9, the Cas9 variants listed herein, and equivalents thereof.See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36(2015), Shmakov et al., Molecular Cell, 60:385-397(2015).

[0251] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains derived from at least two different proteins. Since one protein is located at the amino-terminal (N-terminal) portion or carboxy-terminal (C-terminal) protein of the fusion protein, it can form an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein", respectively. Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods of recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)) (the entire content of which is incorporated herein by reference).

[0252] As used herein, the term "linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties. Typically, the linker is located between or sandwiched between two groups, molecules or other moieties and is linked to each by a covalent bond. In some embodiments, the linker is one amino acid or a plurality of amino acids (e.g., a peptide or protein), such as a 16 amino acid residue "XTEN" linker, or a variant thereof (see, e.g., the Examples, and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 930), SGSETPGTSESA (SEQ ID NO: 931), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 932). In some embodiments, the linker is a peptide linker comprising one or more sequences selected from SEQ ID NOs: 933-994.

[0253] As used herein, the term "uracil glycosylase inhibitor" or "UGI" refers to a protein that can inhibit the base excision repair enzyme uracil DNA glycosylase (UDG).

[0254] As used herein, the "open reading frame" or "ORF" of a gene refers to a sequence consisting of a series of codons that specify the amino acid sequence of the protein encoded by that gene. The ORF begins with a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon, e.g., TAA, TAG or TGA in DNA, or UAA, UAG or UGA in RNA.

[0255] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA associated with a Cas nuclease, such as an RNA-guided DNA binder like a Cas cleaver, Cas nickase, or dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA directs an RNA-guided DNA binder, such as Cas9, to a target sequence, the guide RNA hybridizes to the target sequence, and a drug can bind to the target sequence and, if the drug is a cleaver or nickase, perform cleavage or nicking after binding.

[0256] As used herein, when alignment of a first sequence to a second sequence shows that overall, at least X% of the positions of the second sequence match the first sequence, the first sequence is considered to "comprise a sequence having at least X% identity to" the second sequence. For example, the sequence AAGA comprises a sequence having 100% identity to the sequence AAG, because the alignment gives 100% identity in that there is a match at all three positions of the second sequence. Differences between RNA and DNA (generally, exchanging thymidine for uridine, or vice versa), and the presence of nucleoside analogs such as modified uridines, do not contribute to differences in identity or complementarity between polynucleotides, so long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5'-AXG where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, is considered to be 100% identical to AUG in that both are completely complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. One of ordinary skill in the art will understand which algorithm and parameter settings are appropriate for a given pair of sequences to be aligned. In general, for sequences with lengths and expected identities generally similar and greater than 50% for amino acids or greater than 75% for nucleotides, the Needleman-Wunsch algorithm using the default settings of the Needleman-Wunsch algorithm interface provided by EBI at the www.ebi.ac.uk web server is generally appropriate.

[0257] As used herein, "messenger RNA" or "mRNA" refers to a polynucleotide that contains an open reading frame that can be translated into a polypeptide (i.e., can function as a substrate for translation by ribosomes and aminoacylated tRNAs). The mRNA can contain one or more modifications as provided below.

[0258] As used herein, "gene modification" is a change at the DNA level induced, for example, by the CRISPR / Cas9 gRNA and Cas9 system. Gene modification typically can include insertions, deletions, or substitutions (i.e., base sequence substitutions, i.e., mutations) within a defined sequence or genomic locus. Gene modification changes the nucleic acid sequence of DNA. Gene modification can be at a single nucleotide position. Gene modification can be at multiple nucleotides, for example, 2, 3, 4, 5 or more nucleotides, typically in close proximity to each other, for example, contiguous nucleotides. Gene modification can be within a coding sequence, for example, an exon sequence. Gene modification can be at a position sufficiently close to a splice site, i.e., a splice acceptor site or a splice donor site, and can disrupt splicing. Gene modification can include the insertion of a nucleotide sequence that is not endogenous to the genomic locus, for example, the insertion of a heterologous open reading frame or the coding sequence of a gene. As used herein, gene modification can be used to prevent the translation of an endogenous full-length protein having the amino acid sequence of the full-length protein prior to gene modification of the genomic locus. Prevention of translation of the endogenous full-length protein or gene product includes prevention of translation of a protein or gene product of any length. Translation of the endogenous full-length protein can be prevented, for example, by a frameshift mutation that results in the generation of a premature stop codon, or by the generation of a nonsense mutation. Translation of the endogenous full-length protein can be prevented by disruption of splicing. Translation of the endogenous full-length protein can be prevented by the insertion of a heterologous coding sequence. Translation of the endogenous full-length protein can be prevented, for example, if the endogenous full-length protein contains an undesirable mutation, by introducing changes at one or more positions to change the endogenous full-length protein coding sequence and provide a modified full-length coding sequence that is different from the endogenous sequence present in the cell, for example, by correcting a point mutation. Translation of the endogenous full-length protein can be prevented by altering the splicing of the endogenous full-length protein to produce different proteins by alternative splicing.

[0259] As used herein, "indel" refers to an insertion or deletion mutation consisting of several nucleotides inserted, deleted, or inserted and deleted, for example, at a double-strand break (DSB) site of a target nucleic acid. As used herein, when an insertion occurs due to indel formation, this insertion is a random insertion at the DSB site and may or may not be induced by or based on the template sequence.

[0260] As used herein, "heterologous coding sequence" refers to a coding sequence that has been introduced as an exogenous source into a cell (for example, inserted into a genomic locus such as a safe harbor locus containing a TCR locus). That is, the introduced coding sequence is heterologous at least with respect to its insertion site. A polypeptide expressed from such a heterologous coding sequence gene is referred to as a "heterologous polypeptide". The heterologous coding sequence can be naturally occurring or manipulable and can be wild-type or variant. The heterologous coding sequence can include nucleotide sequences other than those encoding a heterologous polypeptide (for example, an internal ribosome entry site). The heterologous coding sequence can be a coding sequence that naturally exists in the genome as a wild-type or variant (for example, a mutant). For example, a cell contains a coding sequence of interest (as a wild-type or variant), but the same coding sequence or its variant can be introduced as an exogenous source for expression, for example, at a highly expressed locus. The heterologous coding sequence can also be a coding sequence that does not naturally exist in the genome or that expresses a heterologous polypeptide that does not naturally exist in the genome. "Heterologous coding sequence", "exogenous coding sequence", and "transgene" are used interchangeably. In some embodiments, the heterologous coding sequence or transgene includes an exogenous nucleic acid sequence. For example, the nucleic acid sequence is not endogenous to the recipient cell. In some embodiments, the heterologous coding sequence or transgene includes an exogenous nucleic acid sequence, for example, a nucleic acid sequence that does not naturally exist within the recipient cell. For example, the heterologous coding sequence can be heterologous with respect to its insertion site and with respect to its recipient cell.

[0261] As used herein, “reduction or elimination” of protein expression in a cell refers to the partial or complete loss of expression of that protein as compared to an unmodified cell. In some embodiments, surface expression of a protein on a cell is “reduced or eliminated” as compared to an unmodified cell, as demonstrated by a reduction in the fluorescence signal when stained with the same antibody against the protein, as measured by flow cytometry. A cell that “reduces or eliminates” surface expression of a protein by flow cytometry as compared to an unmodified cell may be termed “negative” for expression of that protein, as demonstrated by a fluorescence signal similar to that of a cell stained with an isotype control antibody. “Reduction or elimination” of protein expression can be measured by other known techniques in the art, using appropriate controls known to those of skill in the art.

[0262] As used herein, “knockdown” refers to reducing the expression of a particular gene product (e.g., a protein, mRNA, or both), e.g., as compared to the expression of an unedited target sequence. Knockdown of a protein can be measured by detecting the total cellular amount of the protein in a sample such as a tissue, fluid, or cell population of interest. This can also be measured by measuring an alternative, marker, or activity of the protein. Methods for measuring knockdown of mRNA are known and include analyzing mRNA isolated from a sample of interest. In some embodiments, “knockdown” can refer to some loss of expression of a particular gene product, e.g., a decrease in the amount of transcribed mRNA, or a decrease in the amount of protein expressed by a cell or cell population (including in vivo populations such as those found in a tissue).

[0263] As used herein, "knockout" refers to the absence of expression from a particular gene or the expression of a particular protein in a cell. A knockout can result in a decrease in expression below the detection level of an assay. A knockout can be measured by any of detecting the total cellular amount of a protein in a cell, tissue, or cell population.

[0264] As used herein, "target sequence" or "genomic target sequence" refers to the nucleic acid sequence of a target gene that has complementarity to the guide sequence of a gRNA. The interaction between the target sequence and the guide sequence directs the RNA-guided DNA binder to bind within the target sequence and potentially form a nick or cleavage (depending on the activity of the agent).

[0265] As used herein, "treating" refers to administering or applying a therapeutic agent to a subject for a disease or disorder, including inhibiting the disease, preventing its onset, alleviating one or more of the symptoms of the disease, curing the disease, or preventing one or more of the symptoms of the disease (including recurrence of the symptoms).

[0266] In the following, specific embodiments of the invention will be referred to in detail, examples of which are illustrated in the accompanying drawings. The invention is described in conjunction with the illustrated embodiments, but it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is defined by the appended claims and is intended to cover all alternatives, modifications, and equivalents that may be included within the invention by the embodiments included.

[0267] Before elaborating on the teachings of the present invention, it should be understood that since certain compositions or process steps can vary, the present disclosure is not limited thereto. As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a conjugate" includes plural conjugates, a reference to "a cell" includes plural cells, and the like.

[0268] Numerical ranges include the numbers defining the range. Measured values and measurable values are understood to be approximate, taking into account significant digits and errors associated with the measurement. Also, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" is not intended to be limiting. It should be understood that the foregoing general description and the detailed description of the invention are exemplary and explanatory only and do not limit the present teachings.

[0269] Unless otherwise noted herein, embodiments described herein as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components, and embodiments described herein as "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components. Also, embodiments described herein as "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims). The term "or" is used in an inclusive sense, i.e., equivalent to "and / or", unless the context clearly indicates otherwise.

[0270] The section headings used in this specification are for organization purposes only and should not be construed in any way as limiting the desired subject matter. In the event of any conflict between any incorporated reference and any term defined in this specification or any other explicit content of this specification, this specification shall prevail. Although the present teachings are described in conjunction with various embodiments, this is not intended to limit the present teachings to such embodiments. On the contrary, the present teachings include various alternatives, modifications, and equivalents as would be understood by one of ordinary skill in the art.

[0271] II. Genetically Modified Cells A. Engineered Cell Compositions The present disclosure provides, as disclosed herein, engineered cell compositions in which the surface expression of HLA-A, HLA-B, TRAC, TRBC, and / or MHC class II is reduced or eliminated as compared to unmodified cells. In some embodiments, the engineered cell compositions include genetic modification in the HLA-A, HLA-B, TRAC, TRBC, and / or CIITA genes. In some embodiments, the engineered cell compositions include genetic modification in each of the HLA-A, HLA-B, and CIITA genes. In some embodiments, the engineered cells are allogeneic cells. In some embodiments, the engineered cells having reduced HLA-A, HLA-B, TRAC, TRBC, and / or MHC class II expression are useful for adoptive cell transfer therapy. In some embodiments, the engineered cells include additional genetic modification in the genome of the cells such that desired cells are obtained for the purpose of allogeneic transplantation.

[0272] In some embodiments, engineered cells are provided that contain a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to unmodified cells, the modification comprising at least 1 nucleotide from within genomic coordinates chr6:29942540-29945459. In some embodiments, engineered cells are provided that contain a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to unmodified cells, the modification comprising at least 1 nucleotide from within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494.

[0273] In some embodiments, engineered cells are provided that contain a genetic modification in the HLA gene, wherein the engineered cells have reduced or eliminated surface expression of HLA-A compared to unmodified cells, and the modification comprises at least 1 nucleotide from within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809.

[0274] In some embodiments, engineered cells are provided that contain a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to unmodified cells, the modification comprising at least 5 nucleotides from within genomic coordinates chr6:29942540-29945459. In some embodiments, the engineered cells that have reduced or eliminated surface expression of HLA-A compared to unmodified cells contain a genetic modification in the HLA gene, and the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0275] In some embodiments, engineered cells are provided that contain a genetic modification in the HLA-A gene that reduces or eliminates surface expression of HLA-A compared to unmodified cells, the modification comprising at least 5 contiguous nucleotides within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494. In some embodiments, engineered cells in which surface expression of HLA-A is reduced or eliminated compared to unmodified cells contain a genetic modification in the HLA gene, the modification comprising at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0276] In some embodiments, engineered cells are provided that contain a genetic modification in the HLA gene, wherein the engineered cells have reduced or eliminated surface expression of HLA-A compared to unmodified cells, and the modification comprises at least 5 nucleotides within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809. In some embodiments, engineered cells in which surface expression of HLA-A is reduced or eliminated compared to unmodified cells contain a genetic modification in the HLA gene, the modification comprising at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0277] In some embodiments, there is provided an engineered cell comprising a genetic modification in an HLA gene, wherein the engineered cell has reduced or eliminated surface expression of HLA-A compared to an unmodified cell, and the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates chr6:29942540-29945459.

[0278] In some embodiments, there is provided an engineered cell comprising a genetic modification in an HLA-A gene, wherein the engineered cell has reduced or eliminated surface expression of HLA-A compared to an unmodified cell, and the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494.

[0279] In some embodiments, there is provided an engineered cell comprising a genetic modification in an HLA-A gene, wherein the engineered cell has reduced or eliminated surface expression of HLA-A compared to an unmodified cell, and the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809.

[0280] In some embodiments, an engineered cell comprising a genetic modification in the HLA-A gene, wherein the engineered cell has reduced or eliminated surface expression of HLA-A compared to an unmodified cell, and the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from (a) chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494 or (b) chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809. In some embodiments, the genetic modification comprises at least 5 consecutive nucleotides within those genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least one C to T substitution, or at least one A to G substitution within the genomic coordinates.

[0281] In some embodiments, engineered cells are provided in which surface expression of HLA-A is reduced or eliminated by a genome editing system that binds to an HLA-A genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates selected from (a) chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494 or (b) chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809. In some embodiments, engineered cells are provided in which surface expression of HLA-A is reduced or eliminated by a genome editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the HLA-A genomic target sequence comprises at least 15 contiguous nucleotides within genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein such as N. meningitidis Cas9.

[0282] In some embodiments, engineered cells are provided in which surface expression of HLA-A is reduced or eliminated by a genome editing system that binds to an HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from (a) chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494 or (b) chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809. In some embodiments, the HLA-A genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the HLA-A genomic target sequence comprises at least 15 contiguous nucleotides within the genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. Meningitidis.

[0283] In some embodiments, provided are engineered cells comprising a genetic modification in the TRAC gene that reduces or eliminates surface expression of TRAC as compared to unmodified cells, the modification comprising at least 1 nucleotide from within genomic coordinates chr14:22547462-22551621. In some embodiments, provided are engineered cells comprising a genetic modification in the TRAC gene that reduces or eliminates surface expression of TRAC as compared to unmodified cells, the modification comprising at least 1 nucleotide from within genomic coordinates chr14:22547505-22551621. In some embodiments, provided are engineered cells comprising a genetic modification in the TRAC gene, wherein the engineered cells have reduced or eliminated surface expression of TRAC as compared to unmodified cells, and the modification comprises at least 1 nucleotide from within genomic coordinates from chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, or chr14:22550574-22550598.

[0284] In some embodiments, provided are engineered cells comprising a genetic modification in the TRAC gene that reduces or eliminates surface expression of TRAC as compared to unmodified cells, the modification comprising at least 5 contiguous nucleotides within genomic coordinates chr14:22547462-22551621. In some embodiments, provided are engineered cells comprising a genetic modification in the TRAC gene that reduces or eliminates surface expression of TRAC as compared to unmodified cells, the modification comprising at least 5 contiguous nucleotides within genomic coordinates chr14:22547505-22551621. In some embodiments, the engineered cells with reduced or eliminated surface expression of TRAC as compared to unmodified cells comprise a genetic modification in the TRAC gene, and the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates.

[0285] In some embodiments, there is provided an engineered cell comprising a genetic modification in the TRAC gene, wherein the engineered cell has reduced or eliminated surface expression of TRAC compared to an unmodified cell, and the modification comprises at least 5 contiguous nucleotides within a genomic locus selected from chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, or chr14:22550574-22550598. In some embodiments, the engineered cell having reduced or eliminated surface expression of TRAC compared to an unmodified cell comprises a genetic modification in the TRAC gene, and the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic locus.

[0286] In some embodiments, there is provided an engineered cell comprising a genetic modification in the TRAC gene, wherein the engineered cell has reduced or eliminated surface expression of TRAC compared to an unmodified cell, and the modification comprises at least one C to T substitution, or at least one A to G substitution within the genomic locus chr14:22547505-22551621 or chr14:22547462-22551621.

[0287] In some embodiments, there is provided an engineered cell comprising a genetic modification in the TRAC gene, wherein the engineered cell has reduced or eliminated surface expression of TRAC compared to an unmodified cell, and the modification comprises at least one C to T substitution, or at least one A to G substitution within a genomic locus selected from chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, or chr14:22550574-22550598.

[0288] In some embodiments, there is provided an engineered cell comprising a genetic modification in the TRAC gene, wherein the engineered cell has reduced or eliminated surface expression of TRAC as compared to an unmodified cell, and the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from (a) chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, and chr14:22550574-22550598. In some embodiments, the genetic modification comprises at least 5 consecutive nucleotides within those genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least one C to T substitution, or at least one A to G substitution within the genomic coordinates.

[0289] In some embodiments, there is provided an engineered cell in which surface expression of TRAC has been reduced or eliminated by a genome editing system that binds to a TRAC genomic target sequence comprising at least 10 consecutive nucleotides within genomic coordinates selected from (a) chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, and chr14:22550574-22550598. In some embodiments, there is provided an engineered cell in which surface expression of TRAC has been reduced or eliminated by a genome editing system that binds to a TRAC genomic target sequence comprising at least 5 consecutive nucleotides within the genomic coordinates. In some embodiments, the TRAC genomic target sequence comprises at least 15 consecutive nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein such as N. meningitidis Cas9.

[0290] In some embodiments, engineered cells are provided in which surface expression of TRAC is reduced or eliminated by a genome editing system that binds to a TRAC genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from (a) chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, and chr14:22550574-22550598. In some embodiments, the TRAC genomic target sequence comprises at least 10 contiguous nucleotides within those genomic coordinates. In some embodiments, the TRAC genomic target sequence comprises at least 15 contiguous nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. Meningitidis.

[0291] In some embodiments, engineered cells are provided that comprise a genetic modification in the TRBC gene that reduces or eliminates surface expression of TRBC as compared to an unmodified cell, the modification comprising at least 1 nucleotide from within genomic coordinates selected from chr7:142791756-142802543. In some embodiments, engineered cells are provided that comprise a genetic modification in the TRBC gene that reduces or eliminates surface expression of TRBC as compared to an unmodified cell, the modification comprising at least 1 nucleotide from within genomic coordinates selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543.

[0292] In some embodiments, engineered cells are provided that contain a genetic modification in the TRBC gene that reduces or eliminates surface expression of TRBC compared to unmodified cells, the modification comprising at least one nucleotide from within genomic coordinates from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130.

[0293] In some embodiments, engineered cells are provided that contain a genetic modification in the TRBC gene, wherein the engineered cells have reduced or eliminated surface expression of TRBC compared to unmodified cells, and the modification comprises at least one nucleotide from within genomic coordinates selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213.

[0294] In some embodiments, engineered cells are provided that contain a genetic modification in the TRBC gene that reduces or eliminates surface expression of TRBC as compared to unmodified cells, the modification comprising at least 5 contiguous nucleotides within genomic coordinates selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, engineered cells in which surface expression of TRBC is reduced or eliminated as compared to unmodified cells contain a genetic modification in the TRBC gene, the modification comprising at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0295] In some embodiments, engineered cells are provided that contain a genetic modification in the TRBC gene that reduces or eliminates surface expression of TRBC as compared to unmodified cells, the modification comprising at least 5 contiguous nucleotides within genomic coordinates selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130. In some embodiments, engineered cells in which surface expression of TRBC is reduced or eliminated as compared to unmodified cells contain a genetic modification in the TRBC gene, the modification comprising at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0296] In some embodiments, provided are engineered cells comprising a genetic modification in the TRBC gene, wherein the engineered cells have reduced or eliminated surface expression of TRBC as compared to unmodified cells, and the modification comprises at least 5 consecutive nucleotides within genomic coordinates selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213. In some embodiments, the engineered cells having reduced or eliminated surface expression of TRBC as compared to unmodified cells comprise a genetic modification in the TRBC gene, and the modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic coordinates.

[0297] In some embodiments, provided are engineered cells comprising a genetic modification in the TRBC gene, wherein the engineered cells have reduced or eliminated surface expression of TRBC as compared to unmodified cells, and the modification comprises at least one C to T substitution, or at least one A to G substitution within genomic coordinates selected from.

[0298] In some embodiments, provided are engineered cells comprising a genetic modification in the TRBC gene, wherein the engineered cells have reduced or eliminated surface expression of TRBC as compared to unmodified cells, and the modification comprises at least one C to T substitution, or at least one A to G substitution within genomic coordinates selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543.

[0299] In some embodiments, an engineered cell comprising a genetic modification in the TRBC gene, wherein the engineered cell has reduced or eliminated surface expression of TRBC compared to an unmodified cell, and the modification comprises at least one C to T substitution, or at least one A to G substitution within a genomic locus selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130, is provided.

[0300] In some embodiments, an engineered cell comprising a genetic modification in the TRBC gene, wherein the engineered cell has reduced or eliminated surface expression of TRBC compared to an unmodified cell, and the modification comprises at least one C to T substitution, or at least one A to G substitution within a genomic locus selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213, is provided.

[0301] In some embodiments, an engineered cell comprising a genetic modification in the TRBC gene, wherein the engineered cell has reduced or eliminated surface expression of TRBC compared to an unmodified cell, and the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within a genomic locus selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130. In some embodiments, the genetic modification comprises at least 5 consecutive nucleotides within those genomic loci. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic locus. In some embodiments, the genetic modification comprises at least one C to T substitution, or at least one A to G substitution within the genomic locus.

[0302] In some embodiments, there is provided an engineered cell comprising a genetic modification in the TRBC gene, wherein the engineered cell has reduced or eliminated surface expression of TRBC as compared to an unmodified cell, and the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within a genomic locus selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213. In some embodiments, the genetic modification comprises at least 5 consecutive nucleotides within those genomic loci. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within the genomic locus. In some embodiments, the genetic modification comprises at least one C to T substitution, or at least one A to G substitution within the genomic locus.

[0303] In some embodiments, engineered cells are provided in which surface expression of TRBC is reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130. In some embodiments, engineered cells are provided in which surface expression of TRBC is reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the TRBC genomic target sequence comprises at least 15 contiguous nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. meningitidis Cas9.

[0304] In some embodiments, engineered cells are provided in which surface expression of TRBC is reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213. In some embodiments, engineered cells are provided in which surface expression of TRBC is reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the TRBC genomic target sequences comprise at least 15 contiguous nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. meningitidis Cas9.

[0305] In some embodiments, an engineered cell is provided in which surface expression of TRBC is reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130. In some embodiments, the TRBC genomic target sequence comprises at least 10 contiguous nucleotides within those genomic coordinates. In some embodiments, the TRBC genomic target sequence comprises at least 15 contiguous nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. Meningitidis.

[0306] In some embodiments, engineered cells are provided in which surface expression of TRBC is reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from chr7:142792690-142792714, chr7:142802103-142802127, and chr7:142802106-14280213. In some embodiments, the TRBC genomic target sequence comprises at least 10 contiguous nucleotides within those genomic coordinates. In some embodiments, the TRBC genomic target sequence comprises at least 15 contiguous nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. Meningitidis.

[0307] In some embodiments, engineered cells are provided that comprise a genetic modification at the CIITA locus, wherein the engineered cells have reduced or eliminated surface expression of MHC class II as compared to unmodified cells, and the modification comprises at least 1 nucleotide at genomic coordinates (a) chr16:10877363-10907788 or (b) chr16:10906515-10908136.

[0308] In some embodiments, engineered cells are provided that include genetic modification in the CIITA gene that reduces or eliminates surface expression of MHC class II, compared to unmodified cells, where the modification comprises (a) at least one nucleotide of genomic coordinates selected from chr16:10907504-10907528, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906643-10906667, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, and chr16:10907574-10907598, or (b) at least one nucleotide of genomic coordinates selected from chr16:10906889-10906913, and chr16:10907504-10907528.

[0309] In some embodiments, there is provided an engineered cell comprising a genetic modification in the CIITA gene, wherein the engineered cell has reduced or eliminated surface expression of MHC class II as compared to an unmodified cell, and the modification comprises at least one nucleotide at a genomic locus selected from chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10906643-10906667, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, and chr16:10907508-10907532.

[0310] In some embodiments, there is provided an engineered cell comprising a genetic modification in the CIITA gene, wherein the engineered cell has reduced or eliminated surface expression of MHC class II as compared to an unmodified cell, and the modification comprises at least 5 contiguous nucleotides within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, the engineered cell having reduced or eliminated surface expression of MHC class II as compared to an unmodified cell comprises a genetic modification in the CIITA gene, and the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic locus.

[0311] In some embodiments, engineered cells are provided that comprise genetic modification in the CIITA gene that reduces or eliminates surface expression of MHC class II, compared to unmodified cells, wherein the modification comprises (a) at least 5 contiguous nucleotides within genomic coordinates selected from chr16:10907504-10907528, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906643-10906667, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, and chr16:10907574-10907598, or (b) at least 5 contiguous nucleotides within genomic coordinates selected from chr16:10906889-10906913, or chr16:10907504-10907528.In some embodiments, engineered cells with reduced or eliminated surface expression of MHC class II compared to unmodified cells contain a genetic modification in the CIITA gene, the modification comprising at least 6, 7, 8, 9, or 10 contiguous nucleotides within a genomic locus.

[0312] In some embodiments, provided are engineered cells comprising a genetic modification in the CIITA gene, wherein the engineered cells have reduced or eliminated surface expression of MHC class II compared to unmodified cells, and the modification comprises at least 5 contiguous nucleotides within a genomic locus selected from chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10906643-10906667, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, and chr16:10907508-10907532. In some embodiments, engineered cells with reduced or eliminated surface expression of MHC class II compared to unmodified cells contain a genetic modification in the CIITA gene, the modification comprising at least 6, 7, 8, 9, or 10 contiguous nucleotides within a genomic locus.

[0313] In some embodiments, provided are engineered cells comprising a genetic modification in the CIITA gene, wherein the engineered cells have reduced or eliminated surface expression of MHC class II compared to unmodified cells, and the modification comprises at least one C to T substitution, or at least one A to G substitution, within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136.

[0314] In some embodiments, an engineered cell comprising a genetic modification in the CIITA gene, wherein the engineered cell has reduced or eliminated surface expression of MHC class II as compared to an unmodified cell, and the modification comprises (a) at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from chr16:10907504-10907528, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906643-10906667, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or (b) chr16:10906889-10906913, or at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from chr16:10907504-10907528, there is provided an engineered cell.

[0315] In some embodiments, an engineered cell comprising a genetic modification in the CIITA gene, wherein the engineered cell has reduced or eliminated surface expression of MHC class II as compared to an unmodified cell, and the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10906643-10906667, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, and chr16:10907508-10907532, is provided.

[0316] In some embodiments, an engineered cell is provided that comprises a genetic modification in the CIITA gene and has reduced or eliminated surface expression of MHC class II as compared to an unmodified cell, and the modification comprises at least one nucleotide within genomic coordinates selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136.

[0317] In some embodiments, engineered cells are provided that contain genetic modification in the CIITA gene that reduces or eliminates surface expression of MHC class II, compared to unmodified cells, wherein the modification comprises at least one nucleotide within genomic coordinates selected from (a) chr16:10907504-10907528, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906643-10906667, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or (b) chr16:10906889-10906913, or within genomic coordinates selected from chr16:10907504-10907528.

[0318] In some embodiments, there is provided an engineered cell comprising a genetic modification in the CIITA gene, wherein the engineered cell has reduced or eliminated surface expression of MHC class II as compared to an unmodified cell, and the modification comprises at least one nucleotide within genomic coordinates selected from chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10906643-10906667, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, and chr16:10907508-10907532.

[0319] In some embodiments, an engineered cell that has reduced or eliminated surface expression of MHC class II as compared to an unmodified cell comprises a genetic modification in the CIITA gene, and the genetic modification comprises at least one nucleotide within genomic coordinates selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136.

[0320] In some embodiments, engineered cells with reduced or eliminated surface expression of MHC class II compared to unmodified cells contain genetic modification in the CIITA gene, where the genetic modification comprises at least 1 nucleotide within genomic coordinates selected from chr16:10907504-10907528, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906643-10906667, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or (b) within genomic coordinates selected from chr16:10906889-10906913, or chr16:10907504-10907528.

[0321] In some embodiments, the engineered cells, which have reduced or eliminated surface expression of MHC class II compared to unmodified cells, contain a genetic modification in the CIITA gene, the genetic modification comprising at least one nucleotide within genomic coordinates selected from chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10906643-10906667, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, and chr16:10907508-10907532.

[0322] In some embodiments, engineered cells in which surface expression of MHC class II is reduced or eliminated as compared to unmodified cells comprise a genetic modification in the CIITA gene, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from (a) chr16:10907504-10907528, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906643-10906667, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or (b) chr16:10906889-10906913, or chr16:10907504-10907528. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within those genomic coordinates.In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within a genomic coordinate. In some embodiments, the genetic modification comprises at least one C-to-T substitution, or at least one A-to-G substitution within a genomic coordinate.

[0323] In some embodiments, an engineered cell in which surface expression of MHC class II is reduced or eliminated compared to an unmodified cell comprises a genetic modification in the CIITA gene, wherein the genetic modification comprises an indel, a C-to-T substitution, or an A-to-G substitution within a genomic coordinate selected from chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10906643-10906667, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, and chr16:10907508-10907532. In some embodiments, the genetic modification comprises at least 5 consecutive nucleotides within those genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 consecutive nucleotides within a genomic coordinate. In some embodiments, the genetic modification comprises at least one C-to-T substitution, or at least one A-to-G substitution within a genomic coordinate.

[0324] In some embodiments, the surface expression of MHC class II is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates selected from (a) chr16:10907504-10907528, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906643-10906667, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or (b) chr16:10906889-10906913, or chr16:10907504-10907528, and engineered cells are provided.

[0325] In some embodiments, engineered cells are provided in which surface expression of MHC class II is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates selected from (a) chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10906643-10906667, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, and chr16:10907508-10907532. In some embodiments, engineered cells are provided in which surface expression of MHC class II is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein such as N. meningitidis Cas9.

[0326] In some embodiments, the surface expression of MHC class II is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from (a) chr16:10907504-10907528, chr16:10907508-10907532, chr16:10907539-10907559, chr16:10895658-10895682, chr16:10895668-10895692, chr16:10895750-10895774, chr16:10895753-10895777, chr16:10895754-10895778, chr16:10898684-10898708, chr16:10901529-10901553, chr16:10902121-10902145, chr16:10902701-10902725, chr16:10904726-10904750, chr16:10904760-10904784, chr16:10906493-10906517, chr16:10906515-10906539, chr16:10906631-10906655, chr16:10906636-10906660, chr16:10906643-10906667, chr16:10906770-10906794, chr16:10906788-10906812, chr16:10906789-10906813, chr16:10906816-10906840, chr16:10907148-10907172, chr16:10907254-10907278, chr16:10907331-10907355, chr16:10907477-10907501, chr16:10907497-10907521, chr16:10907503-10907527, chr16:10907574-10907598, or (b) chr16:10906889-10906913, or chr16:10907504-10907528, and engineered cells are provided.

[0327] In some embodiments, engineered cells are provided in which surface expression of MHC class II is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from chr16:10895658-10895682, chr16:10902701-10902725, chr16:10906493-10906517, chr16:10906631-10906655, chr16:10906643-10906667, chr16:10907477-10907501, and chr16:10907497-10907521, chr16:10907504-10907528, and chr16:10907508-10907532. In some embodiments, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within those genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 contiguous nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. Meningitidis.

[0328] In some embodiments, engineered cells are provided that comprise a genetic modification in the AAVS1 gene, the modification comprising at least 1 nucleotide from within genomic coordinates chr19:55115151-55116209.

[0329] In some embodiments, engineered cells are provided that include a genetic modification in the AAVS1 gene, where the modification includes at least one nucleotide from within genomic coordinates selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030.

[0330] In some embodiments, engineered cells are provided that include a genetic modification in the AAVS1 gene, where the modification includes at least 5 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, the engineered cells include a genetic modification in the AAVS1 gene, where the modification includes at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0331] In some embodiments, engineered cells are provided that include a genetic modification in the AAVS1 gene, where the modification includes at least 5 contiguous nucleotides within a genomic locus selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030. In some embodiments, the engineered cells include a genetic modification in the AAVS1 gene, where the modification includes at least 6, 7, 8, 9, or 10 contiguous nucleotides within a genomic locus.

[0332] In some embodiments, engineered cells are provided that include a genetic modification in the AAVS1 gene, where the modification includes at least one C to T substitution or at least one A to G substitution within the genomic locus chr19:55115151-55116209.

[0333] In some embodiments, engineered cells are provided that include genetic modification in the AAVS1 gene, the modification including at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030.

[0334] In some embodiments, engineered cells are provided that include a genetic modification in the AAVS1 gene, where the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within genomic coordinates selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within those genomic coordinates. In some embodiments, the genetic modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least one C to T substitution, or at least one A to G substitution within the genomic coordinates.

[0335] In some embodiments, an engineered cell is provided that has a gene modification in AAVS1 induced by a genome editing system that binds to an AAVS1 genomic target sequence comprising at least 10 contiguous nucleotides within a genomic locus selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030. In some embodiments, an engineered cell is provided that has a gene modification in AAVS1 induced by a genome editing system that binds to an AAVS1 genomic target sequence comprising at least 5 contiguous nucleotides within a genomic locus. In some embodiments, the AAVS1 genomic target sequence comprises at least 15 contiguous nucleotides within those genomic loci. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. meningitidis Cas9.

[0336] In some embodiments, an engineered cell having a gene modification in AAVS1 induced by a genome editing system that binds to an AAVS1 genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from chr19:55115218-55115242, chr19:55115477-55115501, chr19:55115504-55115528, chr19:55115513-55115537, chr19:55115514-55115538, chr19:55115517-55115541, chr19:55115518-55115542, chr19:55115549-55115573, chr19:55115574-55115598, chr19:55115606-55115630, chr19:55115933-55115957, chr19:55116026-55116050, chr19:55116045-55116069, chr19:55116084-55116108, chr19:55115276-55115300, chr19:55115509-55115533, chr19:55115579-55115603, chr19:55115863-55115887, chr19:55115906-55115930, or chr19:55116006-55116030 is provided. In some embodiments, the AAVS1 genomic target sequence comprises at least 10 contiguous nucleotides within those genomic coordinates. In some embodiments, the AAVS1 genomic target sequence comprises at least 15 contiguous nucleotides within those genomic coordinates. In some embodiments, the genome editing system comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein such as N. Meningitidis.

[0337] In some embodiments, for each of a given range of genomic coordinates, the range may include ±10 nucleotides at either end of the designated coordinates. For each of a given range of genomic coordinates, the range may include ±5 nucleotides at either end of the range. For example, when chr16:10923222-10923242 is provided, in some embodiments, the genomic target sequence or gene modification can fall within chr16:10923212-10923252.

[0338] In some embodiments, a given range of genomic coordinates may include target sequences on both strands of the DNA (i.e., the plus (+) strand and the minus (-) strand).

[0339] Gene modifications in the HLA-A, TRAC, TRBC, CIITA, and AAVS1 genes are further described herein.

[0340] In some embodiments, gene modifications at the HLA-A, TRAC, TRBC, CIITA, and AAVS1 gene loci include any one or more of insertions, deletions, substitutions, or deaminations of at least one nucleotide in the target sequence. In some embodiments, engineered cells are provided that include a gene modification in the HLA-A gene and have reduced or eliminated surface expression of HLA-A compared to unmodified cells. In some embodiments, engineered cells are provided that include a gene modification in the TRAC gene and have reduced or eliminated surface expression of TRAC compared to unmodified cells. In some embodiments, engineered cells are provided that include a gene modification in the TRBC1 gene and have reduced or eliminated surface expression of TRBC1 compared to unmodified cells. In some embodiments, engineered cells are provided that include a gene modification in the TRBC2 gene and have reduced or eliminated surface expression of TRBC2 compared to unmodified cells. In some embodiments, engineered cells are provided that include a gene modification in the CIITA gene and have reduced or eliminated surface expression of MHC class II compared to unmodified cells.

[0341] In some embodiments, provided are engineered cells that have reduced or eliminated surface expression of HLA-A, TRAC, TRBC, or MHC class II as compared to unmodified cells, including gene modification in the HLA-A, TRAC, TRBC, or CIITA gene, and the cells further reduce or eliminate expression of the endogenous TCR protein as compared to unmodified cells. In some embodiments, provided are engineered cells that have reduced or eliminated surface expression of HLA-A, TRAC, TRBC, or MHC class II as compared to unmodified cells, including gene modification in the HLA-A, TRAC, TRBC, or CIITA gene, the cells further contain exogenous nucleic acid, and the cells further reduce or eliminate expression of the endogenous TCR protein as compared to unmodified cells. In some embodiments, provided are engineered cells that have reduced or eliminated surface expression of HLA-A, TRAC, TRBC, or MHC class II as compared to unmodified cells, including gene modification in the HLA-A, TRAC, TRBC, or CIITA gene, the cells further reduce or eliminate surface expression of MHC class I, and the cells further reduce or eliminate expression of the endogenous TCR protein as compared to unmodified cells.

[0342] In some embodiments, provided are engineered cells that have reduced or eliminated surface expression of HLA-A, TRAC, TRBC, or MHC class II as compared to unmodified cells, including gene modification in the HLA-A, TRAC, TRBC, or CIITA gene, the cells further contain exogenous nucleic acid, the cells further reduce or eliminate surface expression of MHC class I, and the cells further reduce or eliminate expression of the endogenous TCR protein as compared to unmodified cells. In some embodiments, the engineered cells have reduced or eliminated expression of the TRAC protein as compared to unmodified cells. In some embodiments, the engineered cells have reduced or eliminated expression of the TRBC protein as compared to unmodified cells.

[0343] In some embodiments, provided are engineered cells that have reduced or eliminated surface expression of MHC class II as compared to unmodified cells, including genetic modification in the CIITA gene, wherein the modification comprises at least one nucleotide of an exon within genomic coordinates chr16:10902662-chr16:10923285, the cells further comprise exogenous nucleic acid, the cells further have reduced or eliminated surface expression of HLA-A, and the cells further have reduced or eliminated expression of endogenous TCR protein as compared to unmodified cells. In some embodiments, the engineered cells have reduced or eliminated expression of the TRAC protein as compared to unmodified cells. In some embodiments, the engineered cells have reduced or eliminated expression of the TRBC protein as compared to unmodified cells. In some embodiments, the engineered cells include genetic modification in the HLA-A gene. In some embodiments, the engineered cells include genetic modification that reduces the expression of HLA-A protein on the surface of the engineered cells.

[0344] The engineered cells can be any of the exemplary cell types disclosed herein. In some embodiments, the engineered cells are immune cells. In some embodiments, the engineered cells are hematopoietic stem cells (HSCs). In some embodiments, the engineered cells are induced pluripotent stem cells (iPSCs). In some embodiments, the engineered cells are monocytes, macrophages, mast cells, dendritic cells, or granulocytes. In some embodiments, the engineered cells are monocytes. In some embodiments, the engineered cells are macrophages. In some embodiments, the engineered cells are mast cells. In some embodiments, the engineered cells are dendritic cells.

[0345] In some embodiments, the engineered cell is a granulocyte. In some embodiments, the engineered cell is a lymphocyte. In some embodiments, the engineered cell is a T cell. In some embodiments, the engineered cell is a CD4+ T cell. In some embodiments, the engineered cell is a CD8+ T cell. In some embodiments, the engineered cell is a memory T cell. In some embodiments, the engineered cell is a B cell. In some embodiments, the engineered cell is a plasma B cell. In some embodiments, the engineered cell is a memory B cell.

[0346] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one of the engineered cells disclosed herein. In some embodiments, the pharmaceutical composition comprises a population of any one of the engineered cells disclosed herein. In some embodiments, the cell population is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% negative for surface antigens (e.g., HLA-A, MHC class II (HLA-DP, DQ, DR), or endogenous TCR) as measured by flow cytometry. In some embodiments, an engineered cell population that is at least 65% negative as measured by flow cytometry. In some embodiments, an engineered cell population that is at least 70% negative as measured by flow cytometry. In some embodiments, the engineered cell population is at least 80% negative as measured by flow cytometry. In some embodiments, the engineered cell population is at least 90% negative as measured by flow cytometry. In some embodiments, the engineered cell population is at least 91% negative as measured by flow cytometry. In some embodiments, the engineered cell population is at least 92% negative as measured by flow cytometry. In some embodiments, the engineered cell population is at least 93% negative as measured by flow cytometry. In some embodiments, the engineered cell population is at least 94% negative as measured by flow cytometry. In some embodiments, the engineered cell population is at least 95% negative for endogenous TCR protein as measured by flow cytometry. In some embodiments, the engineered cell population is at least 97% negative for endogenous TCR protein as measured by flow cytometry. In some embodiments, the engineered cell population is at least 98% negative for endogenous TCR protein as measured by flow cytometry.In some embodiments, the engineered cell population is at least 99% negative for endogenous TCR protein as measured by flow cytometry.

[0347] In some embodiments, provided are methods for administering the engineered cells or pharmaceutical compositions disclosed herein to a subject in need thereof. In some embodiments, provided are methods for administering the engineered cells or pharmaceutical compositions disclosed herein to a subject as an ACT therapy. In some embodiments, provided are methods for administering the engineered cells or pharmaceutical compositions disclosed herein to a subject for the treatment of cancer. In some embodiments, provided are methods for administering the engineered cells or pharmaceutical compositions disclosed herein to a subject for the treatment of an autoimmune disease. In some embodiments, provided are methods for administering the engineered cells or pharmaceutical compositions disclosed herein to a subject for the treatment of an infectious disease.

[0348] B. Methods and compositions for reducing or eliminating surface expression of HLA-A, TRAC, TRBC, and MHC class II The present disclosure provides methods and compositions for reducing or eliminating surface expression of HLA-A, TRAC, TRBC, or MHC class II protein on a cell as compared to an unmodified cell by genetically modifying the HLA-A, TRAC, TRBC, or CIITA gene. The resulting genetically modified cells may herein also be referred to as engineered cells. In some embodiments, cells that have already been genetically modified (or engineered) may be starting cells for further genetic modification using the methods or compositions provided herein. In some embodiments, the cells are allogeneic cells. In some embodiments, cells with reduced HLA-A, TRAC, TRBC, or MHC class II expression are useful for adoptive cell transfer therapy. In some embodiments, editing of the HLA-A, TRAC, TRBC, or CIITA gene is combined with additional genetic modification to produce cells that are desirable for the purposes of allogeneic transplantation.

[0349] In some embodiments, the method comprises reducing or eliminating surface expression of HLA-A protein on the surface of the cell, and contacting the cell with a composition comprising an HLA-A guide RNA comprising a guide sequence targeting an HLA-A genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the HLA-A guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the expression of HLA-A protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80. In some embodiments, the method comprises generating an engineered cell having reduced or eliminated surface expression of HLA-A protein compared to an unmodified cell, and contacting the cell with a composition comprising an HLA-A guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the HLA-A guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region.In some embodiments, the RNA-guided DNA binder comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of HLA-A protein on the surface of a cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0350] In some embodiments, the method comprises genetically modifying a cell to reduce or eliminate surface expression of HLA-A protein, and contacting the cell with a composition comprising an HLA-A guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binder, or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is Cas9. In some embodiments, the RNA-guided DNA binder is N. meningitidis Cas9. In some embodiments, the HLA-A guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binder comprises a deaminase region. In some embodiments, the RNA-guided DNA binder comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of HLA-A protein on the surface of a cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0351] In some embodiments, the method comprises contacting a cell with a composition comprising an HLA-A guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459, to generate a genetically modified HLA-A. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binder, or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is Cas9. In some embodiments, the RNA-guided DNA binder is N. meningitidis Cas9. In some embodiments, the HLA-A guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binder comprises a deaminase domain. In some embodiments, the RNA-guided DNA binder comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the HLA-A protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0352] In some embodiments, the method includes inducing a DSB or single-strand break (SSB) in HLA-A and contacting the cell with a composition comprising an HLA-A guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, the method further includes contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is S. pyogenes Cas9. In some embodiments, the HLA-A guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the HLA-A protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0353] In some embodiments, the method includes reducing the expression of HLA-A protein in a cell, delivering a composition to the cell, and contacting the cell with a composition comprising an HLA-A guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, the method further includes contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the HLA-A guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of HLA-A protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0354] In some embodiments, a method of reducing the expression of HLA-A protein on the surface of a cell includes contacting the cell with any one or more of the HLA-A guide RNAs disclosed herein. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0355] In some embodiments, provided is a composition comprising an HLA-A guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, the composition further comprises an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the composition comprises an RNA-guided DNA binding agent that is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the HLA-A guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0356] In some embodiments, provided is a composition comprising an HLA-A guide RNA (gRNA) comprising: a) i) a guide sequence selected from SEQ ID NOs: 2-80, or ii) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 2-80, or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 2-80, or iv) a sequence comprising 10 contiguous nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides, or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence from (iv), or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (iv). In some embodiments, the HLA-A guide RNA (gRNA) is a single guide RNA.

[0357] In some embodiments, provided is a method of generating engineered cells in which surface expression of HLA-A protein is reduced or eliminated as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, the composition comprises an HLA-A guide RNA comprising any one of the guide sequences of SEQ ID NO: 1, 13, 55, 61, 66, 70, and 71. In some embodiments, the composition comprises an HLA-A guide RNA comprising any one of the guide sequences of SEQ ID NO: 13, 55, 61, 66, 70, and 71. In some embodiments, the composition comprises an HLA-A guide RNA comprising any one of the guide sequences of SEQ ID NO: 13, 17, 55, 61, 66, and 70.

[0358] In some embodiments, provided is a method of reducing surface expression of HLA-A protein in engineered cells as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein.

[0359] In some embodiments, the composition further comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates a cytosine (C) to thymine (T) conversion with an HLA-A genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates an adenosine (A) to guanine (G) conversion with an HLA-A genomic target sequence.

[0360] In some embodiments, provided are engineered cells produced by the methods described herein. In some embodiments, the engineered cells produced by the methods and compositions described herein are allogeneic cells. In some embodiments, the method produces a composition comprising engineered cells in which the expression of HLA-A is reduced. In some embodiments, the method produces a composition comprising engineered cells in which the expression of HLA-A protein is reduced. In some embodiments, the method produces a composition comprising engineered cells in which the HLA-A level in the cell nucleus is reduced. In some embodiments, the method produces a composition comprising engineered cells that express a truncated form of HLA-A protein. In some embodiments, the method produces a composition comprising engineered cells that produce an undetectable HLA-A protein. In some embodiments, the engineered cells have a reduced HLA-A expression, reduced HLA-A protein, or reduced HLA-A level in the cell nucleus as compared to unmodified cells. In some embodiments, the engineered cells produced by the methods disclosed herein induce a reduction in the response from CD4+ T cells as measured in an in vitro cell culture assay containing CD4+ T cells as compared to unmodified cells.

[0361] In some embodiments, there are provided engineered cells produced by the methods or compositions disclosed herein, wherein the cells have reduced or eliminated surface expression of HLA-A protein and the cells contain a genetic modification that includes at least 5 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, there are provided engineered cells produced by the methods or compositions disclosed herein, wherein the cells have reduced or eliminated surface expression of HLA-A protein and the cells contain a genetic modification that includes at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, there are provided engineered cells produced by the methods or compositions disclosed herein, wherein the cells have reduced or eliminated surface expression of HLA-A protein and the cells contain a genetic modification that includes at least one C to T substitution or at least one A to G substitution within genomic coordinates chr6:29942540-29945459.

[0362] In some embodiments, the method comprises reducing or eliminating surface expression of the TRAC protein on the surface of the cell, and contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRAC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRAC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101 to 120.

[0363] In some embodiments, the method includes generating engineered cells having reduced or eliminated surface expression of the TRAC protein as compared to unmodified cells, by contacting the cells with a composition comprising a TRAC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the method further includes contacting the cells with an RNA-guided DNA binder, or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is Cas9. In some embodiments, the RNA-guided DNA binder is N. meningitidis Cas9. In some embodiments, the TRAC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binder comprises a deaminase domain. In some embodiments, the RNA-guided DNA binder comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRAC protein on the surface of the cells (i.e., the engineered cells) is thereby reduced. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101-120.

[0364] In some embodiments, the method comprises genetically modifying a cell to reduce or eliminate surface expression of the TRAC protein, and contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRAC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRAC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101-120.

[0365] In some embodiments, the method comprises genetically modifying TRAC, and contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRAC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRAC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101 to 120.

[0366] In some embodiments, the method comprises inducing a DSB or single-strand break (SSB) in TRAC and contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binder, or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is Cas9. In some embodiments, the RNA-guided DNA binder is S. pyogenes Cas9. In some embodiments, the TRAC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binder comprises a deaminase region. In some embodiments, the RNA-guided DNA binder comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRAC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101-120.

[0367] In some embodiments, the method includes reducing the expression of the TRAC protein in the cell, delivering a composition to the cell, and contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the method further includes contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRAC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRAC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101 to 120.

[0368] In some embodiments, a method of reducing the expression of the TRAC protein on the surface of a cell includes contacting the cell with any one or more of the TRAC guide RNAs disclosed herein. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101 to 120.

[0369] In some embodiments, provided is a composition comprising a TRAC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the composition further comprises an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the composition comprises an RNA-guided DNA binding agent that is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRAC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101-120.

[0370] In some embodiments, provided is a composition comprising a TRAC guide RNA (gRNA) comprising: a) i) a guide sequence selected from SEQ ID NOs: 101-120, or ii) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 101-120, or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 101-120, or iv) a sequence comprising 10 contiguous nucleotides of the genomic coordinates listed in Table 2 ± 10 nucleotides, or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence from (iv), or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (v). In some embodiments, the TRAC guide RNA (gRNA) is a single guide RNA (sgRNA).

[0371] In some embodiments, provided is a method of generating engineered cells in which surface expression of the TRAC protein is reduced or eliminated as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, the composition comprises a TRAC guide RNA comprising any one of the guide sequences of SEQ ID NOs: 101, 102, 103, 105, 107, 109, 111, and 115. In some embodiments, the composition comprises a TRAC guide RNA comprising any one of the guide sequences of SEQ ID NOs: 101, 102, 103, 107, and 111.

[0372] In some embodiments, provided is a method of reducing surface expression of the TRAC protein in engineered cells as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments shown herein.

[0373] In embodiments, the composition further comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates a cytosine (C) to thymine (T) conversion with a TRAC genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates an adenosine (A) to guanine (G) conversion with a TRAC genomic target sequence.

[0374] In some embodiments, provided are engineered cells produced by the methods described herein. In some embodiments, the engineered cells produced by the methods and compositions described herein are allogeneic cells. In some embodiments, the method produces a composition comprising engineered cells in which the expression of TRAC is reduced. In some embodiments, the method produces a composition comprising engineered cells in which the expression of the TRAC protein is reduced. In some embodiments, the method produces a composition comprising engineered cells in which the level of TRAC in the cell nucleus is reduced. In some embodiments, the method produces a composition comprising engineered cells that express a truncated form of the TRAC protein. In some embodiments, the method produces a composition comprising engineered cells that do not produce a detectable TRAC protein. In some embodiments, the engineered cells have a reduced expression of TRAC, a reduced TRAC protein, or a reduced level of TRAC in the cell nucleus as compared to non-engineered cells. In some embodiments, the engineered cells produced by the methods disclosed herein induce a reduction in the response from CD4+ T cells as measured in an in vitro cell culture assay containing CD4+ T cells as compared to non-engineered cells.

[0375] In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of the TRAC protein, and the cells comprise a genetic modification that includes at least 5 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of the TRAC protein, and the cells comprise a genetic modification that includes at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of the TRAC protein, and the cells comprise a genetic modification that includes at least one C to T substitution or at least one A to G substitution within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621.

[0376] In some embodiments, the method comprises reducing or eliminating surface expression of the TRBC protein on the cell surface, and contacting the cell with a composition comprising a TRBC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic coordinate selected from genomic coordinates chr7:142791756-142802543. In some embodiments, the method comprises reducing or eliminating surface expression of the TRBC protein on the cell surface, and contacting the cell with a composition comprising a TRBC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic coordinate selected from genomic coordinates (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRBC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRBC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0377] In some embodiments, the method comprises generating engineered cells with reduced or eliminated surface expression of the TRBC protein as compared to non-engineered cells, by contacting the cells with a composition comprising a TRBC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, the method further comprises contacting the cells with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRBC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRBC protein on the surface of the cells (i.e., the engineered cells) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0378] In some embodiments, the method comprises genetically modifying a cell to reduce or eliminate surface expression of the TRBC protein, and contacting the cell with a composition comprising a TRBC guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRBC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRBC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0379] In some embodiments, the method comprises genetically modifying a TRBC and contacting the cell with a composition comprising a TRBC guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRBC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRBC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0380] In some embodiments, the method includes inducing a DSB or single-strand break (SSB) in the TRBC and contacting the cell with a composition comprising a TRBC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, the method further includes contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is S. pyogenes Cas9. In some embodiments, the TRBC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRBC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0381] In some embodiments, the method includes reducing the expression of TRBC protein in a cell, delivering a composition to the cell, and contacting the cell with a composition comprising a TRBC guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, the method further includes contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRBC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of the TRBC protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0382] In some embodiments, a method of reducing the expression of a TRBC protein on the surface of a cell comprises contacting the cell with any one or more of the TRBC guide RNAs disclosed herein. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0383] In some embodiments, a composition is provided that includes a TRBC guide RNA comprising a guide sequence targeting a genomic target that includes at least 10 contiguous nucleotides within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, the composition further includes an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the composition includes an RNA-guided DNA binding agent that is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the TRBC guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent includes a deaminase region. In some embodiments, the RNA-guided DNA binding agent includes deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the TRBC guide RNA includes a guide sequence selected from SEQ ID NOs: 201-265.

[0384] In some embodiments, a composition is provided that includes a TRBC guide RNA (gRNA) comprising: a) i) a guide sequence selected from SEQ ID NOs: 201-265, or ii) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 201-265, or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 201-265, or iv) a sequence comprising 10 contiguous nucleotides of the genomic coordinates listed in Table 3 ± 10 nucleotides, or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence from (iv), or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (v). In some embodiments, the TRBC guide RNA is a single guide RNA (sgRNA).

[0385] In some embodiments, the composition further comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates a conversion of cytosine (C) to thymine (T) with a TRBC genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates a conversion of adenosine (A) to guanine (G) with a TRBC genomic target sequence.

[0386] In some embodiments, provided are engineered cells produced by the methods described herein. In some embodiments, the engineered cells produced by the methods and compositions described herein are allogeneic cells. In some embodiments, the method produces a composition comprising engineered cells in which the expression of TRBC is reduced. In some embodiments, the method produces a composition comprising engineered cells in which the expression of the TRBC protein is reduced. In some embodiments, the method produces a composition comprising engineered cells in which the level of TRBC in the cell nucleus is reduced. In some embodiments, the method produces a composition comprising engineered cells that express a truncated form of the TRBC protein. In some embodiments, the method produces a composition comprising engineered cells that do not produce a detectable TRBC protein. In some embodiments, the engineered cells have a reduced expression of TRBC, a reduced TRBC protein, or a reduced level of TRBC in the cell nucleus as compared to unmodified cells. In some embodiments, the engineered cells produced by the methods disclosed herein induce a reduction in the response from CD4+ T cells as compared to unmodified cells when measured in an in vitro cell culture assay containing CD4+ T cells.

[0387] In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of the TRBC protein, and the cells comprise a genetic modification that includes at least 5 contiguous nucleotides within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of the TRBC protein, and the cells comprise a genetic modification that includes at least 10 contiguous nucleotides within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543. In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of the TRBC protein, and the cells comprise a genetic modification that includes at least one C to T substitution or at least one A to G substitution within a genomic locus selected from (a) chr7:142791862-142793149, (b) chr7:142791756-142792721, or (c) chr7:142801104-142802543.

[0388] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC compared to unmodified cells, the engineered cells comprising a genetic modification in the TRBC gene, the genetic modification comprising at least one contiguous nucleotide within genomic coordinates selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130.

[0389] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC compared to unmodified cells, the engineered cells comprising a genetic modification in the TRBC gene, the genetic modification comprising indels, C to T substitutions, or A to G substitutions within genomic coordinates selected from (a) chr7:142792690-142792714, or chr7:142792693-142792717, or (b) chr7:142791756-142791780, chr7:142791761-142791785, chr7:142791820-142791844, chr7:142791939-142791963, chr7:142791940-142791964, or chr7:142792004-142792028, or (c) chr7:142801104-142801124, chr7:142802103-142802127, or chr7:142802106-142802130.

[0390] In some embodiments, provided is a method of generating engineered cells in which surface expression of the TRBC protein is reduced or eliminated as compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, the composition comprises a TRBC guide RNA comprising any one of the guide sequences of SEQ ID NOs: 215, 216, 223, 224, 229, 230, 246, 259, and 260. In some embodiments, the composition comprises a TRBC guide RNA comprising any one of the guide sequences of SEQ ID NOs: 215, 216, 224, 229, 246, 259, and 260. In some embodiments, the composition comprises a TRBC guide RNA comprising any one of the guide sequences of SEQ ID NOs: 215, 259, and 260.

[0391] In some embodiments, the method comprises reducing or eliminating surface expression of MHC class II proteins on the surface of a cell, and contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the CIITA guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the expression of MHC class II proteins on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0392] In some embodiments, the method includes generating engineered cells with reduced or eliminated surface expression of MHC class II proteins compared to unmodified cells, by contacting the cells with a composition comprising a CIITA guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, the method further includes contacting the cells with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the CIITA guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of MHC class II proteins on the surface of the cells (i.e., the engineered cells) is thereby reduced. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0393] In some embodiments, the method comprises genetically modifying a cell to reduce or eliminate surface expression of MHC class II protein, and contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the CIITA guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of MHC class II protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0394] In some embodiments, the method comprises genetically modifying CIITA and contacting the cell with a composition comprising a CIITA guide RNA targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the CIITA guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of MHC class II protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0395] In some embodiments, the method comprises inducing a DSB or single-strand break (SSB) in CIITA and contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is S. pyogenes Cas9. In some embodiments, the CIITA guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of MHC class II protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0396] In some embodiments, the method includes reducing the expression of CIITA protein in a cell, delivering a composition to the cell, and contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within a genomic locus selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, the method further includes contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the CIITA guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the expression of MHC class II protein on the surface of the cell (i.e., the engineered cell) is thereby reduced. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0397] In some embodiments, a method of reducing the expression of MHC class II protein on the surface of a cell includes contacting the cell with any one or more of the CIITA guide RNAs disclosed herein. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0398] In some embodiments, a composition is provided that includes a CIITA guide RNA comprising a guide sequence targeting a genomic target that includes at least 10 contiguous nucleotides within genomic coordinates selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, the composition further includes an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the composition includes an RNA-guided DNA binding agent that is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the CIITA guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent includes a deaminase region. In some embodiments, the RNA-guided DNA binding agent includes deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the CIITA guide RNA includes a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0399] In some embodiments, a composition is provided that includes a CIITA guide RNA (gRNA) comprising: a) i) a guide sequence selected from SEQ ID NOs: 301, 302, 304-576, or ii) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 301, 302, 304-576, or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 301, 302, 304-576, or iv) a sequence comprising 10 contiguous nucleotides of the genomic coordinates listed in Table 4 ± 10 nucleotides, or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence from (iv), or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (v). In some embodiments, the CIITA guide RNA is a single guide RNA (sgRNA).

[0400] In some embodiments, a method of producing engineered cells in which surface expression of MHC class II proteins is reduced or eliminated compared to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments shown herein. In some embodiments, the composition comprises a CIITA guide RNA comprising any one guide sequence of SEQ ID NOs: 301-302, 320-321, 324, 326, 327, 332, 354, 361, 372, 400, 415, 419-420, 422, 428, 431, 432, 434, 451, 455, 458, 462-464, and 468. In some embodiments, the composition comprises a CIITA guide RNA comprising any one guide sequence of SEQ ID NOs: 301, 302, 320, 372, 414, 419, 422, and 462-463.

[0401] In some embodiments, the composition further comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the composition comprises an RNA-guided DNA binder that generates a cytosine (C) to thymine (T) conversion with a CIITA genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binder that generates an adenosine (A) to guanine (G) conversion with a CIITA genomic target sequence.

[0402] In some embodiments, provided are engineered cells produced by the methods described herein. In some embodiments, the engineered cells produced by the methods and compositions described herein are allogeneic cells. In some embodiments, the method produces a composition comprising engineered cells in which MHC class II expression is reduced. In some embodiments, the method produces a composition comprising engineered cells in which CIITA protein expression is reduced. In some embodiments, the method produces a composition comprising engineered cells in which the level of CIITA in the cell nucleus is reduced. In some embodiments, the method produces a composition comprising engineered cells that express a truncated form of the CIITA protein. In some embodiments, the method produces a composition comprising engineered cells that do not produce a detectable CIITA protein. In some embodiments, the engineered cells have a reduction in MHC class II expression, a reduction in CIITA protein, or a reduction in CIITA level in the cell nucleus as compared to unmodified cells. In some embodiments, the engineered cells produced by the methods disclosed herein induce a reduction in the response from CD4+ T cells as compared to unmodified cells when measured in an in vitro cell culture assay containing CD4+ T cells.

[0403] In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of MHC class II proteins, and the cells comprise a genetic modification that includes at least 5 contiguous nucleotides within genomic coordinates selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of MHC class II proteins, and the cells comprise a genetic modification that includes at least 10 contiguous nucleotides within genomic coordinates selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136. In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells have reduced or eliminated surface expression of MHC class II proteins, and the cells comprise a genetic modification that includes at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from (a) chr16:10877363-10907788, or (b) chr16:10906515-10908136.

[0404] In some embodiments, the method includes generating an engineered cell by contacting the cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, the method further includes contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0405] In some embodiments, the method comprises genetically modifying a cell by contacting the cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0406] In some embodiments, the method comprises genetically modifying AAVS1 by contacting a cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0407] In some embodiments, the method comprises inducing a DSB or single-strand break (SSB) at AAVS1 and contacting the cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA-binding agent, or a nucleic acid encoding an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the RNA-guided DNA-binding agent is S. pyogenes Cas9. In some embodiments, the AAVS1 guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA-binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA-binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0408] In some embodiments, the method comprises delivering a composition to a cell and contacting the cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, the method further comprises contacting the cell with an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase region. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0409] In some embodiments, a method of genetically modifying the AAVS1 gene comprises contacting a cell with any one or more of the AAVS1 guide RNAs disclosed herein. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0410] In some embodiments, provided is a composition comprising an AAVS1 guide RNA comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, the composition further comprises an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the composition comprises an RNA-guided DNA binding agent that is Cas9. In some embodiments, the RNA-guided DNA binding agent is N. meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is an N. meningitidis Cas9 guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments, the RNA-guided DNA binding agent comprises deaminase APOBEC3A (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601 to 774.

[0411] In some embodiments, provided is a composition comprising an AAVS1 guide RNA (gRNA) comprising: a) i) a guide sequence selected from SEQ ID NOs: 601 to 774, or ii) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 601 to 774, or iii) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 601 to 774, or iv) a sequence comprising 10 contiguous nucleotides of the genomic coordinates listed in Table 5 ± 10 nucleotides, or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence from (iv), or vi) a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from (v). In some embodiments, the AAVS1 guide RNA (gRNA) is a single guide RNA.

[0412] In some embodiments, methods of generating engineered cells are provided, the methods including contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, the composition comprises an AAVS1 guide RNA comprising any one guide sequence from SEQ ID NOs: 611, 620, 622, 626, 627, 628, 629, 632, 633, 634, 656, 659, 660, 661, 673, 691, 692, 730, 734, and 746.

[0413] In some embodiments, the composition further comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates a cytosine (C) to thymine (T) conversion with the AAVS1 genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates an adenosine (A) to guanine (G) conversion with the AAVS1 genomic target sequence.

[0414] In some embodiments, engineered cells produced by the methods described herein are provided. In some embodiments, the engineered cells produced by the methods and compositions described herein are allogeneic cells. In some embodiments, the engineered cells produced by the methods disclosed herein induce a reduction in response from CD4+ T cells as measured in an in vitro cell culture assay containing CD4+ T cells as compared to unmodified cells.

[0415] In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells comprise a genetic modification that includes at least 5 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells comprise a genetic modification that includes at least 10 contiguous nucleotides within genomic coordinates chr19:55115151-55116209. In some embodiments, engineered cells produced by the methods or compositions disclosed herein are provided, wherein the cells comprise a genetic modification that includes at least one C to T substitution or at least one A to G substitution within genomic coordinates chr19:55115151-55116209. In some embodiments, the compositions disclosed herein further comprise a pharmaceutically acceptable carrier. In some embodiments, cells produced by the compositions disclosed herein that comprise a pharmaceutically acceptable carrier are provided. In some embodiments, compositions are provided that comprise the cells disclosed herein.

[0416] C. HLA-A guide RNA The methods and compositions provided by the present invention disclose HLA-A guide RNAs useful for reducing the expression of HLA-A protein on the surface of cells. In some embodiments, such guide RNAs direct an RNA-guided DNA binding agent to an HLA-A genomic target sequence and may be referred to herein as "HLA-A guide RNAs". In some embodiments, the HLA-A guide RNA directs an RNA-guided DNA binding agent to a human HLA-A genomic target sequence. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0417] In some embodiments, the methods and compositions disclosed herein include an HLA-A guide RNA comprising a guide sequence that targets an HLA-A genomic target sequence comprising at least 10 nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, the methods and compositions disclosed herein include an HLA-A guide RNA comprising a guide sequence that targets an HLA-A genomic target sequence comprising at least 1 nucleotide within genomic coordinates chr6:29942540-29945459.

[0418] In some embodiments, the methods and compositions disclosed herein include an HLA-A guide RNA comprising a guide sequence that directs an RNA-guided DNA binding agent to induce a double-strand break (DSB) or single-strand break (SSB) in the HLA-A gene, wherein the HLA-A guide RNA target and the HLA-A genomic target sequence comprise at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, the methods and compositions disclosed herein include an HLA-A guide RNA comprising a guide sequence that directs an RNA-guided DNA binding agent to induce a double-strand break (DSB) or single-strand break (SSB) in the HLA-A gene, wherein the HLA-A guide RNA target and the HLA-A genomic target sequence comprise at least 1 nucleotide within genomic coordinates chr6:29942540-29945459.

[0419] In some embodiments, the methods and compositions disclose an HLA-A guide RNA that directs an RNA-guided DNA binding agent to induce a double-strand break (DSB) or single-strand break (SSB) in the HLA-A genomic target sequence. In some embodiments, the methods and compositions disclose an HLA-A guide RNA that directs an RNA-guided DNA binding agent to effect cleavage in the HLA-A genomic target sequence. In embodiments where the RNA-guided DNA cleavage agent is Cas9, cleavage occurs at the third base from the protospacer adjacent motif (PAM) sequence.

[0420] In some embodiments, provided is a composition comprising the HLA-A guide RNA described herein, and an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0421] In some embodiments, provided is a composition comprising an HLA-A single guide RNA (sgRNA) comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, provided is a composition comprising the HLA-A sgRNA described herein, and an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0422] In some embodiments, provided is a composition comprising an HLA-A dual guide RNA (dgRNA) comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6:29942540-29945459. In some embodiments, provided is a composition comprising the HLA-A dgRNA described herein, and an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0423] In some embodiments, the TRAC gRNA comprises a guide sequence selected from any one of SEQ ID NOs: 2 to 80. Exemplary HLA-A guide sequences are shown in Table 1 (SEQ ID NOs: 2 to 80) below, which comprises the corresponding guide RNA sequences 2 to 80.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

[0424] Throughout the present application, the terms "mA", "mC", "mU", or "mG" may be used to denote nucleotides modified with 2'-O-Me. Throughout the present application, the terms A*, C*, U*, or G* may be used to denote nucleotides linked to the following (e.g., 3') nucleotides by phosphorothioate (PS) linkages.

[0425] In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80. In some embodiments, the HLA-A guide RNA comprises a guide sequence that is at least 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 2-80. In some embodiments, the HLA-A guide RNA comprises a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 2-80. In some embodiments, the HLA-A guide RNA comprises a guide sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 2-80.

[0426] In some embodiments, the HLA-A guide RNA comprises a guide sequence that comprises at least 10 consecutive nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides. As used herein, at least 10 consecutive nucleotides of the genomic coordinates ± 10 nucleotides means, for example, that there are at least 10 consecutive nucleotides within the genomic coordinates, and the genomic coordinates include 10 nucleotides in the 5' direction and 10 nucleotides in the 3' direction from the range listed in Table 1. For example, the HLA-A guide RNA may include 10 consecutive nucleotides within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494, including the boundary nucleotides of these ranges. In some embodiments, the HLA guide RNA comprises a guide sequence that is at least 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence that comprises 10 consecutive nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides. In some embodiments, the HLA guide RNA comprises a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence that comprises 10 consecutive nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides.

[0427] In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising at least 15 consecutive nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising at least 24 consecutive nucleotides of the genomic coordinates listed in Table 1 ± 10 nucleotides.

[0428] In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 2. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 3. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 4. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 5. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 6. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 7. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 8. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 9. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 10. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 11. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 12. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 13. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 14. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 15. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 16. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 17. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 18. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 19. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 20. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 21. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 22. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 23. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 24. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 25. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 26. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 27. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 28. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 29.In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 30. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 31. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 32. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 33. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 34. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 35. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 36. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 37. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 38. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 39. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 40. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 41. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 42. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 43. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 44. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 45. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 46. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 47. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 48. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 49. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 50. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 51. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 52. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 53. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 54. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 55. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 56. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 57.In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 58. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 59. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 60. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 61. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 62. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 63. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 64. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 65. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 66. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 67. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 68. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 69. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 70. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 71. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 72. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 73. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 74. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 75. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 76. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 77. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 78. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 79. In some embodiments, the HLA-A guide RNA comprises SEQ ID NO: 80.

[0429] In some embodiments, the HLA-A guide RNA comprises any one of the guide sequences of SEQ ID NOs: 13, 55, 61, 66, and 70-71.

[0430] In some embodiments, the HLA-A guide RNA comprises a sequence listed in Table 1. In some embodiments, the HLA-A guide RNA comprises any one of the sequences of SEQ ID NOs: 2 to 80. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 61 or 66. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 61. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 61. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising any one of the sequences of SEQ ID NOs: 2 to 80. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 61 or 66. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 61. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 66. In some embodiments, the HLA-A guide RNA comprises any one of the sequences of SEQ ID NOs: 1002 to 1080. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 1061 or 1066. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 1061. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 1066. In some embodiments, the HLA-A guide RNA comprises any one of the sequences of SEQ ID NOs: 2002 to 2080, 3001, and 3002. In some embodiments, the HLA-A guide RNA comprises any one of the sequences of SEQ ID NOs: 2061, 2066, 3001, and 3002. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 2061. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 2066. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 3001. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NO: 3002.

[0431] In some embodiments, the HLA-A guide RNA is a single guide RNA (sgRNA) comprising any one of the sgRNA sequences listed in Table 1.

[0432] Additional embodiments of HLA-A guide RNAs are provided herein, including exemplary modifications to the guide RNAs.

[0433] 1. Gene modification to HLA-A In some embodiments, the methods and compositions disclosed herein genetically modify at least one nucleotide of the HLA-A gene in a cell. In some embodiments, the gene modification to HLA-A reduces or eliminates the expression of HLA-A protein on the surface of the genetically modified cell (or engineered cell). The gene modification includes a population of modifications resulting from contact with a genome editing system (e.g., a population of edits resulting from Cas9 and an HLA-A guide RNA, or a population of edits resulting from BC22 and an HLA-A guide RNA).

[0434] In some embodiments, the gene modification includes at least one nucleotide within genomic coordinates chr6:29942540-29945459. In some embodiments, the gene modification includes at least one nucleotide within genomic coordinates selected from any of the genomic coordinates listed in Table 1.

[0435] In some embodiments, the gene modification includes at least one nucleotide within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494.

[0436] In some embodiments, the gene modification comprises at least one nucleotide within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809.

[0437] In some embodiments, the gene modification comprises at least five consecutive nucleotides within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, and chr6:29944470-29944494. In some embodiments, the gene modification comprises at least ten consecutive nucleotides within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944470-29944494. In some embodiments, the gene modification comprises at least one indel, at least one C to T substitution, or at least one A to G substitution within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29944266-29944290, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944470-29944494.

[0438] In some embodiments, the gene modification comprises at least 5 consecutive nucleotides within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809. In some embodiments, the gene modification comprises at least 10 consecutive nucleotides within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809. In some embodiments, the gene modification comprises at least 1 indel, at least 1 C-to-T substitution, or at least 1 A-to-G substitution within genomic coordinates selected from chr6:29942891-29942915, chr6:29942609-29942633, chr6:29942889-29942913, chr6:29944471-29944495, chr6:29944266-29944290, chr6:29942785-29942809.

[0439] In some embodiments, the modification to HLA-A comprises any one or more of insertion, deletion, substitution, or deamination of at least one nucleotide in the target sequence. In some embodiments, the modification to HLA-A comprises insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification to HLA-A comprises deletion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In other embodiments, the modification to HLA-A comprises insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In other embodiments, the modification to HLA-A comprises deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification to HLA-A comprises an indel, which is generally defined in the art as an insertion or deletion of less than 1000 base pairs (bp). In some embodiments, the modification to HLA-A comprises an indel that results in a frameshift mutation in the target sequence. In some embodiments, the modification to HLA-A comprises substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification to HLA-A comprises any one or more of insertion, deletion, or substitution of nucleotides resulting from incorporation of a template nucleic acid. In some embodiments, the modification to HLA-A is not transient.

[0440] In some embodiments, the methods and compositions disclosed herein use an RNA-guided DNA binding agent (e.g., a Cas enzyme) to modify the HLA-A gene in a cell. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent cleaves within the HLA-A gene, and the HLA-A guide RNA targets an HLA-A genomic target sequence that comprises at least 10 contiguous nucleotides within the genomic coordinates chr6:29942540-29945459.

[0441] In some embodiments, the genetic modification to HLA-A results in the utilization of a frameshift stop codon. In some embodiments, the genetic modification to HLA-A results in exon skipping during splicing. In some embodiments, the genetic modification to HLA-A results in a reduction of HLA-A protein expression by the cell. In some embodiments, the modification to HLA-A results in a reduction or elimination of HLA-A protein expression on the surface of the cell.

[0442] In some embodiments, the expression of HLA-A on the surface of the cell is reduced as a result of the genetic modification to HLA-A. In some embodiments, the expression of HLA-A on the surface of the cell is absent as a result of the genetic modification to HLA-A.

[0443] 2. Efficacy of HLA-A guide RNA The efficacy of the HLA guide RNA can be determined by techniques available in the art that evaluate the editing efficiency of the guide RNA, the level of HLA-A mRNA in the target cell, or the level of HLA-A protein. In some embodiments, the reduction or elimination of HLA-A protein on the surface of the cell may be determined by comparison with unmodified cells (i.e., "compared to unmodified cells"). Engineered cells or cell populations can also be compared to a population of unmodified cells.

[0444] "Unmodified cells" (or "a plurality of unmodified cells") refers to control cells (or a plurality of control cells) of the same type of cell in an experiment or test, and their "unmodified" control cells have not been contacted with an HLA-A guide (i.e., non-manipulated cells). Thus, unmodified cells (or cells (plural)) can be cells that have not been contacted with the guide RNA or cells that have been contacted with a guide RNA that does not target HLA-A.

[0445] In some embodiments, the efficacy of the HLA-A guide RNA is determined by measuring the reduction or elimination of HLA-A protein on the surface of the target cells. In some embodiments, HLA-A protein expression is measured by flow cytometry (e.g., using antibodies against HLA-A2 / HLA-A3). In some embodiments, the cell population is enriched (e.g., by FACS or MACS) and is at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, or 94% HLA-A negative when measured by flow cytometry relative to an unmodified cell population. In some embodiments, the cell population is not enriched (e.g., by FACS or MACS) and is at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, or 94% HLA-A negative when measured by flow cytometry relative to an unmodified cell population.

[0446] In some embodiments, the cell population is at least 65% HLA-A negative when measured by flow cytometry as compared to an unmodified cell population. In some embodiments, the cell population is at least 70% HLA-A negative when measured by flow cytometry as compared to an unmodified cell population. In some embodiments, the cell population is at least 80% HLA-A negative when measured by flow cytometry as compared to an unmodified cell population. In some embodiments, the cell population is at least 90% HLA-A negative when measured by flow cytometry as compared to an unmodified cell population. In some embodiments, the cell population is at least 91% HLA-A negative when measured by flow cytometry as compared to an unmodified cell population. In some embodiments, the cell population is at least 92% HLA-A negative when measured by flow cytometry as compared to an unmodified cell population. In some embodiments, the cell population is at least 93% HLA-A negative when measured by flow cytometry as compared to an unmodified cell population. In some embodiments, the cell population is at least 94% HLA-A negative when measured by flow cytometry as compared to an unmodified cell population.

[0447] In some embodiments, an effective HLA-A guide RNA can be determined by measuring the immune cell response of in vitro or in vivo (e.g., CD8+ T cells) immune cells to genetically modified target cells. For example, a reduction in the response from CD8+ T cells indicates an effective HLA-A guide RNA. The CD8+ T cell response can be evaluated by assays that measure CD8+ T cell activation responses, such as CD8+ T cell proliferation, expression of activation markers, and / or cytokine production (IL-2, IFN-γ, TNF-α) (e.g., flow cytometry, ELISA). The CD8+ T cell response can be evaluated in vitro or in vivo. In some embodiments, the CD8+ T cell response can be evaluated by co-culturing genetically modified cells with CD8+ T cells in vitro. In some embodiments, CD8+ T cell activity can be evaluated in an in vivo model, such as a rodent model. In an in vivo model, for example, genetically modified cells can be administered with CD8+ T cells, and the survival of the genetically modified cells indicates the ability to avoid CD8+ T cell lysis. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of CD8+ T cells for 1, 2, 3, 4, 5, or 6 weeks or more. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of CD8+ T cells for at least 1 week to 6 weeks. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of CD8+ T cells for at least 2 to 4 weeks. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of CD8+ T cells for at least 4 to 6 weeks. In some embodiments, the method produces a composition comprising cells that survive in vivo in the presence of CD8+ T cells for more than 6 weeks.

[0448] The effectiveness of the HLA-A guide RNA can also be evaluated by the survival of the cells after editing. In some embodiments, the cells survive for at least 1 week to 6 weeks after editing. In some embodiments, the cells survive for at least 2 weeks after editing. In some embodiments, the cells survive for at least 3 weeks after editing. In some embodiments, the cells survive for at least 4 weeks after editing. In some embodiments, the cells survive for at least 5 weeks after editing. In some embodiments, the cells survive for at least 6 weeks after editing. In some embodiments, the cells survive for at least 12 weeks after editing. The survival rate of the genetically modified cells can be measured using standard techniques including, for example, measures of cell death, live / dead staining by flow cytometry, or cell proliferation.

[0449] D.TRAC guide RNA The methods and compositions provided by the present invention disclose TRAC guide RNAs useful for reducing the expression of TRAC protein on the surface of cells. In some embodiments, such guide RNAs direct an RNA-guided DNA binding agent to a TRAC genomic target sequence, and are sometimes referred to herein as "TRAC guide RNAs". In some embodiments, the TRAC guide RNA directs an RNA-guided DNA binding agent to a human TRAC genomic target sequence. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101 to 120.

[0450] In some embodiments, the methods and compositions disclosed herein comprise a TRAC guide RNA comprising a guide sequence that targets a TRAC genomic target sequence comprising at least 10 nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the methods and compositions disclosed herein comprise a TRAC guide RNA comprising a guide sequence that targets a TRAC genomic target sequence comprising at least 1 nucleotide within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621.

[0451] In some embodiments, the methods and compositions disclosed herein include a TRAC guide RNA comprising a guide sequence that directs an RNA-guided DNA binding agent to induce a double-strand break (DSB) or a single-strand break (SSB) in the TRAC gene, wherein the TRAC guide RNA target and the TRAC genomic target sequence comprise at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the methods and compositions disclosed herein include a TRAC guide RNA comprising a guide sequence that directs an RNA-guided DNA binding agent to induce a double-strand break (DSB) or a single-strand break (SSB) in the TRAC gene, wherein the TRAC guide RNA targets a TRAC genomic target sequence that comprises at least 1 nucleotide within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621.

[0452] In some embodiments, the methods and compositions disclose a TRAC guide RNA that directs an RNA-guided DNA binding agent to induce a double-strand break (DSB) or a single-strand break (SSB) in the TRAC genomic target sequence. In some embodiments, the methods and compositions disclose a TRAC guide RNA that directs an RNA-guided DNA binding agent to effect cleavage in the TRAC genomic target sequence. In embodiments where the RNA-guided DNA cleavage agent is Cas9, the cleavage or "cleavage site" occurs at the third base from the protospacer adjacent motif (PAM) sequence.

[0453] In some embodiments, provided are compositions comprising the TRAC guide RNA described herein, and an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0454] In some embodiments, provided is a composition comprising a TRAC single guide RNA (sgRNA) comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, provided is a composition comprising the TRAC sgRNA described herein and an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0455] In some embodiments, provided is a composition comprising a TRAC dual guide RNA (dgRNA) comprising a guide sequence targeting a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, provided is a composition comprising the TRAC dgRNA described herein and an RNA-guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent.

[0456] In some embodiments, the TRAC gRNA comprises a guide sequence selected from any one of SEQ ID NOs: 101 to 120. Exemplary TRAC guide sequences are shown in Table 2 (SEQ ID NOs: 101 to 120) below, which includes the corresponding guide RNA sequences 101 to 120.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0457] Throughout this application, the terms "mA", "mC", "mU", or "mG" may be used to denote nucleotides modified with 2'-O-Me. Throughout this application, the terms A*, C*, U*, or G* may be used to denote nucleotides linked to the following (e.g., 3') nucleotides by phosphorothioate (PS) bonds.

[0458] In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101-120. In some embodiments, the TRAC guide RNA comprises a guide sequence that is at least 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 101-120. In some embodiments, the TRAC guide RNA comprises a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 101-120. In some embodiments, the TRAC guide RNA comprises a guide sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 101-120.

[0459] In some embodiments, the TRAC guide RNA comprises a guide sequence comprising at least 10 consecutive nucleotides of the genomic coordinates listed in Table 2 plus or minus 10 nucleotides. As used herein, at least 10 consecutive nucleotides of the genomic coordinates plus or minus 10 nucleotides means, for example, at least 10 consecutive nucleotides within the genomic coordinates, and the genomic coordinates include 10 nucleotides in the 5' direction and 10 nucleotides in the 3' direction from the range listed in Table 2. For example, the TRAC guide RNA may comprise 10 consecutive nucleotides within genomic coordinates selected from chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, or chr14:22550574-22550598, including the boundary nucleotides of these ranges. As another example, the TRAC guide RNA may comprise 10 consecutive nucleotides within genomic coordinates selected from chr14:22547481-22547505, chr14:22547471-22547495, chr14:22547470-22547494, or chr14:22547462-22547486, including the boundary nucleotides of these ranges. In some embodiments, the TRAC guide RNA comprises a guide sequence that is at least 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence comprising at least 10 consecutive nucleotides of the genomic coordinates listed in Table 2 plus or minus 10 nucleotides. In some embodiments, the TRAC guide RNA comprises a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from sequences that are 19, 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence comprising 10 consecutive nucleotides of the genomic coordinates listed in Table 2 plus or minus 10 nucleotides.

[0460] In some embodiments, the TRAC guide RNA comprises a guide sequence comprising at least 15 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising at least 24 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2.

[0461] In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 101. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 102. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 103. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 104. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 105. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 106. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 107. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 108. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 109. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 110. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 111. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 112. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 113. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 114. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 115. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 116. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 117. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 118. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 119. In some embodiments, the TRAC guide RNA comprises SEQ ID NO: 120.

[0462] In some embodiments, the TRAC guide RNA comprises nucleotides selected from SEQ ID NOs: 101-103, 107, 111, 117, or 118.

[0463] In some embodiments, the TRAC guide RNA comprises the sequences listed in Table 2. In some embodiments, the TRAC guide RNA comprises any one of the sequences of SEQ ID NOs: 101 to 120. In some embodiments, the TRAC guide RNA comprises any one of the sequences of SEQ ID NOs: 107, 111, and 117 to 120. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 107. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 111. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 117. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 118. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 119. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 120. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising any one of the sequences of SEQ ID NOs: 101 to 120. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising any one of the sequences of SEQ ID NOs: 107, 111, and 117 to 120. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 107. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 111. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 117. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 118. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 119. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 120. In some embodiments, the TRAC guide RNA comprises any one of the sequences of SEQ ID NOs: 1101 to 1120. In some embodiments, the TRAC guide RNA comprises any one of the sequences of SEQ ID NOs: 1107, 1111, and 1117 to 1120. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 1107. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 1111.In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 1117. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 1118. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 1119. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 1120. In some embodiments, the TRAC guide RNA comprises any one of the sequences of SEQ ID NOs: 2101-2120, 3003, and 3004. In some embodiments, the TRAC guide RNA comprises any one of the sequences of SEQ ID NOs: 2107, 2111, 2117-2120, 3003, and 3004. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 2107. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 2111. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 2117. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 2118. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 2119. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 2120. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 3003. In some embodiments, the TRAC guide RNA comprises the sequence of SEQ ID NO: 3004.

[0464] In some embodiments, the TRAC guide RNA is a single guide RNA (sgRNA) comprising any one of the sgRNA sequences listed in Table 2.

[0465] For example, additional embodiments of the TRAC guide RNA are provided herein, including exemplary modifications to the guide RNA.

[0466] 1. Gene Modification to TRAC In some embodiments, the methods and compositions disclosed herein genetically modify at least one nucleotide of the TRAC gene in a cell. In some embodiments, the genetic modification to TRAC reduces or eliminates the expression of the TRAC protein on the surface of the genetically modified cell (or engineered cell). The genetic modification includes a population of modifications resulting from contact with a genome editing system (e.g., a population of edits resulting from Cas9 and a TRAC guide RNA, or a population of edits resulting from BC22 and a TRAC guide RNA).

[0467] In some embodiments, the genetic modification includes at least one nucleotide within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, the genetic modification includes at least one nucleotide within genomic coordinates selected from any of the genomic coordinates listed in Table 2.

[0468] In some embodiments, the genetic modification comprises at least 1 nucleotide within a genomic locus selected from chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, or chr14:22550574-22550598. In some embodiments, the genetic modification comprises at least 5 consecutive nucleotides within a genomic locus selected from chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, or chr14:22550574-22550598. In some embodiments, the genetic modification comprises at least 10 consecutive nucleotides within a genomic locus selected from chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, or chr14:22550574-22550598. In some embodiments, the genetic modification comprises at least 10 consecutive nucleotides within a genomic locus selected from chr14:22547481-22547505, chr14:22547471-22547495, chr14:22547470-22547494, or chr14:22547462-22547486.

[0469] In some embodiments, the genetic modification comprises at least one indel, at least one C to T substitution, or at least one A to G substitution within a genomic locus selected from chr14:22547505-22547529, chr14:22547525-22547549, chr14:22547674-22547698, chr14:22550544-22550568, or chr14:22550574-22550598. In some embodiments, the genetic modification comprises at least one indel, at least one C to T substitution, or at least one A to G substitution within a genomic locus selected from chr14:22547481-22547505, chr14:22547471-22547495, chr14:22547470-22547494, or chr14:22547462-22547486.

[0470] In some embodiments, the modification to TRAC includes any one or more of insertion, deletion, substitution, or deamination of at least one nucleotide in the target sequence. In some embodiments, the modification to TRAC includes insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification to TRAC includes deletion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In other embodiments, the modification to TRAC includes insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In other embodiments, the modification to TRAC includes deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification to TRAC includes an indel, which is generally defined i...

Claims

[Claim 1] The invention described in the specification.

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