Compositions and methods for genome editing

Abstract

Disclosed are compositions and methods for ex vivo genome editing using a Neisseria meningitidis (Nme) CRISPR / Cas9 system. The present disclosure provides engineered cells comprising genetic modifications for use in, for example, adoptive cell transfer therapies.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No. 63 / 352,990, filed on June 16, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML file was created on June 13, 2023, is named "01155-0055-00PCT_SL.xml", and is 20,586,099 bytes in size.

[0005] Introduction and Summary of the Invention

[0006] The present disclosure relates to genome editing using the Neisseria meningitidis CRISPR / Cas9 system.

[0007] The ability to downregulate endogenous T cell receptor (TCR) α and β units, MHC class I and MHC class II loci is crucial for many in vivo and ex vivo applications, for example, when allogeneic cells (derived from a donor) are used for transplantation or, for example, for producing a cell population of inactivated T cells in vitro. Specifically, allogeneic cells are transferred into a subject to great interest in the field of cell therapy. The use of allogeneic cells is limited due to the rejection problem of the recipient's immune cells, which recognize the transplanted cells as foreign cells and attack them. In order to avoid the problem of immune rejection, cell-based therapies focus on autologous methods using the subject's own cells as a source of cells for therapy, which is time-consuming and costly.

[0008] In general, the immune rejection of allogeneic cells is caused by the mismatch of the major histocompatibility complex (MHC) molecules between the donor and the recipient. In the human population, MHC molecules exist in various forms, including, for example, many genetic variants of any given MHC gene, that is, alleles encoding different forms of MHC proteins. The main categories of MHC molecules are called MHC I and MHC II. MHC class I molecules (such as HLA-A, HLA-B and HLA-C in the human) are expressed on all nucleated cells and present antigens to activate cytotoxic T cells (CD8+T cells or CTLs). MHC class II molecules (such as HLA-DP, HLA-DQ and HLA-DR in the human) are only expressed on certain cell types (such as B cells, dendritic cells and macrophages) and present antigens to activate helper T cells (CD4+T cells or Th cells), which then provide signals to produce antibodies to B cells.

[0009] For example, slight differences in MHC alleles between individuals can cause T cells in the recipient to become activated. During T cell development, an individual's T cell pool tolerates its own MHC molecules, but T cells that recognize another individual's MHC molecules can persist in the circulation and are called alloreactive T cells. Alloreactive T cells can become activated, for example, due to the presence of cells expressing another individual's MHC molecules in the body, causing, for example, graft-versus-host disease and transplant rejection.

[0010] The method and composition for reducing the susceptibility of allogeneic cells to rejection are of interest, including, for example, reducing the MHC protein expression of cells to avoid recipient T cell reactions. In practice, the ability of allogeneic cells to be genetically modified for transplantation into a subject is hindered by the need for multiple genome editing to reduce the expression of all MHC proteins while avoiding other harmful recipient immune responses. For example, although the strategy of consuming MHC class I proteins can reduce the activation of CTLs, cells lacking MHC class I on the surface are easily dissolved by natural killer (NK) cells of the immune system, because NK cell activation is regulated by MHC class I specific inhibitory receptors. Therefore, it has been shown that safely reducing or eliminating the expression of MHC class I is challenging. It has also been shown that for reasons including low editing efficiency and low cell viability, the genome editing strategy of consuming MHC class II molecules is difficult, particularly in certain cell types, thereby hindering practical applications as cell therapy.

[0011] Therefore, there is a need for improved methods and compositions to modify cells to overcome the problem of recipient immune rejection and the technical difficulties associated with the multiple genetic 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), making NmeCas9 suitable for messenger RNA (mRNA)-based delivery methods. NmeCas9 has favorable specificity and low off-target cleavage rate.

[0012] Engineered cells comprising genetic modifications in HLA-A, TRAC, TRBC, or CIITA (class II major histocompatibility complex transactivator) can be used in cell therapy. The present disclosure further provides compositions and methods for reducing or eliminating the surface expression of endogenous T cell receptors, MHC class I or class II proteins in cells by genetically modifying the HLA-A, TRAC, TRBC, or CIITA genes.

[0013] In some embodiments, a method is provided for reducing the surface expression of HLA-A proteins in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any embodiment provided herein. In some embodiments, a method is provided for reducing the surface expression of TRAC proteins in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any embodiment provided herein. In some embodiments, a method is provided for reducing the surface expression of TRBC proteins in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any embodiment provided herein. In some embodiments, a method is provided for reducing the surface expression of MHC class II proteins in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any embodiment provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A Shown are the percentage distributions of HLA-A2- and HLA-A3- T cells after HLA-A targeting-guided editing.

[0015] Figure 1B Shown are the percentages of HLA-A2-HLA-A3+(HLA-A-), HLA-A+, HLA-A3+(HLA-A3-), and HLA-A2-, HLA-A3- T cells after editing with the selected guides.

[0016] Figure 1C Shown are the percentages of HLA-BT cells after editing directed by the selected HLA-A.

[0017] Figure 2 Shown are the percentages of HLA-A2-negative T cells after HLA-A-directed editing with a dilution series.

[0018] Figure 3A Shown are the percentage distributions of CD3- T cells after TRAC-directed editing.

[0019] Figure 3B The percentage of editing at the TRAC locus is shown.

[0020] Figure 4 Shown are the percentages of CD3-negative T cells after TRAC-directed editing with a dilution series.

[0021] Figure 5A Shown are the percentage distributions of CD3-T cells after TRBC-guided editing.

[0022] Figure 5B The percentage of editing at the TRBC locus is shown.

[0023] Figure 6 Shown are the percentages of CD3-negative T cells after editing guided by a dilution series of TRBCs.

[0024] Figure 7A Shown are the percentage distributions of HLA-DP, DQ, and DR-T cells after CIITA-guided editing.

[0025] Figure 7B The percentage of editing at the CIITA locus is shown.

[0026] Figure 8 Shown are the percentage of HLA II-DR, DP, DQ negative cells on the left vertical axis and the average editing percentage on the right vertical axis after CIITA-guided editing.

[0027] Figure 9A Shown are the percentages of reads with C to T conversions at the CIITA locus after guiding editing with a dilution series of CIITA.

[0028] Figure 9B Shown are the percentages of HLA-DP, DQ, and DR-negative T cells after editing guided by CIITA in a dilution series.

[0029] Figures 10A-10B Editing expressed as insertion / deletion (indel) frequency at the AAVS1 locus is shown.

[0030] Figures 11A-11B Shown are the indel frequencies at AAVS1 after directing editing with a dilution series of AAVS1.

[0031] Figure 12 Shown are the average editing frequencies at various TRAC sgRNA concentrations.

[0032] Figure 13 The average editing frequency at the CIITA locus is shown.

[0033] Figure 14 Mean percentage of T cells showing negative HLA-A2 surface expression.

[0034] Figure 15 The average editing percentage at the CIITA locus is shown.

[0035] Figures 16A-16L Shown are the average editing percentages at a range of doses of the guide RNA.

[0036] Figures 17A-17D Shown are the average editing percentages of CD8+ T cells negative for the corresponding expression markers at various sgRNA concentrations. DETAILED DESCRIPTION

[0037] The present disclosure provides engineered cells, and methods and compositions for genetically modifying cells to prepare engineered cells and engineered cell colonies, which can be used for, for example, adoptive cell transfer (ACT) therapy. The disclosure provided herein overcomes certain obstacles of the prior art methods by providing methods and compositions for genetically modifying HLA-A, TRAC, TRBC, CIITA or AAVS1 loci to reduce HLA-A, TRAC, TRBC or MHC II class protein expression on cell surfaces. In some embodiments, the disclosure provides engineered cells with surface expression of HLA-A, TRAC, TRBC or MHC II classes reduced or eliminated due to genetic modification of HLA-A, TRAC, TRBC or CIITA genes. In some embodiments, the disclosure provides compositions and methods for reducing or eliminating HLA-A, TRAC, TRBC or MHC II class protein expression, as well as compositions and methods for further reducing the susceptibility of cells to immune rejection. For example, in some embodiments, the methods and compositions include reducing or eliminating the surface expression of HLA-A proteins by genetically modifying HLA-A genes. 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. The engineered cell compositions produced by the methods disclosed herein have desired properties, including, for example, reduced MHC molecule expression, reduced in vitro and in vivo immunogenicity, increased survival, and increased genetic compatibility with larger transplant subjects.

[0038] The term "about" or "approximately" means an acceptable error for a particular value as determined by one skilled in the art, which depends in part on the manner in which the value is measured or determined; or a degree of variation that does not substantially affect the properties of the subject matter or is within a tolerance acceptable in the art (e.g., within 10%, 5%, 2% or 1%). Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​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 scope of 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.

[0039] This article provides the following numbered implementation plans:

[0040] Embodiment 1 is an engineered cell having reduced or eliminated surface expression of HLA-A relative to an unmodified cell, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr6:29942540-29945459.

[0041] Embodiment 2 is the engineered cell of embodiment 1, having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 1, or wherein the genetic modification comprises at least one nucleotide within the genomic coordinate targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80.

[0042] Embodiment 3 is an engineered cell having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises at least one nucleotide within the genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495 and chr6: 29944470-29944494.

[0043] Embodiment 4 is an engineered human cell having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809.

[0044] Embodiment 5 is an engineered human cell having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr6:29942891-29942915; chr6:29942609-29942633; chr6:29944266-29944290; chr6:29942889-29942913; chr6:29944471-29944495 and chr6:29944470-29944494.

[0045] Embodiment 6 is an engineered human cell having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809.

[0046] Embodiment 7 is an engineered cell according to any one of embodiments 1-6, wherein the HLA-A expression 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 the group consisting of chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942915; 9942913; chr6:29944471-29944495 and chr6:29944470-29944494, or selected from chr6:29942891-29942915; chr6:29942609-29942633: chr6:29942889-29942913; chr6:29944471-29944495; chr6:29944266-29944290; chr6:29942785-29942809.

[0047] Embodiment 8 is the engineered cell of any one of embodiments 1-7, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 1.

[0048] Embodiment 9 is the engineered cell of any one of embodiments 1-8, wherein the cell is homozygous for HLA-C.

[0049] Embodiment 10 is the engineered cell of any one of embodiments 1-9, wherein the cell is homozygous for HLA-B and homozygous for HLA-C.

[0050] Embodiment 11 is 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 the group consisting of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; ii. at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; or iii. a sequence selected from the group consisting of SEQ ID NOs: NO: 66, 61, 2-60, 62-65, 67-80; iv. a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 1; v. at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi. a guide sequence at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0051] Embodiment 12 is a method of making an engineered human cell having reduced or eliminated surface expression of an HLA-A protein relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; ii. at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; or iii. a sequence selected from the group consisting of SEQ ID NOs: NO: 66, 61, 2-60, 62-65, 67-80; iv. a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 1; v. at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi. a guide sequence at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0052] Embodiment 13 is a method of reducing surface expression of an HLA-A protein in a human cell relative to an unmodified cell, comprising contacting the cell 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 the group consisting of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; ii. at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80; or iii. a sequence selected from the group consisting of SEQ ID NOs: NO: 66, 61, 2-60, 62-65, 67-80; iv. a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 1; v. at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi. a guide sequence at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0053] Embodiment 14 is the composition or method of any one of embodiments 11-13, wherein the HLA-A guide RNA comprises the guide sequence of any one of SEQ ID NOs: 66, 61, 13, 55, 70, and 71.

[0054] Embodiment 15 is the composition or method of any one of embodiments 11-13, wherein the HLA-A guide RNA comprises the guide sequence of any one of SEQ ID NOs: 61, 66, 13, 17, 55, and 70.

[0055] Embodiment 16 is the composition or method of any one of embodiments 11-13, wherein the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 61.

[0056] Embodiment 17 is the composition or method of any one of embodiments 11-13, wherein the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 66.

[0057] Embodiment 18 is a cell population comprising the engineered cells of any one of embodiments 1-10, or engineered cells produced by the method of any one of embodiments 12-17, or by using the composition of embodiment 11.

[0058] Embodiment 19 is a pharmaceutical composition comprising (a) an engineered cell as described in any one of embodiments 1-10; an engineered cell produced by the method as described in any one of embodiments 12-17 or by using the composition as described in embodiment 11; or (b) a cell population as described in embodiment 18.

[0059] Embodiment 20 is an engineered human cell having reduced or eliminated surface expression of TRAC relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621.

[0060] Embodiment 21 is the engineered cell of embodiment 20, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 2, or wherein the genetic modification comprises at least one nucleotide within the genomic coordinate targeted by a guide RNA comprising the guide sequence of any one of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120.

[0061] Embodiment 22 is an engineered cell as described in embodiment 20 or 21, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: chr14:22550574-22550598; chr14:22550544-22550568; chr14:22547505-22547529; or chr14:22547525-22547549; chr14:22547674-22547698.

[0062] Embodiment 23 is the engineered cell of embodiment 20 or 21, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr14:22547481-22547505.

[0063] Embodiment 24 is the engineered cell of embodiment 20 or 21, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr14:22547471-22547495.

[0064] Embodiment 25 is the engineered cell of embodiment 20 or 21, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr14:22547470-22547494.

[0065] Embodiment 26 is the engineered cell of embodiment 20 or 21, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr14:22547462-22547486.

[0066] Embodiment 27 is an engineered human cell having reduced or eliminated TRAC expression relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr14: 22550574-22550598; chr14: 22550544-22550568; chr14: 22547505-22547529; chr14: 22547525-22547549 or chr14: 22547674-22547698.

[0067] Embodiment 28 is an engineered cell as described in any of embodiments 20-27, wherein the TRAC expression is reduced or eliminated by a genome editing system that binds to a TRAC target sequence, wherein the TRAC target sequence comprises at least 5 contiguous nucleotides within the following genomic coordinates: chr14: 22550574-22550598; chr14: 22550544-22550568; chr14: 22547505-22547529; chr14: 22547525-22547549 or chr14: 22547674-22547698.

[0068] Embodiment 29 is a composition comprising: a) a TRAC guide RNA comprising a guide sequence, wherein the guide sequence i) targets a TRAC genomic target sequence; or ii) a guide RNA-guided DNA binder that induces a double-strand break (DSB) or a single-strand break (SSB) in the TRAC genomic target sequence, wherein the TRAC genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates selected from the group consisting of: chr14: 22550574-22550598; chr14: 22550544-22550568; chr14: 22547505-22547529; chr14: 22547525-22547549 or chr14: 22547674-22547698.

[0069] Embodiment 30 is 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 the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iii) a sequence selected from the group consisting of SEQ ID NOs: or iv) a guide sequence that is at least 95%, 90% or 85% identical to the sequence of NOs: 111, 107, 101-106, 108-110 and 112-120; or iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; v) at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0070] Embodiment 31 is a composition according to embodiment 30, for altering a DNA sequence within the TRAC locus in a cell.

[0071] Embodiment 32 is the composition of embodiment 30, which is used to reduce or eliminate the expression of TRAC protein in a cell.

[0072] Embodiment 33 is a method of making an engineered human cell having reduced or eliminated surface expression of a TRAC protein relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iii) a sequence selected from the group consisting of SEQ ID NOs: or iv) a guide sequence that is at least 95%, 90% or 85% identical to the sequence of NOs: 111, 107, 101-106, 108-110 and 112-120; or iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; v) at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0073] Embodiment 34 is a method of reducing surface expression of a TRAC protein in a human cell relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; or iii) a sequence selected from the group consisting of SEQ ID NOs: or iv) a guide sequence that is at least 95%, 90% or 85% identical to the sequence of NOs: 111, 107, 101-106, 108-110 and 112-120; or iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0074] Embodiment 35 is the composition or method of any one of embodiments 29-34, wherein the TRAC guide RNA comprises the guide sequence of any one of SEQ ID NOs: 111, 107, 101, 102, and 103.

[0075] Embodiment 36 is the composition or method of any one of embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 107.

[0076] Embodiment 37 is the composition or method of any one of embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 111.

[0077] Embodiment 38 is the composition or method of any one of embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 117.

[0078] Embodiment 39 is the composition or method of any one of embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 118.

[0079] Embodiment 40 is the composition or method of any one of embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 119.

[0080] Embodiment 41 is the composition or method of any one of embodiments 29-34, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO: 120.

[0081] Embodiment 42 is a cell population comprising the engineered cells of any one of embodiments 20-28, or engineered cells produced by use of the composition of embodiment 29 or 30, or the method of any one of embodiments 33-41, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of the cell population are CD3- cells.

[0082] Embodiment 43 is a cell population comprising the engineered cells of any one of embodiments 20-28, or engineered cells produced by use of the composition of embodiment 29 or 30, or by the method of any one of embodiments 33-41, or the cell population of embodiment 42, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of the population lack endogenous T cell receptors.

[0083] Embodiment 44 is a cell population comprising the engineered cells of any one of embodiments 20-28, or engineered cells produced by using the composition of embodiment 29 or 30 or by the method of any one of embodiments 33-41, or the cell population of embodiment 42 or 43, wherein the expression of the TRAC gene in the population has been 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% relative to the same unaltered cell population.

[0084] Embodiment 45 is a pharmaceutical composition comprising the engineered cell of any one of embodiments 20-28, or an engineered cell produced by using the composition of embodiment 29 or 30 or by the method of any one of embodiments 33-41, or a cell population of any one of embodiments 42-44.

[0085] Embodiment 46 is an engineered cell having reduced or eliminated surface expression of TRBC relative to an unmodified cell, comprising a genetic modification in the TRBC locus, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr7:142791756-142802543.

[0086] Embodiment 47 is an engineered cell having reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC locus, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543, or wherein the genetic modification comprises at least one nucleotide within the genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 215, 201-214, and 216-265.

[0087] Embodiment 48 is the engineered cell of embodiment 46 or 47, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 3.

[0088] Embodiment 49 is an engineered cell as described in embodiment 46 or 47, wherein the genetic modification comprises at least one nucleotide within the genomic coordinates selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 142791761-142791785; chr7: 142792690-142792714 or chr7: 142792693-142792717; 791820-142791844; chr7:142791939-142791963; chr7:142791940-142791964 or chr7:142792004-142792028; or (c) chr7:142801104-142801124; chr7:142802103-142802127 or chr7:142802106-142802130.

[0089] Embodiment 50 is the engineered cell of embodiment 46 or 47, wherein the genetic modification comprises at least one nucleotide within genomic coordinates selected from the group consisting of: chr7: 142792690-142792714; chr7: 142802103-142802127 and chr7: 142802106-14280213.

[0090] Embodiment 51 is an engineered cell having reduced or eliminated TRBC expression relative to an unmodified cell, comprising a genetic modification in a human TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the following genomic coordinates: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543.

[0091] Embodiment 52 is an engineered human cell having reduced or eliminated TRBC expression relative to an unmodified cell, comprising a genetic modification in the TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 14279 1761-142791785; 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.

[0092] Embodiment 53 is an engineered human cell having reduced or eliminated TRBC expression relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr7: 142792690-142792714; chr7: 142802103-142802127 and chr7: 142802106-14280213.

[0093] Embodiment 54 is an engineered cell as described in any one of embodiments 46-53, wherein the TRBC expression is reduced or eliminated by a genome editing system that binds to a TRBC target sequence, the TRBC target sequence comprising at least 5 contiguous nucleotides within the following genomic coordinates: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 142791 761-142791785; 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.

[0094] Embodiment 55 is an engineered cell as described in any of embodiments 46-54, wherein the TRBC expression is reduced or eliminated by a genome editing system that binds to a TRBC target sequence, wherein the TRBC target sequence comprises at least 5 contiguous nucleotides within the following genomic coordinates: chr7: 142792690-142792714; chr7: 142802103-142802127 and chr7: 142802106-14280213.

[0095] Embodiment 56 is a composition comprising: (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 the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; iii) a sequence selected from the group consisting of SEQ ID NOs: NOs: 215, 201-214, and 216-265; iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 3; v) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of the sequence from (iv); or vi) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0096] Embodiment 57 is the composition of embodiment 56 for altering a DNA sequence within the TRBC locus in a cell.

[0097] Embodiment 58 is the composition of embodiment 56, which is used to reduce or eliminate the expression of TRBC protein in a cell.

[0098] Embodiment 59 is a method of making an engineered human cell having reduced or eliminated surface expression of a TRBC protein relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; iii) a sequence selected from the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; NOs: 215, 201-214, and 216-265; iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 3; v) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of the sequence from (iv); or vi) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0099] Embodiment 60 is a method of reducing surface expression of a TRBC protein in a human cell relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; iii) a sequence selected from the group consisting of SEQ ID NOs: NOs: 215, 201-214, and 216-265; iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 3; v) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of the sequence from (iv); or vi) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0100] Embodiment 61 is a method or composition as described in any one of embodiments 56-60, wherein the TRBC guide RNA comprises the guide sequence of any one of SEQ ID NOs: 215, 216, 223, 224, 229, 230, 246, 259 and 260.

[0101] Embodiment 62 is a method or composition as described in any one of embodiments 56-61, wherein the TRBC guide RNA comprises the guide sequence of any one of SEQ ID NOs: 215, 216, 224, 229, 246, 259 and 260.

[0102] Embodiment 63 is a method or composition as described in any one of embodiments 56-62, wherein the TRBC guide RNA comprises the guide sequence of any one of SEQ ID NOs: 215, 259 and 260.

[0103] Embodiment 64 is a cell population comprising the engineered cells of any one of embodiments 46-55, or engineered cells produced by use of the composition of embodiment 56 or by the method of any one of embodiments 59-63, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of the cell population are CD3- cells.

[0104] Embodiment 65 is a population of cells comprising the engineered cells of any one of embodiments 46-55, or engineered cells produced by use of the composition of embodiment 56, or by the method of any one of embodiments 59-63, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of the population lack endogenous T cell receptors.

[0105] Embodiment 66 is a cell population comprising the engineered cells of any one of embodiments 46-55, or engineered cells produced by using the composition of embodiment 56 or by the method of any one of embodiments 59-63, wherein the expression of the TRBC gene in the population has been 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% relative to the same unaltered cell population.

[0106] Embodiment 67 is a pharmaceutical composition comprising (a) an engineered cell as described in any one of embodiments 46-55, or an engineered cell produced by using the composition as described in embodiment 56 or by the method as described in any one of embodiments 59-63, or (b) a cell population as described in any one of embodiments 64-66.

[0107] Embodiment 68 is an engineered cell having reduced or eliminated surface expression of MHC class II relative to unmodified cells, comprising a genetic modification in the CIITA gene, wherein the genetic modification comprises at least one nucleotide within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136.

[0108] Embodiment 69 is the engineered cell of embodiment 68, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 4.

[0109] Embodiment 70 is an engineered cell as described in embodiment 68, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: (a) chr16: 10907504-10907528; chr16: 10906643-10906667; chr16: 10907508-10907532; chr16: 10907539-10907559; chr16: 10895658-10895682; chr16: 10895668-10895692; chr16: 1 0895750-10895774; chr16: 10895753-10895777; chr16: 10895754-10895778; chr16: 10898684-10898708; chr16: 10901529 -10901553; chr16: 10902121-10902145; chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-1090478 4; 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: 1090714 8-10907172; 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.

[0110] Embodiment 71 is an engineered cell as described in embodiment 68, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: 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.

[0111] Embodiment 72 is an engineered cell having reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA locus, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the following genomic coordinates: (a) chr16: 10907504-10907528; chr16: 10906643-10906667; chr16: 10907508-10907532; chr16: 10907539-10907559; chr16: 108 95658-10895682; chr16: 10895668-10895692; chr16: 10895750-10895774; chr16: 10895753-10895777; chr16: 10895754-10895 778; chr16: 10898684-10898708; chr16: 10901529-10901553; chr16: 10902121-10902145; chr16: 10902701-10902725; chr16: 1 0904726-10904750; chr16: 10904760-10904784; chr16: 10906493-10906517; chr16: 10906515-10906539; chr16: 10906631-109 06655; 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) chr16:10907504-10907528.

[0112] Embodiment 73 is an engineered cell having reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA locus, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr16: 10907504-10907528; chr16: 10906643-10906667; chr16: 10895658-1089 5682; chr16:10902701-10902725; chr16:10906493-10906517; chr16:10906631-10906655; chr16:10907477-10907501; and chr16:10907497-10907521; chr16:10907504-10907528; chr16:10907508-10907532.

[0113] Embodiment 74 is an engineered cell as described in any of embodiments 68-73, wherein the MHC class II expression is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence, the CIITA genomic target sequence comprising at least 5 contiguous nucleotides within the following genomic coordinates: (a) chr16: 10907504-10907528; chr16: 10906643-10906667; chr16: 10907508-10907532; chr16: 10907539-10907559; chr16: 10895658-10907582; chr16: 10895682; 95668-10895692; chr16: 10895750-10895774; chr16: 10895753-10895777; chr16: 10895754-10895778; chr16: 10898684- 10898708; chr16: 10901529-10901553; chr16: 10902121-10902145; chr16: 10902701-10902725; chr16: 10904726-1090475 0; chr16: 10904760-10904784; chr16: 10906493-10906517; chr16: 10906515-10906539; chr16: 10906631-10906655; chr1 6: 10906636-10906660; chr16: 10906770-10906794; chr16: 10906788-10906812; chr16: 10906789-10906813; chr16: 10906 816-10906840;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.

[0114] Embodiment 75 is an engineered cell as described in any of embodiments 68-74, wherein the MHC class II expression is reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence, the CIITA genomic target sequence comprising at least 5 contiguous nucleotides within the following genomic coordinates: 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.

[0115] 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 comprises: i) a guide sequence selected from the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or ii) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or iii) a sequence selected from the group consisting of SEQ ID NOs: or iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 4; or v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides of the sequence from (iv); or vi) a guide sequence at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0116] Embodiment 77 is the composition of embodiment 76, which is used to alter the DNA sequence within the CIITA gene in a cell.

[0117] Embodiment 78 is a composition according to embodiment 76, which is used to reduce or eliminate the expression of CIITA in a cell.

[0118] Embodiment 79 is a method of making an engineered human cell having reduced or eliminated surface expression of an MHC class II protein relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or ii) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or iii) at least 25 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 4; or v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides of the sequence from (iv); or vi) a guide sequence at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0119] Embodiment 80 is a method of reducing surface expression of an MHC class II protein in a human cell relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or ii) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or iii) a sequence selected from the group consisting of SEQ ID NOs: or iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 4; or v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides of the sequence from (iv); or vi) a guide sequence at least 95%, 90% or 85% identical to a sequence selected from (iv).

[0120] Embodiment 81 is a method or composition as described in any one of embodiments 76-80, wherein the CIITA guide RNA comprises the guide sequence of any one of SEQ ID NO: 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.

[0121] Embodiment 82 is a method or composition as described in any one of embodiments 76-81, wherein the CIITA guide RNA comprises the guide sequence of any one of SEQ ID NO: 538.

[0122] Embodiment 83 is a method or composition as described in any one of embodiments 76-81, wherein the CIITA guide RNA comprises the guide sequence of any one of SEQ ID NOs: 301, 422, 302, 320, 372, 414, 419, 462, and 463.

[0123] Embodiment 84 is a cell population comprising the engineered cells of any one of embodiments 68-75, or engineered cells produced by use of the composition of embodiment 76 or by the method of any one of embodiments 79-83, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of the cell population are negative for MHC class II molecules as measured by flow cytometry.

[0124] Embodiment 85 is a cell population comprising the engineered cells of any one of embodiments 68-75, or engineered cells produced by use of the composition of embodiment 76 or by the method of any one of embodiments 79-83, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of the cell population are negative for MHC class II molecules as measured by next generation sequencing (NGS).

[0125] Embodiment 86 is a cell population comprising the engineered cells of any one of embodiments 68-75, or engineered cells produced by use of the composition of embodiment 76 or by the method of any one of embodiments 79-83, wherein the expression of MHC class II molecules in the population has been 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% relative to an unaltered population of the same cells.

[0126] Embodiment 87 is a pharmaceutical composition comprising (a) an engineered cell as described in any one of embodiments 68-75, or an engineered cell produced by using the composition as described in embodiment 76 or by the method as described in any one of embodiments 79-83, or (b) a cell population as described in any one of embodiments 84-86.

[0127] Embodiment 88 is an engineered cell comprising a genetic modification in the AAVS1 locus, wherein the genetic modification comprises at least one nucleotide within the genomic coordinates selected from the group consisting of: chr19:55115151-55116209.

[0128] Embodiment 89 is an engineered cell as described in embodiment 88, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 5.

[0129] Embodiment 90 is the engineered cell of embodiment 88, wherein the genetic modification comprises at least one nucleotide within the genomic coordinates selected from the group consisting of: 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: 55115574-551155 98; chr19: 55115606-55115630; chr19: 55115933-55115957; chr19: 55116026- 55116050; chr19: 55116045-55116069; chr19: 55116084-55116108; chr19: 5511 5276-55115300; chr19: 55115509-55115533; chr19: 55115579-55115603; chr19: 55115863-55115887; chr19: 55115906-55115930 or chr19: 55116006-55116030.

[0130] Embodiment 91 is an engineered cell comprising a genetic modification in the AAVS1 gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: 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: 551155 574-55115598;chr19:55115606-55115630;chr19:55115933-55115957;chr19:55 116026-55116050; 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.

[0131] Embodiment 92 is an engineered cell as described in any of embodiments 88-91, wherein the genetic modification is induced by a genome editing system that binds to an AAVS1 genomic target sequence, the AAVS1 genomic target sequence comprising at least 5 contiguous nucleotides within the following genomic coordinates: chr19: 55115218-55115242; chr19: 55115477-55115501; chr19: 55115504-55115528; chr19: 55115513-55115537; chr19: 55115514-55115538; chr19: 55115517-55115541; chr19: 55115518-55115542; chr19: 55115549-5511555 73; chr19: 55115574-55115598; chr19: 55115606-55115630; chr19: 55115933-551159 57; chr19: 55116026-55116050; chr19: 55116045-55116069; chr19: 55116084-5511610 8;chr19:55115276-55115300;chr19:55115509-55115533;chr19:55115579-55115603:chr19:55115863-55115887;chr19:55115906-55115930 or chr19:55116006-55116030.

[0132] Embodiment 93 is a composition comprising: a) an AAVS1 guide RNA comprising a guide sequence that i) targets an AAVS1 genomic target sequence; or ii) a guide RNA-guided DNA binder that induces a double-strand break (DSB) or a single-strand break (SSB) in an AAVS1 genomic target sequence comprising at least 10 contiguous nucleotides within a genomic coordinate selected from the group consisting of: chr19:5 5115218-55115242; chr19: 55115477-55115501; chr19: 55115504-55115528; chr19: 55115513 -55115537; chr19: 55115514-55115538; chr19: 55115517-55115541; chr19: 55115518-551155 42; chr19: 55115549-55115573; chr19: 55115574-55115596; chr19: 55115606-55115630; chr 19:55115933-55115957;chr19:55116026-55116050;chr19:55116045-55116069;chr19:5511 6084-55116108; chr19: 55115276-55115300; chr19: 55115509-55115533; chr19: 55115579-55115603; chr19: 55115863-55115887; chr19: 55115906-55115930 or chr19: 55116006-55116030.

[0133] Embodiment 94 is a composition comprising: a) an AAVS1 guide RNA (gRNA) and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, 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 contiguous nucleotides of a sequence selected from SEQ ID NOs: 601-774; iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 601-774; iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 5; v) at least 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence from (iv); or vi) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0134] Embodiment 95 is a method of making an engineered human cell, comprising contacting the cell with: (a) an AAVS1 guide RNA and optionally (b) an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, 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 contiguous nucleotides of a sequence selected from SEQ ID NOs: 601-774; iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 601-774; iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 5; v) at least 20, 21, 22, 23, or 24 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from (iv).

[0135] Embodiment 96 is a method or composition as described in any one of embodiments 93-95, wherein the AAVS1 guide RNA comprises the guide sequence of any one 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.

[0136] Embodiment 97 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-96, wherein the genetic modification comprises 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 contiguous nucleotides within the genomic coordinates.

[0137] Embodiment 98 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-97, wherein the genetic modification comprises at least 5, 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0138] Embodiment 99 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-98, wherein the genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates.

[0139] Embodiment 100 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-99, wherein the genomic target sequence comprises at least 15 contiguous nucleotides within the genomic coordinates.

[0140] Embodiment 101 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-100, wherein the genomic target sequence comprises at least 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides within the genomic coordinates.

[0141] Embodiment 102 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-101, wherein the genetic modification comprises an insertion / deletion.

[0142] Embodiment 1Q3 is an engineered cell as described in any one of embodiments 1-102, wherein the genetic modification comprises the insertion of a heterologous coding sequence.

[0143] Embodiment 104 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-103, wherein the genetic modification comprises at least one A to G substitution within the genomic coordinates.

[0144] Embodiment 105 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-104, wherein the genetic modification comprises at least one C to T substitution within the genomic coordinates.

[0145] Embodiment 106 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-105, wherein the cell has a genetic modification in the CIITA gene.

[0146] Embodiment 107 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-106, wherein the cell has reduced expression of TRAC protein on the surface of the cell.

[0147] Embodiment 108 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-107, wherein the cell has reduced expression of TRBC protein on the surface of the cell.

[0148] Embodiment 109 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-108, wherein the cell has reduced expression of MHC class II molecules on the surface of the cell.

[0149] Embodiment 110 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-109, wherein the engineered cell is an immune cell.

[0150] Embodiment 111 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-110, wherein the cell is a primary cell.

[0151] Embodiment 112 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-111, wherein the engineered cell is a monocyte, a macrophage, a mast cell, a dendritic cell, or a granulocyte.

[0152] Embodiment 113 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-112, wherein the engineered cell is a lymphocyte.

[0153] Embodiment 114 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-113, wherein the cell is a T cell.

[0154] Embodiment 115 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-114, wherein the cell is a CD8+ T cell.

[0155] Embodiment 116 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-114, wherein the cell is a CD4+ T cell.

[0156] Embodiment 117 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-113, wherein the cell is a natural killer (NK) cell.

[0157] Embodiment 118 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-113, wherein the cell is a macrophage.

[0158] Embodiment 119 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-113, wherein the cell is a B cell.

[0159] Embodiment 120 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-113, wherein the cell is a plasma B cell.

[0160] Embodiment 121 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-113, wherein the cell is a memory B cell.

[0161] Embodiment 122 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-109, wherein the cell is a stem cell.

[0162] Embodiment 123 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-109, wherein the cell is a progenitor cell.

[0163] Embodiment 124 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-109, wherein the cell is an HSC.

[0164] Embodiment 125 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-109, wherein the cell is an iPSC.

[0165] Embodiment 126 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-121, wherein the cell is an activated cell.

[0166] Embodiment 127 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-121, wherein the cell is a non-activated cell.

[0167] Embodiment 128 is a population as described in embodiment 18 or a pharmaceutical composition as described in 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 as measured by flow cytometry.

[0168] Embodiment 129 is a population as described in any of embodiments 42-44 and 64-66 or a pharmaceutical composition as described in 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% negative for endogenous TCR protein as measured by flow cytometry.

[0169] Embodiment 130 is a population as described in any of embodiments 84-86 or a pharmaceutical composition as described in 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 as measured by flow cytometry.

[0170] Embodiment 131 is the population of embodiment 18 or the pharmaceutical composition of 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 cells in the population comprise the genetic modification in the HLA-A gene as measured by next generation sequencing (NGS).

[0171] Embodiment 132 is a population as described in any of embodiments 42-44 or a pharmaceutical composition as described in 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 cells in the population comprise the genetic modification in the TRAC gene as measured by next generation sequencing (NGS).

[0172] Embodiment 133 is a population as described in any of embodiments 64-66 or a pharmaceutical composition as described in 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 cells in the population comprise the genetic modification in the TRBC gene as measured by next generation sequencing (NGS).

[0173] Embodiment 134 is a population as described in any of embodiments 84-86 or a pharmaceutical composition as described in 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 cells in the population comprise the genetic modification in the CIITA gene as measured by next generation sequencing (NGS).

[0174] Embodiment 135 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-134, wherein the cell is an allogeneic cell.

[0175] Embodiment 136 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-135, wherein the cell is a primary cell.

[0176] Embodiment 137 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-136, wherein the cell is a CD4+ T cell.

[0177] Embodiment 138 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a CD8+ T cell.

[0178] Embodiment 139 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a memory T cell.

[0179] Embodiment 140 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a I) cell.

[0180] Embodiment 141 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a plasma B cell.

[0181] Embodiment 142 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a memory B cell.

[0182] Embodiment 143 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-136, wherein the cell is a natural killer (NK) cell.

[0183] Embodiment 144 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a macrophage.

[0184] Embodiment 145 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a stem cell.

[0185] Embodiment 146 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a pluripotent stem cell (PSC).

[0186] Embodiment 147 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a hematopoietic stem cell (HSC).

[0187] Embodiment 148 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-136, wherein the cell is an induced pluripotent stem cell (iPSC).

[0188] Embodiment 149 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-136, wherein the cell is a mesenchymal stem cell (MSC).

[0189] Embodiment 150 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-136, wherein the cell is a neural stem cell (NSC).

[0190] Embodiment 151 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a limbal stem cell (LSC).

[0191] Embodiment 152 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a progenitor cell, such as an endothelial progenitor cell or a neural progenitor cell.

[0192] Embodiment 153 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-136, wherein the cell is a tissue-specific primary cell.

[0193] Embodiment 154 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-136, wherein the cell is selected from the group consisting of: a chondrocyte, a myocyte, and a keratinocyte.

[0194] Embodiment 155 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-136, wherein the cell is an activated cell.

[0195] Embodiment 156 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 1-136, wherein the cell is a non-activated cell.

[0196] Embodiment 157 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-156, wherein the cell is engineered with a genome editing system.

[0197] Embodiment 158 ​​is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 7-157, wherein the genome editing system comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0198] Embodiment 159 is an engineered cell, cell population, pharmaceutical composition, or method as described in embodiment 158, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid is Neisseria meningitidis Cas9 (NmeCas9).

[0199] Embodiment 160 is an engineered cell, cell population, pharmaceutical composition or method as described in embodiment 159, wherein the NmeCas9 is Nme1Cas9, Nme2Cas9 or Nme3Cas9.

[0200] Embodiment 161 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 158-160, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid has double-stranded endonuclease activity.

[0201] Embodiment 162 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 158-161, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid has nickase activity.

[0202] Embodiment 163 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 158-161, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid comprises a dCas9 DNA binding domain.

[0203] Embodiment 164 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 158-160, wherein the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is an A to G base editor.

[0204] Embodiment 165 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 158-160, wherein the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is a C to T base editor.

[0205] Embodiment 166 is an engineered cell, cell population, pharmaceutical composition, or method as described in any 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.

[0206] Embodiment 167 is an engineered cell as described in embodiment 158, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid comprises APOBEC3A deaminase (A3A) and Neisseria meningitidis Cas9 nickase.

[0207] Embodiment 168 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-167, wherein the guide RNA is provided to the cell in a vector.

[0208] Embodiment 169 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-168, wherein the RNA-guided DNA binder is provided to the cell in a vector, optionally in the same vector as the guide RNA.

[0209] Embodiment 170 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-169, wherein the exogenous nucleic acid is provided to the cell in a vector.

[0210] Embodiment 171 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 168-170, wherein the vector is a viral vector.

[0211] Embodiment 172 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 168-170, wherein the vector is a non-viral vector.

[0212] Embodiment 173 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 168-171, wherein the vector is a lentiviral vector.

[0213] Embodiment 174 is an engineered cell, cell population, pharmaceutical composition, or method as described in any one of embodiments 168-171, wherein the vector is a retroviral vector.

[0214] Embodiment 175 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 168-171, wherein the vector is AAV.

[0215] Embodiment 176 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-175, wherein the guide RNA is provided to the cell in a lipid nucleic acid assembly composition, optionally in the same lipid nucleic acid assembly composition as the RNA-guided DNA binder.

[0216] Embodiment 177 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-176, wherein the exogenous nucleic acid is provided to the cell in a lipid nucleic acid assembly composition.

[0217] Embodiment 178 is an engineered cell, cell population, pharmaceutical composition, or method as described in embodiment 176 or 177, wherein the lipid nucleic acid assembly composition is a lipid nanoparticle (LNP).

[0218] Embodiment 179 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-178, wherein the exogenous nucleic acid is integrated into the genome of the cell.

[0219] Embodiment 180 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-179, wherein the exogenous nucleic acid is integrated into the genome of the cell by homologous recombination (HR).

[0220] Embodiment 181 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-180, wherein the exogenous nucleic acid is integrated into a safe harbor locus in the genome of the cell.

[0221] Embodiment 182 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-181, wherein the guide RNA is a single guide RNA.

[0222] Embodiment 183 is an engineered cell, cell population, pharmaceutical composition, or method as described in embodiment 182, wherein the single guide RNA comprises the nucleotide sequence of SEQ ID NO: 900 3' to the guide sequence.

[0223] Embodiment 184 is the engineered cell, cell population, pharmaceutical composition, or method of embodiment 182 or 183, wherein the single guide RNA comprises a guide region and a conserved region, wherein the conserved region comprises one or more of: (a) a shortened repeat / anti-repeat region, wherein the shortened repeat / anti-repeat region lacks 2-24 nucleotides, wherein (i) relative to SEQ ID NO: 900, one or more of nucleotides 37-48 and 53-64 are deleted and optionally one or more of nucleotides 37-64 are substituted; and (ii) nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b) a shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2-10, optionally 2-8, nucleotides, wherein (i) relative to SEQ ID NO: 900, one or more of nucleotides 37-48 and 53-64 are deleted and optionally one or more of nucleotides 37-64 are substituted; NO: 900, one or more of nucleotides 82-86 and 91-95 are deleted and optionally one or more of positions 82-96 are substituted; and (ii) nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (c) a shortened hairpin 2 region, wherein the shortened hairpin 2 lacks 2-18, optionally 2-16 nucleotides, wherein (i) relative to SEQ ID NO: 900, one or more of nucleotides 113-121 and 126-134 are deleted and optionally one or more of nucleotides 113-134 are substituted; and (ii) nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides; wherein relative to SEQ ID NO: 900, one or two of nucleotides 144-145 are optionally deleted; wherein at least 10 nucleotides are modified nucleotides.

[0224] Embodiment 185 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-184, wherein the guide RNA comprises at least one modification.

[0225] Embodiment 186 is an engineered cell, cell population, pharmaceutical composition or method as described in embodiment 185, wherein the modification comprises a modified nucleotide selected from the group consisting of 2'-O-methyl (2'-OMe) modified nucleotides, 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, phosphorothioate (PS) linkages between nucleotides, or reverse abasic modified nucleotides.

[0226] Embodiment 187 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-186, 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.

[0227] Embodiment 188 is an engineered cell, cell population, pharmaceutical composition, or method as described in embodiment 187, wherein the 5' end modification comprises at least one PS linkage, and wherein one or more of the following situations are present: i. there is one PS linkage, and the linkage is between the first nucleotide and the second nucleotide; ii. there are two PS linkages between the first three nucleotides; iii. there is a PS linkage between any one or more of the first four nucleotides; and iv. there is a PS linkage between any one or more of the first five nucleotides.

[0228] Embodiment 189 is an engineered cell, cell population, pharmaceutical composition, or method as described in embodiment 187 or 188, wherein the 5' end modification further comprises at least one nucleotide modified with 2'-OMe, 2'-O-moe, inverted abasic, or 2'-F.

[0229] Embodiment 190 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 187-189, wherein the 5' end modification comprises: i. modification of one or more of the first 1-4 nucleotides, wherein the modification is a PS linkage, an inverted abasic nucleotide, 2'-OMe, 2'-O-moe, or 2'-F; ii. modification of the first nucleotide with 2'-OMe, 2'-O-moe, or 2'-F, and optionally one or two modifications to the next nucleotide or the first nucleoside of the 3' tail. PS linkage with 2'-OMe, 2'-O-moe or 2'-F; iii. modification of the first or second nucleotide with 2'-OMe, 2'-O-moe or 2'-F, and optionally one or more PS linkages; iv. modification of the first, second or third nucleotide with 2'-OMe, 2'-O-moe or 2'-F, and optionally one or more PS linkages; or v. modification of the first, second, third or fourth nucleotide with 2'-OMe, 2'-O-moe or 2'-F, and optionally one or more PS linkages.

[0230] Embodiment 191 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 187-180, wherein the 3' end modification comprises at least one PS linkage, and wherein one or more of the following situations are present: i. there is one PS linkage, and the linkage is between the last nucleotide and the second to last nucleotide; ii. there are two PS linkages between the last three nucleotides; and iii. there is a PS linkage between any one or more of the last four nucleotides.

[0231] Embodiment 192 is an engineered cell, cell population, pharmaceutical composition or method as described in embodiment 191, wherein the 3' end modification further comprises at least one nucleotide modified with 2'-OMe, 2'-O-moe, inverted abasic or 2'-F.

[0232] Embodiment 193 is an engineered cell, cell population, pharmaceutical composition or method as described in embodiment 192, wherein the 3' end modification comprises: i. modification of one or more of the last 1-4 nucleotides, wherein the modification is a PS linkage, an inverted abasic nucleotide, 2'-OMe, 2'-O-moe or 2'-F; ii. modification of the last nucleotide with 2'-OMe, 2'-O-moe or 2'-F, and optionally one or two PS linkages to the next nucleotide or the first nucleotide of the 3' tail; iii. modification of the last nucleotide with 2'-OMe, 2'-O-moe or 2'-F, and optionally one or two PS linkages to the next nucleotide or the first nucleotide of the 3' tail; iv ... '-O-moe or 2'-F to the last or second to last nucleotide, and optionally one or more PS linkages; iv. modification of the last, second to last, or third to last nucleotide with 2'-OMe, 2'-O-moe or 2'-F, and optionally one or more PS linkages; or v. modification of the last, second to last, third to last, or fourth to last nucleotide with 2'-OMe, 2'-O-moe or 2'-F, and optionally one or more PS linkages.

[0233] Embodiment 194 is the engineered cell, cell population, pharmaceutical composition or method of embodiment 193, further comprising a 3' tail comprising a 2'-O-Me modified nucleotide.

[0234] Embodiment 195 is an engineered cell, cell population, pharmaceutical composition, or method as described in any of embodiments 1-194, wherein the guide RNA comprises a 5' end modification or a 3' end modification.

[0235] Embodiment 196 is a method or composition as described in any one of embodiments 1-195, wherein the guide RNA comprises:

[0236] a guide sequence, wherein the guide sequence comprises: 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; a shortened repeat / anti-repeat region, wherein nucleotides 38-48 and 53-63 are deleted relative to SEQ ID NO: 900, comprising: 2′-O-Me modified nucleotides at nucleotides 25, 29, 30, 31, 32, 37, 49-52, 64, 65, 69, 70, and 73; a shortened hairpin 1 region, wherein nucleotides 38-48 and 53-63 are deleted relative to SEQ ID NO: 900, comprising: 2′-O-Me modified nucleotides at nucleotides 25, 29, 30, 31, 32, 37, 49-52, 64, 65, 69, 70, and 73; NO:900 has deleted nucleotides 86 and 91, comprises: 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 1 region and the shortened hairpin 2 region; the shortened hairpin 2 region, wherein nucleotides 112-120 and 127-135 are deleted relative to SEQ ID NO:900, comprises: 2'-O-Me modified nucleotides at nucleotides 104, 110, 111, 122-125, 142 and 143, a PS linkage between nucleotides 141-142 and 142-143, wherein one or two of nucleotides 144-145 are optionally deleted relative to SEQ ID NO:900.

[0237] Embodiment 197 is a method or composition as described in any one of embodiments 1-196, wherein the guide RNA comprises modified nucleotides of any one of SEQ ID NOs: 904-909, 911, 995-997, and 1081-1089.

[0238] Embodiment 198 is a method or composition as described in any one of embodiments 1-197, wherein the guide RNA comprises modified nucleotides of SEQ ID NO: 995.

[0239] Embodiment 199 is a method or composition as described in any one of embodiments 1-198, wherein the guide RNA comprises modified nucleotides of SEQ ID NO: 1083.

[0240] Embodiment 200 is a method or composition as described in any one of embodiments 1-199, wherein the guide RNA is modified according to the pattern of any one of SEQ ID NOs: 904-909, 911 and 995-997, wherein each N in the pattern is any natural or non-natural nucleotide, wherein the Ns collectively are any one of the guide sequences of Tables 1-5.

[0241] Embodiment 201 is a method or composition as described in any of embodiments 1-200, wherein the guide RNA is modified according to the pattern of SEQ ID NO: 995, wherein each N in the pattern is any natural or non-natural nucleotide, wherein the Ns collectively are any one of the guide sequences of Tables 1-5.

[0242] Embodiment 202 is a method of administering to a subject in need thereof the engineered cell, cell population, or pharmaceutical composition of any one of embodiments 1-10, 18-28, 42-55, 64-75, 84-92, and 97-195.

[0243] Embodiment 203 is a method of administering to a subject an engineered cell, cell population, or pharmaceutical composition of any one of embodiments 1-10, 18-28, 42-55, 64-75, 84-92, and 97-195 as adoptive cell transfer (ACT) therapy.

[0244] Embodiment 204 is a method of treating a disease or condition comprising administering to a subject in need thereof an engineered cell, cell population, or pharmaceutical composition of any one of embodiments 1-10, 18-28, 42-55, 64-75, 84-92, and 97-195.

[0245] Embodiment 205 is the engineered cell, cell population, pharmaceutical composition, or method of any one of embodiments 1-10, 18-28, 42-55, 64-75, 84-92, and 97-195 for administration to a subject as adoptive cell transfer (ACT) therapy.

[0246] Embodiment 206 is the engineered cell, cell population, pharmaceutical composition, or method of 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.

[0247] Embodiment 207 is the engineered cell, cell population, pharmaceutical composition, or method of 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.

[0248] Embodiment 208 is the engineered cell, cell population, pharmaceutical composition, or method of 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.

[0249] I. Definition

[0250] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings:

[0251] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the terms listed before that term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also includes BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, CBBA, CABA, etc., are expressly included. Unless otherwise apparent from the context, those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination.

[0252] As used herein, the term "kit" refers to a packaged set of related components, such as one or more polynucleotides or compositions and one or more related materials, such as a delivery device (e.g., a syringe), solvents, solutions, buffers, instructions, or desiccant.

[0253] As used herein, "allogeneic" cells refer to cells derived from a donor subject of the same species as the recipient subject, wherein the donor subject and the recipient subject have genetic dissimilarity, e.g., genes that are not identical at one or more loci. Thus, for example, a cell is allogeneic to the subject to which the cell is administered. As used herein, cells that have been removed or isolated from a donor and will not be reintroduced into the original donor are considered allogeneic cells.

[0254] As used herein, "autologous" cells refer to cells that are derived from the same subject into which the material is subsequently reintroduced. Thus, for example, if cells are removed from a subject and then reintroduced into the same subject, the cells are considered autologous.

[0255] As used herein, "CIITA" or "CIITA" or "C2TA" refers to the nucleic acid sequence or protein sequence of "class II major histocompatibility complex transactivator"; the human gene has accession number NC_000016.10 (range 10866208..10941562), reference GRCh38.p13. The CIITA protein in the cell nucleus acts as a positive regulator of MHC class II gene transcription and is required for MHC class II protein expression.

[0256] As used herein, "MHC" or "one or more MHC molecules" or "MHC proteins" or "one or more MHC complexes" refers to a major histocompatibility complex molecule (or plural), and includes, for example, MHC class I and MHC class II molecules. In humans, MHC molecules are referred to as "human leukocyte antigen" complexes or "HLA molecules" or "HLA proteins." The use of the terms "MHC" and "HLA" is not intended to be limiting; 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.

[0257] As used herein in the context of the HLA-A protein, the term "HLA-A" refers to an MHC class I protein molecule, which is a heterodimer composed of a heavy chain (encoded by the HLA-A gene) and a light chain (i.e., beta-2 microglobulin). As used herein in the context of nucleic acids, the term "HLA-A" or "HLA-A gene" refers to the gene that encodes the heavy chain of the HLA-A protein molecule. The HLA-A gene is also known as "HLA class I histocompatibility, A alpha chain"; the human gene has the accession number NC_000006.12 (29942532..29945870). It is known that the HLA-A gene has thousands of different forms (also known as "alleles") in the population (and an individual may have two different alleles of the HLA-A gene). A public database of HLA-A alleles, including 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."

[0258] As used herein in the context of nucleic acids, "HLA-B" refers to the gene encoding the heavy chain of the HLA-B protein molecule. The HLA-B gene is also known as the "HLA class I histocompatibility, Bα chain"; the human gene has accession number NC_000006.12 (31353875..31357179).

[0259] As used herein in the context of nucleic acids, "HLA-C" refers to the gene encoding the heavy chain of the HLA-C protein molecule. The HLA-C gene is also known as the "HLA class I histocompatibility, Cα chain"; the human gene has accession number NC_000006.12 (31268749..31272092).

[0260] As used herein in the context of TRAC proteins, the term "TRAC" 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 region, TCRA, IMD7, TRCA, and TRA are genetic synonyms for TRAC.

[0261] The term "TRBC" (or "TRBC1 / 2") is used to refer to the nucleic acid sequence or amino acid sequence of a "T cell receptor beta chain," such as TRBC1 and TRBC2. As used herein in the context of TRBC proteins, the terms "TRBC1" and "TRBC2" refer to two homologous proteins that comprise the T cell receptor beta chain. As used herein in the context of nucleic acids, "TRBC1" and "TRBC2" refer to the genes encoding the T cell receptor beta chain. The human wild-type TRBC1 sequence is available at: NCBI Gene ID: 28639; Ensembl: ENSG00000211751. T cell receptor beta constant region, 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 region, V_segment translation product and TCRBC2 are gene synonyms of TRBC2.

[0262] As used herein, the term "AAVS1" refers to the genomic location at chr19:50900000-58617616 according to hg38.

[0263] As used herein, the term "within genomic coordinates" includes the boundaries of a given genomic coordinate range. For example, if chr6:29942854-chr6:29942913 is given, the coordinates chr6:29942854-chr6:29942913 are encompassed. Throughout this application, reference to genomic coordinates is based on the genome annotation in the human genome GRCh38 (also known as hg38) assembly from the Genome Reference Consortium, available on the National Center for Biotechnology Information website. 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 corresponding coordinates in another assembly of the human genome, including conversion to an earlier assembly generated by the same institution or using the same algorithm (e.g., from GRCh38 to GRCh37), as well as conversion of 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 at the National Center for Biotechnology Information website; UCSC LiftOver, available at the UCSC Genome Brower website; and Assembly Converter, available at the Ensembl.org website.

[0264] As used herein, the term "homozygous" refers to having two identical alleles of a particular gene.

[0265] As used herein, the term "subject" is intended to include living organisms in which an immune response can be elicited, including, for example, mammals, primates, humans.

[0266] "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds comprising nucleosides or nucleoside analogs having nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. The nucleic acid "backbone" can be composed of a variety of linkages, including one or more of the following: sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA; PCT No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of the nucleic acid can be ribose, deoxyribose, or similar compounds having substitutions (e.g., 2' methoxy or 2' halide substitutions). The nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., modified uridine, such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, etc.); inosine; a derivative of a purine or pyrimidine (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purine, deaza- or aza-pyrimidine, a pyrimidine base having a substituent at the 5- or 6-position (e.g., 5-methylcytosine), a purine base having a substituent at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thiopyrimidine, 4-aminopyrimidine, 4-dimethylhydrazine-pyrimidine and O 4 -alkylpyrimidines; U.S. Patent No. 5,378,825 and PCT No. WO 93 / 13121. For a general discussion, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., eds., 11th ed., 1992. Nucleic acids may include one or more "abasic" residues, in which the backbone does not include a nitrogenous base for one or more positions 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 include both conventional components and substitutions (e.g., conventional bases with 2'-methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), an analog containing one or more LNA nucleotide monomers having a bicyclic furanose unit locked in an RNA-mimicking sugar conformation that enhances 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 analog in RNA and thymine or its analog in DNA.

[0267] "Guide RNA", "gRNA" and "guide" for short are used interchangeably herein and refer to guidance, for example, that guides an RNA-guided DNA binder to a target DNA, and can be a single guide RNA or a combination of crRNA and trRNA (also referred to as tracrRNA). Exemplary gRNAs include modified or unmodified class II Cas nuclease guide RNAs. CrRNA and trRNA can be associated as a single RNA molecule (single guide RNA, sgRNA), or in two separate RNA strands (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. TrRNA can be a naturally occurring sequence, or a trRNA sequence that has modifications or changes compared to a naturally occurring sequence.

[0268] As used herein, "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and acts as a DNA binder guided by RNA to guide the guide RNA to the target sequence for binding or modification (e.g., cleavage). A "guide sequence" may also be referred to as a "targeting sequence" or "spacer sequence". The length of the guide sequence may be 19, 20, 21, 22, 23, 24, or 25 nucleotides, for example, in the case of Neisseria meningitidis. In some embodiments, the Nme Cas9 guide sequence comprises at least 22, 23, or 24 adjacent nucleotides of a sequence selected from SEQ ID NO: 2-80, 101-120, 201, 265, 301, 302, 304-576, or 601-774. In some embodiments, the target sequence is, for example, in a gene or on a chromosome and is complementary to the guide sequence. In some embodiments, the complementarity or degree of 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 24-contiguous nucleotide 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 target region may be 100% complementary or identical. In other embodiments, the guide sequence and target region may contain at least one mismatch, i.e., one nucleotide that is not identical or complementary, depending on the reference sequence. For example, the guide sequence and target sequence may contain 1-2, preferably no more than 1, mismatches, wherein the total length of the target sequence is 19, 20, 21, 22, 23, or 24 or more nucleotides. In some embodiments, the guide sequence and target region may contain 1-2 mismatches, wherein the guide sequence comprises at least 24 nucleotides or more. In some embodiments, the guide sequence and target region may contain 1-2 mismatches, wherein the guide sequence comprises 24 nucleotides. That is, the guide sequence and target region may form a duplex region of 1 base pair or more. In certain embodiments, the duplex region may include 1-2 mismatches, such that the guide strand is not fully complementary to the target sequence. The positions of mismatches are known in the art, as provided, for example, in the following: PAM distal mismatches tend to be better tolerated than PAM proximal matches. Mismatch tolerances at other positions are known in the art (see, for example, Edraki et al., 2019. Mol. Cell, 73: 1-13).

[0269] The target sequence of the RNA-guided DNA binder includes the positive and negative strands of the genomic DNA (i.e., a given sequence and the reverse complement of the sequence), because the nucleic acid substrate of the RNA-guided DNA binder is a double-stranded nucleic acid. Therefore, when the guide sequence is referred to as "complementary to the target sequence", it should be understood that the guide sequence can guide the guide RNA to bind to the reverse complement of the target sequence. Therefore, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., a target sequence that does not include a PAM) except that U replaces T in the guide sequence.

[0270] As used herein, "RNA-guided DNA binder" means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a 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 lyases / nickases and inactivated forms thereof ("dCas DNA binders"). "Cas nucleases" are also referred to as "Cas proteins" and encompass Cas lyases, Cas nickases, and dCas DNA binders. Cas lyases / nickases and dCas DNA binders include Csm or Cmr complexes of type III CRISPR systems, their Cas10, Csm1, or Cmr2 subunits, Cascade complexes of type I CRISPR systems, their Cas3 subunits, and class 2 Cas nucleases.

[0271] As used herein, "Class 2 Cas nucleases" are single-chain polypeptides with RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas lyases / nickases (e.g., H840A, D10A, or N863A variants of Spy Cas9, and D16A and H588A of NmeCas9 (e.g., Nme2 Cas9), which further have RNA-guided DNA lyase or nickase activity; and Class 2 dCas DNA binders, in which the lyase / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9 (1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. The Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated by reference in its entirety. See, for example, Zetsche, Tables S1 and S3. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0272] Several Cas9 heterologs have been obtained from Neisseria meningitidis (Esvelt et al., NAT. METHODS, Vol. 10, 2013, 1116-1121; Hou et al., PNAS, Vol. 110, 2013, pp. 15644-15649) (NmelCas9, Nme2Cas9 and Nme3Cas9). Nme2Cas9 heterologs function efficiently in mammalian cells, recognize N4CCPAM, and can be used for in vivo editing with homologous gRNA (Ran et al., NATURE, Vol. 520, 2015, pp. 186-191; Kim et al., NAT. COMMUN., Vol. 8, 2017, p. 14500). Nme2Cas9 can be specific and selective, for example, capable of low off-target editing (Lee et al., MOL THER., Vol. 24, 2016, pp. 645-654; Kim et al., 2017). See also, for example, WO / 2020081568 (e.g., pp. 28 and 42), which describes the Nme2Cas9 D16A nickase, the contents of which are hereby incorporated by reference in their entirety. Throughout, "NmeCas9" or "Nme Cas9" is general and encompasses any type of NmeCas9, including Nme1Cas9, Nme2Cas9, and Nme3Cas9.

[0273] Exemplary nucleotide and polypeptide sequences of Cas9 molecules are provided in Table 7. Methods for identifying alternative nucleotide sequences encoding Cas9 polypeptide sequences, including alternative naturally occurring variants, are known in the art. Sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any of the Cas9 nucleic acid sequences or nucleic acid sequences encoding the amino acid sequences provided herein are also contemplated.

[0274] As used herein, the term "editor" refers to a factor comprising a polypeptide capable of modifying a DNA sequence. In some embodiments, the editor is a lyase, such as a Cas9 lyase. In some embodiments, the editor is capable of deaminating bases within a DNA molecule, and it 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 an APOBEC3A deaminase (A3A). In some embodiments, the editor comprises a Cas9 nickase fused to an APOBEC3A deaminase (A3A). In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase and a UGI. In some embodiments, the editor lacks UGI.

[0275] As used herein, "cytidine deaminase" means a polypeptide or polypeptide complex capable of having cytidine deaminase activity that catalyzes the hydrolytic deamination of cytidine or deoxycytidine, typically producing uridine or deoxyuridine. Cytidine deaminase encompasses enzymes in the cytidine deaminase superfamily, and in particular enzymes of the APOBEC family (APOBEC 1, APOBEC2, APOBEC4, and APOBEC3 enzyme subgroups), activation-induced cytidine deaminase (AID or AICDA), and CMP deaminase (see, e.g., 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 contemplated. Variants include proteins having sequences that differ from the wild-type protein by one or more mutations (i.e., substitutions, deletions, insertions) (such as one or more single-point substitutions). For example, a shortened sequence can be used, for example, by deleting the N-terminus, the C-terminus, or internal amino acids, preferably one to four amino acids at the C-terminus of the sequence. As used herein, the term "variant" refers to allelic variants, splice variants, and natural or artificial mutants homologous to a reference sequence. Variants are "functional" because they exhibit catalytic activity for DNA editing.

[0276] As used herein, the term "APOBEC3A" refers to a cytidine deaminase, such as a protein expressed by the human A3A gene. APOBEC3A has catalytic DNA editing activity. The amino acid sequence of APOBEC3A has been 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 proteins having a sequence that differs from the wild-type APOBEC3A protein by one or several mutations (i.e., substitutions, deletions, insertions) (such as one or more single-point substitutions). For example, a shortened APOBEC3A sequence can be used, for example by deleting one to four amino acids 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, splice variants, and natural or artificial mutants that are homologous to the APOBEC3A reference sequence. A variant is "functional" because it exhibits catalytic activity for DNA editing. In some embodiments, APOBEC3A (such as human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, APOBEC3A (such as human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).

[0277] As used herein, "nickase" is an enzyme that produces single-strand breaks (also referred to as "nicks") in double-stranded DNA, i.e., cuts one strand of the DNA double helix but does not cut the other strand. As used herein, "RNA-guided DNA nickase" means a polypeptide or polypeptide complex with DNA nickase activity, wherein the DNA nickase activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA nickases include Cas nickases. Class 2 Cas nickases include polypeptides in which the HNH or RuvC catalytic domain is inactivated, such as Cas9 (e.g., H840A, D10A or N863A variants of SpyCas9 or D16A variants of NmeCas9). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domains of Neisseria meningitidis or RuvC or RuvC-like domains include Nme2Cas9 D16A (HNH nickase) and Nme2Cas9 H588A (RuvC nickase). Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9 (1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015) is homologous to Cas9 and contains a RuvC-like protein domain. Zetsche's Cpf1 sequence is incorporated by reference in its entirety. See, e.g., Zetsche, Tables S1 and S3. "Cas9" encompasses Streptococcus pyogenes (SPy) Cas9, variants of Cas9 listed herein, and equivalents thereof. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60: 385-397 (2015).

[0278] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be located at the amino terminal (N-terminal) portion or the carboxyl terminal (C-terminal) protein of the fusion protein, thereby forming an "amino terminal fusion protein" or a "carboxyl terminal fusion protein", respectively. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising peptide linkers. Expression and purification methods for recombinant proteins are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0279] As used herein, the term "linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties. Typically, a linker is located between or on either side of two groups, molecules, or other moieties and is connected to each other via covalent bonds. In some embodiments, the linker is one or more amino acids (e.g., a peptide or protein), such as a 16-amino acid residue "XTEN" linker or a variant thereof (see, e.g., Examples; and Schellenberger et al., A recombinant polypeptide extends the in vivo half-life of peptides and proteins in an unmanageable 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.

[0280] As used herein, the term "uracil glycosylase inhibitor" or "UGI" refers to a protein that is capable of inhibiting the uracil-DNA glycosylase (UDG) base excision repair enzyme.

[0281] 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 the gene. An 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).

[0282] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, e.g., a Cas lyase, a Cas nickase, or a dCas DNA binding agent (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binding agent (such as Cas9) to a target sequence, and the guide RNA hybridizes to the target sequence and the agent binds to the target sequence; where the agent is a lyase or nickase, cleavage or nicking may occur after binding.

[0283] As used herein, a first sequence is said to "comprise a sequence that has at least X% identity to a second sequence" if an alignment of the first sequence with a second sequence shows that X% or more positions throughout the second sequence match the first sequence. For example, the sequence AAGA comprises a sequence that has 100% identity to the sequence AAG because the alignment would give 100% identity due to matches at all three positions of the second sequence. Differences between RNA and DNA (typically the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs (such as modified uridine) do not result in differences in identity or complementarity between polynucleotides as long as the relevant nucleotides (such as thymidine, uridine or modified uridine) have the same complementary sequence (e.g., adenosine for all thymidine, uridine or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as complementary sequences). Thus, for example, the sequence 5'-AXG (wherein X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine) is considered 100% identical to AUG because both are fully 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 skilled in the art will understand which algorithm and parameter settings to choose for a given pair of sequences to be aligned; for sequences of substantially similar length with an expected amino acid identity of >50% or a nucleotide identity of >75%, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI on the www.ebi.ac.uk website server is generally appropriate.

[0284] "Messenger RNA" or "mRNA" is used herein to refer to a polynucleotide and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNAs). The mRNA may comprise one or more modifications as provided below.

[0285] As used herein, " genetic modification " is the change of DNA level, for example, induced by CRISPR / Cas9 gRNA and Cas9 system.Genetic modification can comprise insertion, deletion or replacement (that is, base sequence replacement, i.e. mutation), usually in the defined sequence or genomic locus.Genetic modification changes the nucleic acid sequence of DNA.Genetic modification can be at a single nucleotide position.Genetic modification can be at multiple nucleotides, such as 2, 3, 4, 5 or more nucleotides, usually very close to each other, such as adjacent nucleotides.Genetic modification can be in a coding sequence (such as exon sequence).Genetic modification can be at a splice site, i.e., close enough to a splice acceptor site or a splice donor site to destroy splicing.Genetic modification can include insertion of a nucleotide sequence that is not endogenous to the genomic locus, such as inserting a heterologous open reading frame or the coding sequence of a gene.As used herein, genetic modification can be used to prevent the translation of a full-length protein with the amino acid sequence of an endogenous full-length protein before the genetic modification of the genomic locus.Preventing the translation of endogenous full-length protein or gene product includes preventing the translation of a protein or gene product of any length. The translation of endogenous full-length protein can be stopped, for example, by causing a frame shift mutation resulting in the generation of premature stop codons or by the generation of nonsense mutations. The translation of endogenous full-length protein can be stopped by destroying splicing. The translation of endogenous full-length protein can be stopped by inserting a heterologous coding sequence. For example, when the endogenous full-length protein contains an undesired mutation, the endogenous full-length protein coding sequence can be changed by making changes at one or more positions to provide a modified full-length coding sequence different from the endogenous sequence present in the cell (such as correction of a point mutation) to stop the translation of the endogenous full-length protein. The translation of the endogenous full-length protein can be stopped by changing the splicing of the endogenous full-length protein to produce different proteins by alternative splicing.

[0286] As used herein, "indel" refers to an insertion or deletion mutation consisting of a number of nucleotides that are inserted, deleted, or both inserted and deleted at a double-strand break (DSB) site in a target nucleic acid. As used herein, when indel formation results in an insertion, the insertion is a random insertion at the DSB site and may or may not be directed by or based on a template sequence.

[0287] As used herein, a "heterologous coding sequence" refers to a coding sequence that has been introduced into a cell as an exogenous source (e.g., inserted into a genomic locus (such as a safe harbor locus, including a TCR locus)). That is, the introduced coding sequence is heterologous at least with respect to its insertion site. The polypeptide expressed from this heterologous coding sequence gene is referred to as a "heterologous polypeptide." A heterologous coding sequence may be naturally occurring or engineered, and may be wild type or variant. A heterologous coding sequence may include a nucleotide sequence (e.g., an internal ribosome entry site) that is different from the sequence encoding the heterologous polypeptide. A heterologous coding sequence may be a coding sequence that naturally occurs in the genome as a wild type or variant (e.g., a mutant). For example, although a cell contains a coding sequence of interest (as a wild type or as a variant), the same coding sequence or a variant thereof may be introduced as an exogenous source for expression, for example, at a highly expressed locus. A heterologous coding sequence may also be a coding sequence that is not naturally present in the genome, or a coding sequence that expresses a heterologous polypeptide that is not naturally present in the genome. "Heterologous coding sequence," "exogenous coding sequence," and "transgene" may be used interchangeably. In some embodiments, a heterologous coding sequence or transgene comprises an exogenous nucleic acid sequence, e.g., a nucleic acid sequence that is not endogenous to the recipient cell. In some embodiments, a heterologous coding sequence or transgene comprises an exogenous nucleic acid sequence, e.g., a nucleic acid sequence that does not naturally occur in the recipient cell. For example, a heterologous coding sequence can be heterologous with respect to its insertion site and with respect to its recipient cell.

[0288] As used herein, the expression of "reduction or elimination" of a protein on a cell refers to the partial or complete loss of protein expression relative to unmodified cells. In some embodiments, the surface expression of the protein on the cell is measured by flow cytometry and has a surface expression of "reduction" or "elimination" relative to unmodified cells, as demonstrated by a reduction in fluorescence signal after staining with the same antibody for the protein. Cells with surface expression of a protein that is "reduced" or "eliminated" by flow cytometry relative to unmodified cells can be referred to as being "negative" for the expression of the protein, as demonstrated by a fluorescence signal similar to that of cells stained with an isotype control antibody. "Reduction" or "elimination" of protein expression can be measured by other known techniques of the technology with appropriate controls known to those skilled in the art.

[0289] As used herein, "knock low" refers to, for example, a decrease in the expression of a specific gene product (e.g., protein, mRNA, or both) compared to the expression of an unedited target sequence. The knock low of a protein can be measured by detecting the total cell mass of the protein from a sample (such as a tissue, fluid, or cell colony of interest). It can also be measured by measuring a substitute, marker, or activity for the protein. Methods for measuring mRNA knock low are known and include analyzing mRNA separated from a sample of interest. In some embodiments, "knock low" may refer to some losses of specific gene product expression, such as a reduction in the amount of mRNA transcription or a reduction in the amount of protein expressed by a cell or cell colony (including a colony in vivo, such as a colony found in a tissue).

[0290] As used herein, "knockout" refers to the loss of expression of a particular gene or protein in a cell. Knockout can result in a decrease in expression below the level of detection in an assay. Knockout can be measured by measuring the total cellular amount of the protein in a cell, tissue, or cell population.

[0291] As used herein, "target sequence" or "genomic target sequence" refers to a nucleic acid sequence in a target gene that has complementarity with the guide sequence of a gRNA. The interaction of the target sequence with the guide sequence directs the RNA-guided DNA binding agent to bind within the target sequence and, depending on the activity of the agent, to produce a nick or cleavage within the target sequence.

[0292] As used herein, "treatment" refers to any administration or application of a therapeutic agent to a disease or condition in a subject, and includes inhibiting the disease, arresting its progression, alleviating one or more symptoms of the disease, curing the disease, or preventing one or more symptoms of the disease, including recurrence of symptoms.

[0293] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Although the invention has been described in conjunction with the illustrated embodiments, it should be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the present invention as defined by the appended claims and the included embodiments.

[0294] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, as these may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates and reference to "a cell" includes a plurality of cells, and so forth.

[0295] Numerical ranges are inclusive of the digits defining the range. Measured and measurable values ​​are understood to be approximate, taking into account significant digits and errors associated with the measurements. Furthermore, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and do not limit the teachings.

[0296] Unless otherwise noted in the specification, embodiments in the specification that "comprise" various components are also considered to be "consisting of" or "consisting essentially of" the listed components; embodiments in the specification that "consisting of" various components are also considered to be "comprising" or "consisting essentially of" the listed components; and embodiments in the specification that "consisting essentially of" various components are also considered to be "consisting of" or "comprising" the listed components (this interchangeability does not apply to the use of these terms in the claims). Unless the context clearly indicates otherwise, the term "or" is used in an inclusive sense, that is, equivalent to "and / or".

[0297] The section headings used herein are for organizational purposes only and should not be construed as limiting the intended subject matter in any way. If any material incorporated by reference conflicts with any term defined in this specification or any other explicit content of this specification, the present specification controls. Although the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be apparent to those skilled in the art.

[0298] II. Genetically Modified Cells

[0299] A. Engineered Cell Compositions

[0300] The present disclosure provides engineered cell compositions having reduced or eliminated surface expression of HLA-A, HLA-B, TRAC, TRBC, and / or MHC II classes relative to unmodified cells as disclosed herein. In some embodiments, the engineered cell compositions comprise genetic modifications in HLA-A, HLA-B, TRAC, TRBC, and / or CIITA genes. In some embodiments, the engineered cell compositions comprise genetic modifications in each of HLA-A, HLA-B, and CIITA genes. In some embodiments, the engineered cells are allogeneic cells. In some embodiments, engineered cells with reduced expression of HLA-A, HLA-B, TRAC, TRBC, and / or MHC II classes can be used for adoptive cell transfer therapy. In some embodiments, the engineered cells comprise additional genetic modifications in the genome of the cell to produce cells desired for allogeneic transplantation purposes.

[0301] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least one nucleotide from within genomic coordinates chr6:29942540-29945459. In some embodiments, engineered cells are provided having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least one nucleotide from within a genomic coordinate selected from the group consisting of: chr6:29942891-29942915; chr6:29942609-29942633; chr6:29944266-29944290; chr6:29942889-29942913; chr6:29944471-29944495 and chr6:29944470-29944494.

[0302] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least one nucleotide from within a genomic coordinate selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809.

[0303] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of HLA-A relative to an unmodified cell, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least 5 contiguous nucleotides within the genomic coordinates chr6: 29942540-29945459. In some embodiments, an engineered cell that has reduced or eliminated surface expression of HLA-A relative to an unmodified cell comprises a genetic modification in the HLA-A gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0304] In some embodiments, an engineered cell having reduced or eliminated surface expression of HLA-A relative to an unmodified cell is provided, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least 5 contiguous nucleotides within a genomic coordinate selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495; and chr6: 29944470-29944494. In some embodiments, an engineered cell having reduced or eliminated surface expression of HLA-A relative to an unmodified cell comprises a genetic modification in the HLA-A gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within a genomic coordinate.

[0305] In some embodiments, an engineered cell having reduced or eliminated surface expression of HLA-A relative to an unmodified cell is provided, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least 5 contiguous nucleotides within a genomic coordinate selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809. In some embodiments, an engineered cell having reduced or eliminated surface expression of HLA-A relative to an unmodified cell comprises a genetic modification in the HLA-A gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within a genomic coordinate.

[0306] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates chr6:29942540-29945459.

[0307] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from the group consisting of: chr6:29942891-29942915; chr6:29942609-29942633; chr6:29944266-29944290; chr6:29942889-29942913; chr6:29944471-29944495, and chr6:29944470-29944494.

[0308] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809.

[0309] In some embodiments, the engineered cells having reduced or eliminated surface expression of HLA-A relative to unmodified cells comprise a genetic modification in the HLA-A gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the following genomic coordinates: (a) chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942 In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within the 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.

[0310] In some embodiments, engineered cells are provided that have surface expression of HLA-A reduced or eliminated by a genome editing system that binds to an HLA-A genomic target sequence comprising at least 10 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942 In some embodiments, engineered cells are provided that have surface expression of HLA-A reduced or eliminated by a genome editing system that binds to an HLA-A genomic target sequence comprising at least 5 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis Cas9.

[0311] In some embodiments, engineered cells are provided that have surface expression of HLA-A 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 the group consisting of: (a) chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-299429 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis.

[0312] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRAC relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the modification comprises at least one nucleotide from within genomic coordinates chr14: 22547462-22551621. In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRAC relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the modification comprises at least one nucleotide from within genomic coordinates chr14: 22547505-22551621. In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRAC relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the modification comprises at least one nucleotide from within the following genomic coordinates: chr14: 22547505-22547529; chr14: 22547525-22547549; chr14: 22547674-22547698; chr14: 22550544-22550568 or chr14: 22550574-22550598.

[0313] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRAC relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the modification comprises at least 5 contiguous nucleotides within the genomic coordinates chr14: 22547462-22551621. In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRAC relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the modification comprises at least 5 contiguous nucleotides within the genomic coordinates chr14: 22547505-22551621. In some embodiments, engineered cells that have reduced or eliminated surface expression of TRAC relative to unmodified cells comprise a genetic modification in the TRAC gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0314] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TRAC relative to an unmodified cell, comprising a genetic modification in the TRAC gene, wherein the modification comprises at least 5 contiguous nucleotides within a genomic coordinate selected from the group consisting of: chr14: 22547505-22547529; chr14: 22547525-22547549; chr14: 22547674-22547698; chr14: 22550544-22550568, or chr14: 22550574-22550598. In some embodiments, an engineered cell that has reduced or eliminated surface expression of TRAC relative to an unmodified cell comprises a genetic modification in the TRAC gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within a genomic coordinate.

[0315] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRAC relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the modification comprises 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.

[0316] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRAC relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within the genomic coordinates selected from the group consisting of: chr14: 22547505-22547529; chr14: 22547525-22547549; chr14: 22547674-22547698; chr14: 22550544-22550568 or chr14: 22550574-22550598.

[0317] In some embodiments, the engineered cells having reduced or eliminated surface expression of TRAC relative to unmodified cells comprise a genetic modification in the TRAC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the following genomic coordinates: (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 contiguous nucleotides within the 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.

[0318] In some embodiments, engineered cells are provided that have surface expression of TRAC reduced or eliminated by a genome editing system that binds to a TRAC genomic target sequence, the TRAC genomic target sequence comprising at least 10 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr14: 22547505-22547529; chr14: 22547525-22547549; chr14: 22547674-22547698; chr14: 22550544-22550568 and chr14: 22550574-22550598. In some embodiments, engineered cells are provided that have surface expression of TRAC reduced or eliminated by a genome editing system that binds to a TRAC genomic target sequence, the TRAC genomic target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates. In some embodiments, the TRAC 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis Cas9.

[0319] In some embodiments, engineered cells are provided that have surface expression of TRAC reduced or eliminated by a genome editing system that binds to a TRAC genomic target sequence, the TRAC genomic target sequence comprising at least 5 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (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 the genomic coordinates. In some embodiments, the TRAC 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis.

[0320] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least one nucleotide from within a genomic coordinate selected from the group consisting of: chr7: 142791756-142802543. In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least one nucleotide from within a genomic coordinate selected from the group consisting of: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543.

[0321] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least one nucleotide from within the following genomic coordinates: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 142791761-1 42791785; 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.

[0322] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least one nucleotide from within genomic coordinates selected from the group consisting of: chr7:142792690-142792714; chr7:142802103-142802127 and chr7:142802106-14280213.

[0323] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises 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 that have reduced or eliminated surface expression of TRBC relative to unmodified cells comprise a genetic modification in the TRBC gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates.

[0324] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 142791761- In some embodiments, the engineered cells having reduced or eliminated surface expression of TRBC relative to unmodified cells comprise a genetic modification in the TRBC gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0325] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr7: 142792690-142792714; chr7: 142802103-142802127, and chr7: 142802106-14280213. In some embodiments, engineered cells that have reduced or eliminated surface expression of TRBC relative to unmodified cells comprise a genetic modification in the TRBC gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates.

[0326] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in a TRBC gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from the group consisting of:

[0327] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within the following genomic coordinates: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543.

[0328] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 142792690-142792714; 91761-142791785; 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.

[0329] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of TRBC relative to unmodified cells, comprising a genetic modification in the TRBC gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within the genomic coordinates selected from the group consisting of: chr7: 142792690-142792714; chr7: 142802103-142802127 and chr7: 142802106-14280213.

[0330] In some embodiments, the engineered cells having reduced or eliminated surface expression of TRBC relative to unmodified cells comprise a genetic modification in the TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 142791 In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within the 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.

[0331] In some embodiments, the engineered cells having reduced or eliminated surface expression of TRBC relative to unmodified cells comprise a genetic modification in the TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the following genomic coordinates: chr7: 142792690-142792714; chr7: 142802103-142802127; and chr7: 142802106-14280213. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within the 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.

[0332] In some embodiments, engineered cells are provided that have surface expression of TRBC reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 10 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; 1-142791785: 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 having surface expression of TRBC reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates. In some embodiments, the TRBC 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis Cas9.

[0333] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC by a genome editing system that binds to a TRBC genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates selected from the group consisting of chr7:142792690-142792714; chr7:142802103-142802127; and chr7:142802106-14280213. In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of TRBC 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 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 Neisseria meningitidis Cas9.

[0334] In some embodiments, engineered cells are provided that have surface expression of TRBC reduced or eliminated by a genome editing system that binds to a TRBC genomic target sequence comprising at least 5 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 14279176 In some embodiments, the TRBC genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the TRBC 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis.

[0335] In some embodiments, engineered cells are provided that have surface expression of TRBC 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 the group consisting of: 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 the genomic coordinates. In some embodiments, the TRBC 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis.

[0336] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of MHC class II relative to unmodified cells, comprising a genetic modification in the CIITA locus, wherein the modification comprises at least one nucleotide of the following genomic coordinates: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136.

[0337] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one nucleotide selected from the group consisting of: (a) chr16: 10907504-10907528; chr16: 10907508-10907532; chr16: 10907539-10907559; chr16: 10895658-10895682; chr16: 10895668-10895692; chr16: 10895750-10895774; chr 16: 10895753-10895777; chr16: 10895754-10895778; chr16: 10898684-10898708; chr16: 10901529-10901553; chr16: 10902 121-10902145; chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-10904784; chr16: 10906493-10906 517;chr16:10906515-10906539;chr16:10906631-10906655;chr16:10906636-10906660:chr16:10906643-10906667:chr1 6: 10906770-10906794; chr16: 10906788-10906812; chr16: 10906789-10906813; chr16: 10906816-10906840; chr16: 1090714 8-10907172; chr16:10907254-10907278: chr16:10907331-10907355: chr16:10907477-10907501; chr16:10907497-10907521; chr16:10907503-10907527; and chr16:10907574-10907598; or (b) chr16:10906889-10906913; and chr16:10907504-10907528.

[0338] In some embodiments, cells are provided that have reduced or eliminated MHC expression relative to unmodified cells. Class II surface-expressing engineered cells comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one nucleotide selected from the following genomic coordinates: 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.

[0339] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least 5 contiguous nucleotides within a genomic coordinate selected from: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. In some embodiments, an engineered cell that has reduced or eliminated surface expression of MHC class II relative to an unmodified cell comprises a genetic modification in the CIITA gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within a genomic coordinate.

[0340] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of MHC class II relative to unmodified cells, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: (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: 109 02121-10902145: chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-10904784; chr16: 10906493-109 06517; chr16: 10906515-10906539; chr16: 10906631-10906655; chr16: 10906636-10906660; chr16: 10906643-10906667; ch r16: 10906770-10906794; chr16: 10906788-10906812; chr16: 10906789-10906813; chr16: 10906816-10906840; chr16: 10907 148-10907172; 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, an engineered cell having reduced or eliminated surface expression of MHC class II relative to unmodified cells comprises a genetic modification in the CIITA gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates.

[0341] In some embodiments, cells are provided that have reduced or eliminated MHC expression relative to unmodified cells. Class II surface-expressing engineered cells comprising a genetic modification in the CIITA gene, wherein the modification comprises at least 5 contiguous nucleotides within the following genomic coordinates: 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, an engineered cell having reduced or eliminated surface expression of MHC class II relative to unmodified cells comprises a genetic modification in the CIITA gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within genomic coordinates.

[0342] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of MHC class II relative to unmodified cells, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136.

[0343] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of MHC class II relative to unmodified cells, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from the group consisting of: (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;c hr16: 10902121-10902145; chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-10904784; chr16: 109064 93-10906517; chr16: 10906515-10906539; chr16: 10906631-10906655; chr16: 10906636-10906660; chr16: 10906643-1090666 7; chr16: 10906770-10906794; chr16: 10906788-10906812; chr16: 10906789-10906813; chr16: 10906816-10906840; chr16: 10 907148-10907172; 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.

[0344] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within the genomic coordinates selected from the group consisting of: 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.

[0345] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of MHC class II relative to unmodified cells, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one nucleotide within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136.

[0346] In some embodiments, engineered cells are provided having reduced or eliminated surface expression of MHC class II relative to unmodified cells, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: (a) chr16: 10907504-10907528: chr16: 10907508-10907532: chr16: 10907539-10907559: chr16: 10895658-10895682: chr16: 10895668-10895692: chr16: 10895750-10895774: chr16: 10895757-10907576: chr16: 10895758-109075777: chr16: 10895750-10907577 r16:10895753-10895777:chr16:10895754-10895778:chr16:10898684-10898708:chr16:10901529-10901553:chr16:1090 2121-10902145: chr16: 10902701-10902725: chr16: 10904726-10904750: chr16: 10904760-10904784: chr16: 10906493-1090 6517: chr16: 10906515-10906539: chr16: 10906631-10906655: chr16: 10906636-10906660: chr16: 10906643-10906667: chr 16:10906770-10906794:chr16:10906788-10906812:chr16:10906789-10906813:chr16:10906816-10906840:chr16:109071 48-10907172: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.

[0347] In some embodiments, cells are provided that have reduced or eliminated MHC expression relative to unmodified cells. Class II surface-expressing engineered cells comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one nucleotide within the following genomic coordinates: 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.

[0348] In some embodiments, the engineered cells having reduced or eliminated surface expression of MHC class II relative to unmodified cells comprise a genetic modification in the CIITA gene comprising at least one nucleotide within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136.

[0349] In some embodiments, the engineered cells having reduced or eliminated surface expression of MHC class II relative to unmodified cells comprise a genetic modification in the CIITA gene comprising at least one nucleotide within the following genomic coordinates: 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: 1090212 1-10902145; chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-10904784; chr16: 10906493-109065 17; chr16: 10906515-10906539; chr16: 10906631-10906655; chr16: 10906636-10906660: chr16: 10906643-10906667: chr16 :10906770-10906794;chr16:10906788-10906812;chr16:10906789-10906813;chr16:10906816-10906840:chr16:1090714 8-10907172: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.

[0350] In some embodiments, the cells have reduced or eliminated MHC Class II surface-expressing engineered cells comprise a genetic modification in the CIITA gene comprising at least one nucleotide within the following genomic coordinates: 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.

[0351] In some embodiments, the engineered cells having reduced or eliminated surface expression of MHC class II relative to unmodified cells comprise a genetic modification in the CIITA gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the following genomic coordinates: (a) chr16: 10907504-10907528; chr16: 10907508-10907532; chr16: 10907539-10907559; chr16: 10895658-10907568; chr16: 10895668-10907569; chr16: 10895750-10907528; 895774; chr16: 10895753-10895777; chr16: 10895754-10895778; chr16: 10898684-10898708; chr16: 10901529-10901553; chr 16: 10902121-10902145; chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-10904784; chr16: 1090649 3-10906517; 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: 109 In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within the 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.

[0352] In some embodiments, the engineered cells having reduced or eliminated surface expression of MHC class II relative to unmodified cells comprise a genetic modification in the CIITA gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr16: 10895658-10895682; chr16: 10902701-10902725; chr16: 10906493-10906517; c In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within the 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.

[0353] In some embodiments, engineered cells are provided that have surface expression of MHC class II reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence comprising at least 10 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr16: 10907504-10907528; chr16: 10907508-10907532; chr16: 10907539-10907559; chr16: 10895658-10895682; chr16: 10895668-10895692; chr16: 10895750-10895774; chr16: 10895 753-10895777; chr16: 10895754-10895778; chr16: 10898684-10898708; chr16: 10901529-10901553; chr16: 10902121-109 02145; chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-10904784; chr16: 10906493-10906517; c hr16: 10906515-10906539; chr16: 10906631-10906655; chr16: 10906636-10906660; chr16: 10906643-10906667; chr16: 1 0906770-10906794; 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.

[0354] In some embodiments, engineered cells are provided that have MHC reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence. Class II surface expression, the CIITA genomic target sequence comprises at least 10 contiguous nucleotides within the following genomic coordinates: (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 that have surface expression of MHC class II reduced or eliminated by a genome editing system that is bound to a CIITA genomic target sequence, wherein the CIITA genomic target sequence comprises at least 5 adjacent nucleotides within the genomic coordinates. In some embodiments, the CIITA genomic target sequence comprises at least 15 adjacent nucleotides within the 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 Neisseria meningitidis Cas9.

[0355] In some embodiments, engineered cells are provided that have surface expression of MHC class II reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence comprising at least 5 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr16: 10907504-10907528; chr16: 10907508-10907532; chr16: 10907539-10907559; chr16: 10895658-10895682; chr16: 10895668-10895692; chr16: 10895750-10895774; chr16: 10895750-10907528; 53-10895777; chr16: 10895754-10895778; chr16: 10898684-10898708; chr16: 10901529-10901553; chr16: 10902121-109 02145; chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-10904784; chr16: 10906493-10906517; c hr16: 10906515-10906539; chr16: 10906631-10906655; chr16: 10906636-10906660; chr16: 10906643-10906667; chr16: 1 0906770-10906794; 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.

[0356] In some embodiments, engineered cells are provided that have MHC reduced or eliminated by a genome editing system that binds to a CIITA genomic target sequence. Class II surface expression, the CIITA genomic target sequence comprises at least 5 contiguous nucleotides within the following genomic coordinates: 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 the genomic coordinates. In some embodiments, the CIITA 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis.

[0357] In some embodiments, an engineered cell is provided that comprises a genetic modification in the AAVS1 gene, wherein the modification comprises at least one nucleotide from within genomic coordinates chr19:55115151-55116209.

[0358] In some embodiments, an engineered cell is provided that comprises a genetic modification in the AAVS1 gene, wherein the modification comprises at least one nucleotide from within a genomic coordinate selected from the group consisting of: 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: 55115574-55115575 5115598; chr19: 55115606-55115630; chr19: 55115933-55115957; chr19: 551160 26-55116050; chr19: 55116045-55116069; chr19: 55116084-55116108; chr19: 55 115276-55115300; chr19: 55115509-55115533; chr19: 55115579-55115603; chr19: 55115863-55115887; chr19: 55115906-55115930 or chr19: 55116006-55116030.

[0359] In some embodiments, an engineered cell is provided that comprises a genetic modification in the AAVS1 gene, wherein the modification comprises at least 5 contiguous nucleotides from within genomic coordinates chr19: 55115151-55116209. In some embodiments, an engineered cell comprises a genetic modification in the AAVS1 gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0360] In some embodiments, an engineered cell is provided that comprises a genetic modification in the AAVS1 gene, wherein the modification comprises at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: 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: 55115574-55115575 5115598; chr19: 55115606-55115630; chr19: 55115933-55115957; chr19: 551160 26-55116050; chr19: 55116045-55116069; chr19: 55116084-55116108; chr19: 55 In some embodiments, the engineered cell comprises a genetic modification in the AAVS1 gene, wherein the modification comprises at least 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0361] In some embodiments, engineered cells are provided that comprise a genetic modification in the AAVS1 gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within genomic coordinates chr19:55115151-55116209.

[0362] In some embodiments, an engineered cell is provided that comprises a genetic modification in the AAVS1 gene, wherein the modification comprises at least one C to T substitution or at least one A to G substitution within the genomic coordinates selected from the group consisting of: 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: 5511 5574-55115598; chr19: 55115606-55115630; chr19: 55115933-55115957; chr19: 5 5116026-55116050; 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.

[0363] In some embodiments, an engineered cell is provided that comprises a genetic modification in the AAVS1 gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: 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: 5511 5574-55115598; chr19: 55115606-55115630; chr19: 55115933-55115957; chr19: 5 5116026-55116050; chr19: 55116045-55116069; chr19: 55116084-55116108; chr19 In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within the 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.

[0364] In some embodiments, engineered cells are provided that have a genetic 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 coordinate selected from the group consisting of: chr19: 55115218-55115242; chr19: 55115477-55115501; chr19: 55115504-55115528; chr19: 55115513-55115537; chr19: 55115514-55115538; chr19: 55115517-55115541; chr19: 55115518-55115542; chr19: 55115549-5511555 73; chr19: 55115574-55115598; chr19: 55115606-55115630; chr19: 55115933-551159 57; chr19: 55116026-55116050; chr19: 55116045-55116069; chr19: 55116084-5511610 8; chr19:55115276-55115300; chr19:55115509-55115533; chr19:55115579-55115603; chr19:55115863-55115887; chr19:55115906-55115930 or chr19:55116006-55116030. In some embodiments, engineered cells are provided having a genetic modification in AAVS1 induced by a genome editing system that binds to an AAVS1 genomic target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates. In some embodiments, the AAVS1 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis Cas9.

[0365] In some embodiments, an engineered cell is provided having a genetic 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 coordinate selected from the group consisting of: chr19: 55115218-55115242; chr19: 55115477-55115501; chr19: 55115504-55115528; chr19: 55115513-55115537; chr19: 55115514-55115538; chr19: 55115517-55115541; chr19: 55115518-55115542; chr19: 55115549-5511555 73; chr19: 55115574-55115598; chr19: 55115606-55115630; chr19: 55115933-551159 57; chr19: 55116026-55116050; chr19: 55116045-55116069; chr19: 55116084-5511610 8; chr19:55115276-55115300; chr19:55115509-55115533; chr19:55115579-55115603; chr19:55115863-55115887; chr19:55115906-55115930 or chr19:55116006-55116030. In some embodiments, the AAVS1 genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the AAVS1 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 binder. In some embodiments, the RNA-guided DNA binder comprises a Cas9 protein, such as Neisseria meningitidis.

[0366] In some embodiments, for each given genomic coordinate range, the range may encompass + / - 10 nucleotides on either end of the specified coordinates. For each given genomic coordinate range, the range may encompass + / - 5 nucleotides on either end of the range. For example, if chr16: 10923222-10923242 is given, in some embodiments, the genomic target sequence or genetic modification may fall within chr16: 10923212-10923252.

[0367] In some embodiments, a given range of genomic coordinates may encompass target sequences on both DNA strands (ie, the positive (+) strand and the negative (-) strand).

[0368] Further described herein are genetic modifications in the HLA-A, TRAC, TRBC, CIITA, and AAVS1 genes.

[0369] In some embodiments, the genetic modification in the HLA-A, TRAC, TRBC, CIITA or AAVS1 locus comprises any one or more of an insertion, deletion, substitution or deamination of at least one nucleotide in the target sequence. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of HLA-A relative to an unmodified cell, comprising a genetic modification in the HLA-A gene. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TRAC relative to an unmodified cell, comprising a genetic modification in the TRAC gene. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TRBC1 relative to an unmodified cell, comprising a genetic modification in the TRBC1 gene. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of TRBC2 relative to an unmodified cell, comprising a genetic modification in the TRBC2 gene. In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA gene.

[0370] In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of HLA-A, TRAC, TRBC, or MHC class II relative to unmodified cells, comprising a genetic modification in the HLA-A, TRAC, TRBC, or CIITA genes, wherein the cells further have reduced or eliminated expression of endogenous TCR proteins relative to unmodified cells. In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of HLA-A, TRAC, TRBC, or MHC class II relative to unmodified cells, comprising a genetic modification in the HLA-A, TRAC, TRBC, or CIITA genes, wherein the cells further comprise an exogenous nucleic acid and further have reduced or eliminated expression of endogenous TCR proteins relative to unmodified cells. In some embodiments, engineered cells are provided that have reduced or eliminated surface expression of HLA-A, TRAC, TRBC, or MHC class II relative to unmodified cells, comprising a genetic modification in an HLA-A, TRAC, TRBC, or CIITA gene, wherein the cells further have reduced or eliminated surface expression of MHC class I, and wherein the cells further have reduced or eliminated expression of endogenous TCR proteins relative to unmodified cells.

[0371] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of HLA-A, TRAC, TRBC or MHC class II relative to an unmodified cell, comprising a genetic modification in the HLA-A, TRAC, TRBC or CIITA gene, wherein the cell further comprises an exogenous nucleic acid, and wherein the cell further has reduced or eliminated surface expression of MHC class I, and wherein the cell further has reduced or eliminated expression of endogenous TCR proteins relative to an unmodified cell. In some embodiments, the engineered cell has reduced or eliminated expression of TRAC proteins relative to an unmodified cell. In some embodiments, the engineered cell has reduced or eliminated expression of TRBC proteins relative to an unmodified cell.

[0372] In some embodiments, an engineered cell is provided that has reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA gene, wherein the modification comprises at least one nucleotide of an exon within the genomic coordinates chr16:10902662-chr16:10923285, and wherein the cell further comprises an exogenous nucleic acid, and wherein the cell further has reduced or eliminated surface expression of HLA-A, and wherein the cell further has reduced or eliminated expression of endogenous TCR proteins relative to unmodified cells. In some embodiments, the engineered cell has reduced or eliminated expression of TRAC proteins relative to unmodified cells. In some embodiments, the engineered cell has reduced or eliminated expression of TRBC proteins relative to unmodified cells. In some embodiments, the engineered cell comprises a genetic modification in the HLA-A gene. In some embodiments, the engineered cell comprises a genetic modification that reduces expression of HLA-A proteins on the surface of the engineered cell.

[0373] The engineered cells may be any exemplary cell type disclosed herein. In some embodiments, the engineered cells are immune cells. In some embodiments, the engineered cells are hematopoietic stem cells (HSC). In some embodiments, the engineered cells are induced pluripotent stem cells (iPSC). 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.

[0374] In some embodiments, the engineered cells are granulocytes. In some embodiments, the engineered cells are lymphocytes. In some embodiments, the engineered cells are T cells. In some embodiments, the engineered cells are CD4+ T cells. In some embodiments, the engineered cells are CD8+ T cells. In some embodiments, the engineered cells are memory T cells. In some embodiments, the engineered cells are B cells. In some embodiments, the engineered cells are plasma B cells. In some embodiments, the engineered cells are memory B cells.

[0375] In some embodiments, the disclosure provides a pharmaceutical composition comprising any engineered cell disclosed herein. In some embodiments, the pharmaceutical composition comprises the colony of any engineered cell disclosed herein. In some embodiments, cell colony 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% to surface antigen (for example, HLA-A, MHCII class (HLA-DP, DQ, DR) or endogenous TCR) is negative, as measured by flow cytometry. In some embodiments, engineered cell colony is at least 65% negative, as measured by flow cytometry. In some embodiments, engineered cell colony is at least 70% negative, as measured by flow cytometry. In some embodiments, engineered cell colony 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% endogenous TCR protein negative, as measured by flow cytometry. In some embodiments, the engineered cell population is at least 97% endogenous TCR protein negative, as measured by flow cytometry. In some embodiments, the engineered cell population is at least 98% endogenous TCR protein negative, as measured by flow cytometry. In some embodiments, the engineered cell population is at least 99% endogenous TCR protein negative, as measured by flow cytometry.

[0376] In some embodiments, methods of administering the engineered cells or pharmaceutical compositions disclosed herein to a subject in need thereof are provided. In some embodiments, methods of administering the engineered cells or pharmaceutical compositions disclosed herein to a subject as ACT therapy are provided. In some embodiments, methods of administering the engineered cells or pharmaceutical compositions disclosed herein to a subject as a treatment for cancer are provided. In some embodiments, methods of administering the engineered cells or pharmaceutical compositions disclosed herein to a subject as a treatment for an autoimmune disease are provided. In some embodiments, methods of administering the engineered cells or pharmaceutical compositions disclosed herein to a subject as a treatment for an infectious disease are provided.

[0377] B. Methods and Compositions for Reducing or Eliminating Surface Expression of HLA-A, TRAC, TRBC, and MHC Class II

[0378] The present disclosure provides methods and compositions for reducing or eliminating the surface expression of HLA-A, TRAC, TRBC or MHC class II proteins on cells relative to unmodified cells by genetically modifying HLA-A, TRAC, TRBC or CIITA genes. The resulting genetically modified cells may also be referred to as engineered cells herein. In some embodiments, genetically modified (or engineered) cells may be starting cells that are further genetically modified using the methods or compositions provided herein. In some embodiments, cells are allogeneic cells. In some embodiments, cells with reduced HLA-A, TRAC, TRBC or MHC class II expression can be used for adoptive cell transfer therapy. In some embodiments, the editing of HLA-A, TRAC, TRBC or CIITA genes is combined with additional genetic modifications to produce cells desired for allogeneic transplantation purposes.

[0379] In some embodiments, the method comprises reducing or eliminating surface expression of an HLA-A protein on the surface of a cell, comprising contacting the cell with a composition comprising an HLA-A guide RNA comprising a guide sequence that targets 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 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 Neisseria meningitidis Cas9. In some embodiments, the HLA-A guide RNA is Neisseria 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 an 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., 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 preparing an engineered cell having reduced or eliminated surface expression of HLA-A protein relative to an unmodified cell, the method comprising 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 Neisseria meningitidis Cas9. In some embodiments, the HLA-A guide RNA is Neisseria 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 an 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., engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from SEQ ID NOs: 2-80.

[0380] In some embodiments, the method comprises genetically modifying a cell to reduce or eliminate surface expression of an HLA-A protein, comprising contacting the cell with a composition comprising an HLA-A guide RNA comprising a guide sequence that targets 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 Neisseria meningitidis Cas9. In some embodiments, the HLA-A guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of HLA-A protein on the surface of the cell (ie, engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 2-80.

[0381] In some embodiments, the method comprises genetically modifying HLA-A, comprising contacting a cell with a composition comprising an HLA-A guide RNA comprising a guide sequence that targets 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 Neisseria meningitidis Cas9. In some embodiments, the HLA-A guide RNA is Neisseria 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 APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, 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 SEOID NOs: 2-80.

[0382] In some embodiments, the method comprises inducing a DSB or single-strand break (SSB) in HLA-A, comprising contacting a cell with a composition comprising an HLA-A guide RNA comprising a guide sequence that targets 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 Streptococcus pyogenes Cas9. In some embodiments, the HLA-A guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of HLA-A protein on the surface of the cell (ie, engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 2-80.

[0383] In some embodiments, the method comprises reducing the expression of an HLA-A protein in a cell, comprising delivering a composition to the cell, comprising contacting the cell with a composition comprising an HLA-A guide RNA comprising a guide sequence that targets 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 Neisseria meningitidis Cas9. In some embodiments, the HLA-A guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of HLA-A protein on the surface of the cell (ie, engineered cell) is thereby reduced. In some embodiments, the HLA-A guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 2-80.

[0384] In some embodiments, the method of reducing HLA-A protein expression on the surface of a cell comprises contacting the cell with any one or more 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.

[0385] In some embodiments, a composition is provided comprising an HLA-A guide RNA comprising a guide sequence that targets 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 binder or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the composition comprises an RNA-guided DNA binder, and the binder is Cas9. In some embodiments, the RNA-guided DNA binder is Neisseria meningitidis Cas9. In some embodiments, the HLA-A guide RNA is a Neisseria 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 an APOBEC3A deaminase (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.

[0386] In some embodiments, a composition is provided, comprising: a) an HLA-A guide RNA (gRNA) comprising 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 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 ± 10 nucleotides of the genomic coordinates listed in Table 1; or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides from a sequence of (iv); or vi) a guide sequence 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.

[0387] In some embodiments, a method of making an engineered cell having reduced or eliminated surface expression of an HLA-A protein relative to an unmodified cell is provided, the method comprising contacting the cell with a composition of any embodiment provided herein. In some embodiments, the composition comprises an HLA-A guide RNA comprising a guide sequence of any one of SEQ ID NOs: 1, 13, 55, 61, 66, 70, and 71. In some embodiments, the composition comprises an HLA-A guide RNA comprising a guide sequence of any one of SEQ ID NOs: 13, 55, 61, 66, 70, and 71. In some embodiments, the composition comprises an HLA-A guide RNA comprising a guide sequence of any one of SEQ ID NOs: 13, 17, 55, 61, 66, and 70.

[0388] In some embodiments, a method is provided for reducing surface expression of an HLA-A protein in an engineered cell relative to an unmodified cell, the method comprising contacting the cell with a composition of any embodiment provided herein.

[0389] 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 for an HLA-A genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binder that generates adenosine (A) to guanine (G) conversion for an HLA-A genomic target sequence.

[0390] 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 methods produce compositions comprising engineered cells having reduced HLA-A expression. In some embodiments, the methods produce compositions comprising engineered cells having reduced HLA-A protein expression. In some embodiments, the methods produce compositions comprising engineered cells having reduced HLA-A levels in the cell nucleus. In some embodiments, the methods produce compositions comprising engineered cells expressing truncated forms of HLA-A proteins. In some embodiments, the methods produce compositions comprising engineered cells that do not produce detectable HLA-A proteins. In some embodiments, the engineered cells have reduced HLA-A expression, reduced HLA-A proteins, or reduced HLA-A levels in the cell nucleus compared to unmodified cells. In some embodiments, the engineered cells produced by the methods disclosed herein elicit a reduced response from CD4+ T cells compared to unmodified cells as measured in an in vitro cell culture assay containing CD4+ T cells.

[0391] In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of an HLA-A protein, and wherein the cell comprises a genetic modification comprising at least 5 contiguous nucleotides within the genomic coordinates chr6: 29942540-29945459. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of an HLA-A protein, and wherein the cell comprises a genetic modification comprising at least 10 contiguous nucleotides within the genomic coordinates chr6: 29942540-29945459. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of an HLA-A protein, and wherein the cell comprises a genetic modification comprising at least one C to T substitution or at least one A to G substitution within the genomic coordinates chr6: 29942540-29945459.

[0392] In some embodiments, the method comprises reducing or eliminating surface expression of a TRAC protein on the surface of a cell, comprising contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence that targets 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 Neisseria meningitidis Cas9. In some embodiments, the TRAC guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRAC protein on the surface of the cell (ie, engineered cell) is thereby reduced.In some embodiments, the TRAC guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 101-120.

[0393] In some embodiments, the method comprises preparing an engineered cell having reduced or eliminated surface expression of a TRAC protein relative to an unmodified cell, the method comprising contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence that targets 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 Neisseria meningitidis Cas9. In some embodiments, the TRAC guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of a TRAC protein on the surface of a cell (i.e., an engineered cell) is thereby reduced. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101-120.

[0394] In some embodiments, the method comprises genetically modifying a cell to reduce or eliminate surface expression of a TRAC protein, comprising contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence that targets 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 Neisseria meningitidis Cas9. In some embodiments, the TRAC guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRAC protein on the surface of the cell (ie, engineered cell) is thereby reduced.In some embodiments, the TRAC guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 101-120.

[0395] In some embodiments, the method comprises genetically modifying TRAC, comprising contacting a cell with a composition comprising a TRAC guide RNA comprising a guide sequence that targets 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 Neisseria meningitidis Cas9. In some embodiments, the TRAC guide RNA is Neisseria 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 APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRAC protein on the surface of the cell (ie, engineered cell) is thereby reduced.In some embodiments, the TRAC guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 101-120.

[0396] In some embodiments, the method comprises inducing a DSB or single-strand break (SSB) in TRAC, comprising contacting a cell with a composition comprising a TRAC guide RNA comprising a guide sequence that targets 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 Streptococcus pyogenes Cas9. In some embodiments, the TRAC guide RNA is Neisseria 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 APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRAC protein on the surface of the cell (ie, engineered cell) is thereby reduced.In some embodiments, the TRAC guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 101-120.

[0397] In some embodiments, the method comprises reducing the expression of a TRAC protein in a cell, comprising delivering a composition to the cell, comprising contacting the cell with a composition comprising a TRAC guide RNA comprising a guide sequence that targets 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 Neisseria meningitidis Cas9. In some embodiments, the TRAC guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRAC protein on the surface of the cell (ie, engineered cell) is thereby reduced.In some embodiments, the TRAC guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 101-120.

[0398] In some embodiments, the method of reducing TRAC protein expression on the surface of a cell comprises contacting the cell with any one or more TRAC guide RNAs disclosed herein. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101-120.

[0399] In some embodiments, a composition comprising a TRAC guide RNA is provided, wherein the TRAC guide RNA comprises a guide sequence that targets a genomic target, wherein the genomic target comprises at least 10 contiguous nucleotides within the genomic coordinates chr14: 22547505-22551621 or chr14: 22547462-22551621. In some embodiments, the composition further comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the composition comprises an RNA-guided DNA binder, and the binder is Cas9. In some embodiments, the RNA-guided DNA binder is Neisseria meningitidis Cas9. In some embodiments, the TRAC guide RNA is Neisseria 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 APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 101-120.

[0400] In some embodiments, a composition is provided, comprising: a) a TRAC guide RNA (gRNA) comprising 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 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 ± 10 nucleotides of the genomic coordinates listed in Table 2; or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides from a sequence of (iv); or vi) a guide sequence 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).

[0401] In some embodiments, a method of making an engineered cell having reduced or eliminated surface expression of a TRAC protein relative to an unmodified cell is provided, the method comprising contacting the cell with a composition of any embodiment provided herein. In some embodiments, the composition comprises a TRAC guide RNA comprising a guide sequence of any one of SEQ ID NOs: 101, 102, 103, 105, 107, 109, 111, and 115. In some embodiments, the composition comprises a TRAC guide RNA comprising a guide sequence of any one of SEQ ID NOs: 101, 102, 103, 107, and 111.

[0402] In some embodiments, methods are provided for reducing surface expression of a TRAC protein in an engineered cell relative to an unmodified cell, the method comprising contacting the cell with a composition of any embodiment provided herein.

[0403] 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 for a TRAC genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binder that generates adenosine (A) to guanine (G) conversion for a TRAC genomic target sequence.

[0404] 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 methods produce compositions comprising engineered cells having reduced TRAC expression. In some embodiments, the methods produce compositions comprising engineered cells having reduced TRAC protein expression. In some embodiments, the methods produce compositions comprising engineered cells having reduced TRAC levels in the nucleus. In some embodiments, the methods produce compositions comprising engineered cells expressing a truncated form of TRAC protein. In some embodiments, the methods produce compositions comprising engineered cells that do not produce detectable TRAC protein. In some embodiments, compared to unmodified cells, engineered cells have reduced TRAC expression, reduced TRAC protein, or reduced TRAC levels in the nucleus. In some embodiments, as measured in an in vitro cell culture assay containing CD4+T cells, compared to unmodified cells, the engineered cells produced by the methods disclosed herein induce a reduction response from CD4+T cells.

[0405] In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of a TRAC protein, and wherein the cell comprises a genetic modification comprising at least 5 contiguous nucleotides within the genomic coordinates chr14: 22547505-22551621 or chr14: 22547462-22551621. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of a TRAC protein, and wherein the cell comprises a genetic modification comprising at least 10 contiguous nucleotides within the genomic coordinates chr14: 22547505-22551621 or chr14: 22547462-22551621. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of a TRAC protein, and wherein the cell comprises a genetic modification comprising 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.

[0406] In some embodiments, the method comprises reducing or eliminating surface expression of a TRBC protein on a surface of a cell, comprising 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 coordinate selected from the group consisting of chr7: 142791756-142802543. In some embodiments, the method comprises reducing or eliminating surface expression of a TRBC protein on a surface of a cell, comprising 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 coordinate selected from the group consisting of: (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 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 Neisseria meningitidis Cas9. In some embodiments, the TRBC guide RNA is Neisseria 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 APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the expression of 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.

[0407] In some embodiments, the method comprises preparing an engineered cell having reduced or eliminated surface expression of a TRBC protein relative to an unmodified cell, the method comprising 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 genomic coordinates selected from the group consisting of: (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 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 Neisseria meningitidis Cas9. In some embodiments, the TRBC guide RNA is a Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRBC protein on the surface of the cell (i.e., engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0408] In some embodiments, the method comprises genetically modifying a cell to reduce or eliminate surface expression of a TRBC protein, comprising 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 genomic coordinates selected from the group consisting of: (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 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 Neisseria meningitidis Cas9. In some embodiments, the TRBC guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRBC protein on the surface of the cell (i.e., engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0409] In some embodiments, the method comprises genetically modifying TRBC, comprising contacting a 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 genomic coordinates selected from the group consisting of: (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 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 Neisseria meningitidis Cas9. In some embodiments, the TRBC guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRBC protein on the surface of the cell (i.e., engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0410] In some embodiments, the method comprises inducing a DSB or single-strand break (SSB) in TRBC, comprising 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 coordinate selected from the group consisting of: (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 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 Streptococcus pyogenes Cas9. In some embodiments, the TRBC guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRBC protein on the surface of the cell (i.e., engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0411] In some embodiments, the method comprises reducing expression of a TRBC protein in a cell, comprising delivering a composition to the cell, comprising 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 genomic coordinates selected from the group consisting of: (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 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 Neisseria meningitidis Cas9. In some embodiments, the TRBC guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, expression of TRBC protein on the surface of the cell (i.e., engineered cell) is thereby reduced. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0412] In some embodiments, the method of reducing TRBC protein expression on the surface of a cell comprises contacting the cell with any one or more TRBC guide RNAs disclosed herein. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0413] In some embodiments, a composition comprising a TRBC guide RNA is provided, the TRBC guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates selected from the group consisting of: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543. In some embodiments, the composition further comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the composition comprises an RNA-guided DNA binder, the binder being Cas9. In some embodiments, the RNA-guided DNA binder is Neisseria meningitidis Cas9. In some embodiments, the TRBC guide RNA is Neisseria 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 APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the TRBC guide RNA comprises a guide sequence selected from SEQ ID NOs: 201-265.

[0414] In some embodiments, a composition is provided, comprising: a) a TRBC guide RNA (gRNA) comprising 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 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 ± 10 nucleotides of the genomic coordinates listed in Table 3; or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides from a sequence of (iv); or vi) a guide sequence 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).

[0415] 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 for a TRBC genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binder that generates adenosine (A) to guanine (G) conversion for a TRBC genomic target sequence.

[0416] 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 methods produce compositions comprising engineered cells having reduced TRBC expression. In some embodiments, the methods produce compositions comprising engineered cells having reduced TRBC protein expression. In some embodiments, the methods produce compositions comprising engineered cells having reduced TRBC levels in the cell nucleus. In some embodiments, the methods produce compositions comprising engineered cells expressing a truncated form of TRBC protein. In some embodiments, the methods produce compositions comprising engineered cells that do not produce detectable TRBC protein. In some embodiments, the engineered cells have reduced TRBC expression, reduced TRBC protein, or reduced TRBC levels in the cell nucleus compared to unmodified cells. In some embodiments, the engineered cells produced by the methods disclosed herein induce a reduced response from CD4+ T cells compared to unmodified cells, as measured in an in vitro cell culture assay containing CD4+ T cells.

[0417] In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of a TRBC protein, and wherein the cell comprises a genetic modification comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of a TRBC protein, and wherein the cell comprises a genetic modification comprising at least 10 contiguous nucleotides within genomic coordinates selected from the group consisting of: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of a TRBC protein, and wherein the cell comprises a genetic modification comprising at least one C to T substitution or at least one A to G substitution within genomic coordinates selected from the group consisting of: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543.

[0418] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of TRBC relative to an unmodified cell, the engineered cell comprising a genetic modification in a TRBC gene, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 1427917 61-142791785; 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.

[0419] In some embodiments, an engineered cell is provided having reduced or eliminated surface expression of TRBC relative to an unmodified cell, the engineered cell comprising a genetic modification in the TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 1 42791761-142791785; 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.

[0420] In some embodiments, a method of making an engineered cell having reduced or eliminated surface expression of a TRBC protein relative to an unmodified cell is provided, the method comprising contacting the cell with a composition of any embodiment provided herein. In some embodiments, the composition comprises a TRBC guide RNA comprising a guide sequence of any one 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 a guide sequence of any one of SEQ ID NOs: 215, 216, 224, 229, 246, 259, and 260. In some embodiments, the composition comprises a TRBC guide RNA comprising a guide sequence of any one of SEQ ID NOs: 215, 216, 224, 229, 246, 259, and 260.

[0421] In some embodiments, the method comprises reducing or eliminating surface expression of MHC class II proteins on the surface of a cell, comprising contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. 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 Neisseria meningitidis Cas9. In some embodiments, the CIITA guide RNA is Neisseria 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 an 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., 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.

[0422] In some embodiments, the method comprises preparing an engineered cell having reduced or eliminated surface expression of an MHC class II protein relative to an unmodified cell, the method comprising contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. 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 Neisseria meningitidis Cas9. In some embodiments, the CIITA guide RNA is Neisseria 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 an 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., 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.

[0423] In some embodiments, the method comprises genetically modifying a cell to reduce or eliminate surface expression of an MHC class II protein, comprising contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. 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 Neisseria meningitidis Cas9. In some embodiments, the CIITA guide RNA is Neisseria 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 an 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., 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.

[0424] In some embodiments, the method comprises genetically modifying CIITA, comprising contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. 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 Neisseria meningitidis Cas9. In some embodiments, the CIITA guide RNA is Neisseria 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 an 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., 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.

[0425] In some embodiments, the method comprises inducing a DSB or single-strand break (SSB) in CIITA, comprising 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 the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. 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 Streptococcus pyogenes Cas9. In some embodiments, the CIITA guide RNA is Neisseria 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 an 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., 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.

[0426] In some embodiments, the method comprises reducing the expression of CIITA protein in a cell, comprising delivering a composition to the cell, comprising contacting the cell with a composition comprising a CIITA guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. 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 Neisseria meningitidis Cas9. In some embodiments, the CIITA guide RNA is Neisseria 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 an 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., 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.

[0427] In some embodiments, the method of reducing the expression of MHC class II protein on the surface of a cell comprises contacting the cell with any one or more 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.

[0428] In some embodiments, a composition comprising a CIITA guide RNA is provided, the CIITA guide RNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. In some embodiments, the composition further comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the composition comprises an RNA-guided DNA binder, the binder being Cas9. In some embodiments, the RNA-guided DNA binder is Neisseria meningitidis Cas9. In some embodiments, the CIITA guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the CIITA guide RNA comprises a guide sequence selected from SEQ ID NOs: 301, 302, 304-576.

[0429] In some embodiments, a composition is provided, comprising: a) a CIITA guide RNA (gRNA) comprising 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 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 ± 10 nucleotides of the genomic coordinates listed in Table 4; or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides from a sequence of (iv); or vi) a guide sequence 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).

[0430] In some embodiments, a method of making an engineered cell having reduced or eliminated surface expression of an MHC class II protein relative to an unmodified cell is provided, the method comprising contacting the cell with a composition of any embodiment provided herein. In some embodiments, the composition comprises a CIITA guide RNA comprising a guide sequence of any one 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 a guide sequence of any one of SEQ ID NOs: 301, 302, 320, 372, 414, 419, 422, and 462-463.

[0431] 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 for a CIITA genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binder that generates adenosine (A) to guanine (G) conversion for a CIITA genomic target sequence.

[0432] In some embodiments, an engineered cell produced by the methods described herein is provided. In some embodiments, the engineered cell produced by the methods and compositions described herein is an allogeneic cell. In some embodiments, the method produces a composition comprising an engineered cell, and the engineered cell has reduced MHC class II expression. In some embodiments, the method produces a composition comprising an engineered cell, and the engineered cell has reduced CIITA protein expression. In some embodiments, the method produces a composition comprising an engineered cell, and the engineered cell has reduced CIITA levels in the cell nucleus. In some embodiments, the method produces a composition comprising an engineered cell, and the engineered cell expresses a truncated form of CIITA protein. In some embodiments, the method produces a composition comprising an engineered cell, and the engineered cell does not produce detectable CIITA protein. In some embodiments, compared to unmodified cells, the engineered cell has reduced MHC class II expression, reduced CIITA protein, or reduced CIITA levels in the cell nucleus. In some embodiments, the engineered cells produced by the methods disclosed herein elicit a reduced response from CD4+ T cells compared to unmodified cells as measured in an in vitro cell culture assay containing CD4+ T cells.

[0433] In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of an MHC class II protein, and wherein the cell comprises a genetic modification comprising at least 5 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of an MHC class II protein, and wherein the cell comprises a genetic modification comprising at least 10 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell has reduced or eliminated surface expression of an MHC class II protein, and wherein the cell comprises a genetic modification comprising at least one C to T substitution or at least one A to G substitution within the genomic coordinates selected from the group consisting of: (a) chr16: 10877363-10907788 or (b) chr16: 10906515-10908136.

[0434] In some embodiments, the method includes preparing an engineered cell, comprising contacting the cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence that targets 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 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 Neisseria meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0435] In some embodiments, the method comprises genetically modifying a cell, comprising contacting the cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence that targets 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 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 Neisseria meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0436] In some embodiments, the method comprises genetically modifying AAVS1, comprising contacting a cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence that targets 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 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 Neisseria meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0437] In some embodiments, the method comprises inducing a DSB or single-strand break (SSB) in AAVS1, comprising contacting a cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence that targets 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 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 Streptococcus pyogenes Cas9. In some embodiments, the AAVS1 guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 601-774.

[0438] In some embodiments, the method comprises delivering a composition to a cell, comprising contacting the cell with a composition comprising an AAVS1 guide RNA comprising a guide sequence that targets 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 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 Neisseria meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0439] In some embodiments, a method of genetically modifying an AAVS1 gene comprises contacting a cell with any one or more AAVS1 guide RNAs disclosed herein. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 601-774.

[0440] In some embodiments, a composition comprising an AAVS1 guide RNA is provided, the AAVS1 guide RNA comprising a guide sequence that targets 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 binder or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the composition comprises an RNA-guided DNA binder, the binder being Cas9. In some embodiments, the RNA-guided DNA binder is Neisseria meningitidis Cas9. In some embodiments, the AAVS1 guide RNA is a Neisseria 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 an APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the AAVS1 guide RNA comprises a guide sequence selected from SEQ ID NOs: 601-774.

[0441] In some embodiments, a composition is provided, comprising: a) an AAVS1 guide RNA (gRNA) comprising i) a guide sequence selected from SEQ ID NOs: 601-774; or ii) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 601-774; or iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 601-774; or iv) a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 5; or v) at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides from a sequence of (iv); or vi) a guide sequence 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.

[0442] In some embodiments, a method of making an engineered cell is provided, the method comprising contacting the cell with a composition of any embodiment provided herein. In some embodiments, the composition comprises an AAVS1 guide RNA comprising a guide sequence from any of the following: SEQ ID NO: 611, 620, 622, 626, 627, 628, 629, 632, 633, 634, 656, 659, 660, 661, 673, 691, 692, 730, 734, and 746.

[0443] 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 for the AAVS1 genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binder that generates adenosine (A) to guanine (G) conversion for the AAVS1 genomic target sequence.

[0444] 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 reduced response from CD4+ T cells compared to unmodified cells as measured in an in vitro cell culture assay containing CD4+ T cells.

[0445] In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell comprises a genetic modification comprising at least 5 contiguous nucleotides within the genomic coordinates chr19: 55115151-55116209. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell comprises a genetic modification comprising at least 10 contiguous nucleotides within the genomic coordinates chr19: 55115151-55116209. In some embodiments, an engineered cell produced by a method or composition disclosed herein is provided, wherein the cell comprises a genetic modification comprising at least one C to T substitution or at least one A to G substitution within the genomic coordinates chr19: 55115151-55116209. In some embodiments, the composition disclosed herein further comprises a pharmaceutically acceptable carrier. In some embodiments, a cell produced by a composition disclosed herein comprising a pharmaceutically acceptable carrier is provided. In some embodiments, a composition comprising a cell disclosed herein is provided.

[0446] C. HLA-A guide RNA

[0447] The methods and compositions provided herein disclose HLA-A guide RNAs that can be used to reduce HLA-A protein expression on the surface of a cell. In some embodiments, such guide RNAs guide an RNA-guided DNA binder 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 RNAs guide an RNA-guided DNA binder 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.

[0448] In some embodiments, the methods and compositions disclosed herein comprise 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 comprise an HLA-A guide RNA comprising a guide sequence that targets an HLA-A genomic target sequence comprising at least one nucleotide within genomic coordinates chr6: 29942540-29945459.

[0449] In some embodiments, the methods and compositions disclosed herein comprise an HLA-A guide RNA comprising a guide sequence that guides an RNA-directed DNA-binding agent to induce a double-strand break (DSB) or a single-strand break (SSB) in the HLA-A gene, wherein the HLA-A guide RNA targets an HLA-A genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates chr6: 29942540-29945459. In some embodiments, the methods and compositions disclosed herein comprise an HLA-A guide RNA comprising a guide sequence that guides an RNA-directed DNA-binding agent to induce a double-strand break (DSB) or a single-strand break (SSB) in the HLA-A gene, wherein the HLA-A guide RNA targets an HLA-A genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates chr6: 29942540-29945459.

[0450] In some embodiments, the methods and compositions disclose an HLA-A guide RNA that guides an RNA-guided DNA binder to induce a double-strand break (DSB) or a single-strand break (SSB) in an HLA-A genomic target sequence. In some embodiments, the methods and compositions disclose an HLA-A guide RNA that guides an RNA-guided DNA binder to cleave an HLA-A genomic target sequence. In embodiments where the RNA-guided DNA cleavage agent is Cas9, cleavage occurs at the third base of a protospacer adjacent motif (PAM) sequence.

[0451] In some embodiments, compositions are provided that include an HLA-A guide RNA described herein and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0452] In some embodiments, a composition is provided comprising an HLA-A single guide RNA (sgRNA) comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6: 29942540-29945459. In some embodiments, a composition is provided comprising an HLA-A sgRNA described herein and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0453] In some embodiments, a composition is provided comprising an HLA-A dual-guide RNA (dgRNA), wherein the HLA-AdgRNA comprises a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr6: 29942540-29945459. In some embodiments, a composition is provided comprising an HLA-AdgRNA described herein and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0454] In some embodiments, the TRAC gRNA comprises a guide sequence selected from any one of SEQ ID NOs: 2-80. Exemplary HLA-A guide sequences and corresponding guide RNA sequences 2-80 are shown below in Table 1 (SEQ ID NOs: 2-80).

[0455] Table 1. Exemplary HLA-A guide sequences

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473] Throughout this application, the terms "mA," "mC," "mU," or "mG" may be used to denote a nucleotide that has been modified with 2'-O-Me. Throughout this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3') nucleotide using a phosphorothioate (PS) bond.

[0474] 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 contiguous 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.

[0475] In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising at least 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 1. As used herein, at least 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates means, for example, at least 10 contiguous nucleotides within the genomic coordinates, wherein 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, an HLA-A guide RNA may comprise 10 contiguous nucleotides within a genomic coordinate selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495 and chr6: 29944470-29944494, including the border nucleotides of these ranges. In some embodiments, an HLA-A guide RNA comprises a guide sequence that is at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 1. 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 at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 1.

[0476] In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising at least 15 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 1. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising at least 24 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 1.

[0477] 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.

[0478] In some embodiments, the HLA-A guide RNA comprises the guide sequence of any one of the following: SEQ ID NOs: 13, 55, 61, 66, and 70-71.

[0479] In some embodiments, the HLA-A guide RNA comprises a sequence listed in Table 1. In some embodiments, the HLA-A guide RNA comprises a sequence of any one of SEQ ID NOs: 2-80. In some embodiments, the HLA-A guide RNA comprises a sequence of SEQ ID NOs: 61 or 66. In some embodiments, the HLA-A guide RNA comprises a sequence of SEQ ID NO: 61. In some embodiments, the HLA-A guide RNA comprises a sequence of SEQ ID NO: 61. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising a sequence of any one of SEQ ID NOs: 2-80. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising a sequence of SEQ ID NOs: 61 or 66. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising a sequence of SEQ ID NO: 61. In some embodiments, the HLA-A guide RNA comprises a guide sequence comprising a sequence of SEQ ID NO: 66. In some embodiments, the HLA-A guide RNA comprises the sequence of any one of SEQ ID NOs: 1002-1080. In some embodiments, the HLA-A guide RNA comprises the sequence of SEQ ID NOs: 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 the sequence of any one of SEQ ID NOs: 2002-2080, 3001, and 3002. In some embodiments, the HLA-A guide RNA comprises the sequence of any one 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.

[0480] In some embodiments, the HLA-A guide RNA is a single guide RNA (sgRNA) comprising the sequence of any one of the sgRNA sequences listed in Table 1.

[0481] Additional embodiments of HLA-A guide RNAs are provided herein, including, for example, exemplary modifications to the guide RNA.

[0482] 1. Genetic modification of HLA-A

[0483] In some embodiments, the methods and compositions disclosed herein are genetically modified to at least one nucleotide of the HLA-A gene in the cell. In some embodiments, the genetic modification of HLA-A reduces or eliminates the expression of HLA-A proteins on the surface of genetically modified cells (or engineered cells). Genetic modification encompasses modification populations produced by contact with genome editing systems (e.g., editor populations produced by Cas9 and HLA-A guide RNA, or editor populations produced by BC22 and HLA-A guide RNA).

[0484] In some embodiments, the genetic modification comprises at least one nucleotide within genomic coordinates chr6: 29942540-29945459. In some embodiments, the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any of the genomic coordinates listed in Table 1.

[0485] In some embodiments, the genetic modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495, and chr6: 29944470-29944494.

[0486] In some embodiments, the genetic modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809.

[0487] In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495, and chr6: 29944470-29944494. In some embodiments, the genetic modification comprises at least 10 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944470-29944494. In some embodiments, the genetic modification comprises at least one insertion / deletion, at least one C to T substitution, or at least one A to G substitution within the genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944470-29944494.

[0488] In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: 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 10 contiguous nucleotides within genomic coordinates selected from the group consisting of: 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 one insertion / deletion, at least one C to T substitution, or at least one A to G substitution within the genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809.

[0489] In some embodiments, the modification of HLA-A comprises any one or more of an insertion, deletion, substitution, or deamination of at least one nucleotide in the target sequence. In some embodiments, the modification of HLA-A comprises an insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification of HLA-A comprises a deletion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In other embodiments, the modification of HLA-A comprises an 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 of HLA-A comprises a 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 of HLA-A comprises an insertion / deletion, which is generally defined in the art as an insertion or deletion of less than 1000 base pairs (bp). In some embodiments, the modification of HLA-A comprises an insertion / deletion resulting in a frameshift mutation in the target sequence. In some embodiments, the modification of HLA-A comprises a 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 of HLA-A comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from incorporation into a template nucleic acid. In some embodiments, the modification of HLA-A is not transient.

[0490] In some embodiments, the methods and compositions disclosed herein use an RNA-guided DNA binder (e.g., a Cas enzyme) to modify the HLA-A gene in a cell. In some embodiments, the RNA-guided DNA binder is Cas9. In some embodiments, the RNA-guided DNA binder cuts within the HLA-A gene, wherein the HLA-A guide RNA targets an HLA-A genomic target sequence comprising at least 10 contiguous nucleotides within genomic coordinates chr6: 29942540-29945459.

[0491] In some embodiments, the genetic modification of HLA-A results in the utilization of an out-of-frame stop codon. In some embodiments, the genetic modification of HLA-A results in exon skipping during splicing. In some embodiments, the genetic modification of HLA-A results in reduced HLA-A protein expression in cells. In some embodiments, the modification of HLA-A results in reduced or eliminated HLA-A protein expression on the cell surface.

[0492] In some embodiments, HLA-A expression on the cell surface is reduced due to genetic modification of HLA- A. In some embodiments, HLA-A expression on the cell surface is absent due to genetic modification of HLA-A.

[0493] 2. Efficacy of HLA-A guide RNA

[0494] The efficacy of the HLA-A guide RNA can be determined by techniques available in the art that assess the editing efficiency of the guide RNA, the level of HLA-A mRNA, or the level of HLA-A protein in the target cell. In some embodiments, the reduction or elimination of HLA-A protein on the cell surface can be determined by comparison with unmodified cells (or "relative to unmodified cells"). Engineered cells or cell populations can also be compared to unmodified cell populations.

[0495] An "unmodified cell" (or "unmodified cells") refers to a control cell (or cells) of the same cell type in an experiment or test, wherein the "unmodified" control cell has not been contacted with an HLA-A guide (i.e., an unengineered cell). Thus, an unmodified cell (or cells) can be a cell that has not been contacted with a guide RNA, or a cell that has been contacted with a guide RNA that does not target HLA-A.

[0496] In some embodiments, the efficacy of HLA-A guide RNA is determined by measuring the reduction or elimination of HLA-A proteins on the target cell surface. In some embodiments, HLA-A protein expression is measured by flow cytometry (for example, using antibodies for HLA-A2 / HLA-A3). In some embodiments, the cell colony is enriched (for example, by FACS or MACS) relative to an unmodified cell colony and is at least 65%, 70%, 80%, 90%, 91%, 92%, 93% or 94% HLA-A negative as measured by flow cytometry. In some embodiments, the cell colony is not enriched (for example, by FACS or MACS) relative to an unmodified cell colony and is at least 65%, 70%, 80%, 90%, 91%, 92%, 93% or 94% HLA-A negative as measured by flow cytometry.

[0497] In some embodiments, the cell population is at least 65% HLA-A negative relative to an unmodified cell population as measured by flow cytometry. In some embodiments, the cell population is at least 70% HLA-A negative relative to an unmodified cell population as measured by flow cytometry. In some embodiments, the cell population is at least 80% HLA-A negative relative to an unmodified cell population as measured by flow cytometry. In some embodiments, the cell population is at least 90% HLA-A negative relative to an unmodified cell population as measured by flow cytometry. In some embodiments, the cell population is at least 91% HLA-A negative relative to an unmodified cell population as measured by flow cytometry. In some embodiments, the cell population is at least 92% HLA-A negative relative to an unmodified cell population as measured by flow cytometry. In some embodiments, the cell population is at least 93% HLA-A negative relative to an unmodified cell population as measured by flow cytometry. In some embodiments, the cell population is at least 94% HLA-A negative relative to an unmodified cell population as measured by flow cytometry.

[0498] In some embodiments, effective HLA-A guide RNA can be determined by measuring the response of immune cells (e.g., CD8+ T cells) to genetically modified target cells in vitro or in vivo. For example, a reduced response from CD8+ T cells indicates an effective HLA-A guide RNA. CD8+ T cell responses can be evaluated by measuring CD8+ T cell activation responses (e.g., CD8+ T cell proliferation, expression of activation markers, and / or cytokine production (IL-2, IFN-γ, TNF-α)) analysis (e.g., flow cytometry, ELISA). CD8+ T cell responses can be assessed in vitro or in vivo. In some embodiments, CD8+ T cell responses 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 (e.g., a rodent model). In an in vivo model, for example, genetically modified cells can be administered together with CD8+ T cells; the survival of 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 for greater than 1, 2, 3, 4, 5, or 6 weeks or longer in the presence of CD8+T cells. In some embodiments, the method produces a composition comprising cells that survive in vivo for at least one to six weeks in the presence of CD8+T cells. In some embodiments, the method produces a composition comprising cells that survive in vivo for at least two to four weeks in the presence of CD8+T cells. In some embodiments, the method produces a composition comprising cells that survive in vivo for at least four to six weeks in the presence of CD8+T cells. In some embodiments, the method produces a composition comprising cells that survive in vivo for more than six weeks in the presence of CD8+T cells.

[0499] The efficacy of the HLA-A guide RNA can also be assessed by the survival of the cells after editing. In some embodiments, the cells survive for at least one to six weeks after editing. In some embodiments, the cells survive for at least two weeks after editing. In some embodiments, the cells survive for at least three weeks after editing. In some embodiments, the cells survive for at least four weeks after editing. In some embodiments, the cells survive for at least five weeks after editing. In some embodiments, the cells survive for at least six weeks after editing. In some embodiments, the cells survive for at least twelve weeks after editing. Standard techniques can be used (including, for example, by measurement of cell death, by flow cytometry live / dead staining, or cell proliferation) to measure the viability of genetically modified cells.

[0500] D.TRAC guide RNA

[0501] The methods and compositions provided herein disclose TRAC guide RNAs that can be used to reduce TRAC protein expression on the surface of a cell. In some embodiments, such guide RNAs guide an RNA-guided DNA binder to a TRAC genomic target sequence and may be referred to herein as "TRAC guide RNAs." In some embodiments, the TRAC guide RNAs guide an RNA-guided DNA binder to a human TRAC genomic target sequence. In some embodiments, the TRAC guide RNA comprises a guide sequence selected from SEQ ID NOs: 101-120.

[0502] 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 one nucleotide within genomic coordinates chr14: 22547505-22551621 or chr14: 22547462-22551621.

[0503] In some embodiments, the methods and compositions disclosed herein comprise a TRAC guide RNA comprising a guide sequence that guides an RNA-guided DNA-binding agent to induce a double-strand break (DSB) or a single-strand break (SSB) in a TRAC gene, wherein the TRAC guide RNA targets a TRAC genomic target sequence comprising at least 10 contiguous 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 guides an RNA-guided DNA-binding agent to induce a double-strand break (DSB) or a single-strand break (SSB) in a TRAC gene, wherein the TRAC guide RNA targets a TRAC genomic target sequence comprising at least one nucleotide within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621.

[0504] In some embodiments, the methods and compositions disclose a TRAC guide RNA that guides an RNA-guided DNA binder to induce a double-strand break (DSB) or a single-strand break (SSB) in a TRAC genomic target sequence. In some embodiments, the methods and compositions disclose a TRAC guide RNA that guides an RNA-guided DNA binder to cleave in a 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 of the protospacer adjacent motif (PAM) sequence.

[0505] In some embodiments, compositions are provided that include a TRAC guide RNA described herein and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0506] In some embodiments, a composition is provided comprising a TRAC single guide RNA (sgRNA), the TRAC sgRNA comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621. In some embodiments, a composition is provided comprising a TRAC sgRNA described herein and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0507] In some embodiments, a composition is provided comprising a TRAC dual-guide RNA (dgRNA) comprising a guide sequence that targets a genomic target comprising at least 10 contiguous nucleotides within genomic coordinates chr14: 22547505-22551621 or chr14: 22547462-22551621. In some embodiments, a composition is provided comprising a TRAC dgRNA described herein and an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0508] In some embodiments, the TRAC gRNA comprises a guide sequence selected from any one of SEQ ID NOs: 101- 120. Exemplary TRAC guide sequences and corresponding guide RNA sequences 101-120 are shown below in Table 2 (SEQ ID NOs: 101-120).

[0509] Table 2. Exemplary TRAC guide sequences.

[0510]

[0511]

[0512]

[0513]

[0514]

[0515] Throughout this application, the terms "mA," "mC," "mU," or "mG" may be used to denote a nucleotide that has been modified with 2'-O-Me. Throughout this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3') nucleotide using a phosphorothioate (PS) bond.

[0516] In some embodiments, the TRAC guide RNA comprises a guide sequence selected from the group consisting of 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 contiguous nucleotides of a sequence selected from the group consisting of 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 the group consisting of 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 the group consisting of SEQ ID NOs: 101-120.

[0517] In some embodiments, the TRAC guide RNA comprises a guide sequence comprising at least 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2. As used herein, at least 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates means, for example, at least 10 contiguous nucleotides within the genomic coordinates, wherein 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 can comprise 10 contiguous nucleotides within the genomic coordinates selected from the group consisting of: 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 can comprise 10 contiguous nucleotides within a genomic coordinate selected from the group consisting of: 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 contiguous nucleotides of a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2. 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 at least 19, 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence comprising 10 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2.

[0518] 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.

[0519] 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.

[0520] In some embodiments, the TRAC guide RNA comprises nucleotides selected from SEQ ID NOs: 101-103, 107, 111, 117, or 118.

[0521] In some embodiments, the TRAC guide RNA comprises a sequence listed in Table 2. In some embodiments, the TRAC guide RNA comprises the sequence of any one of SEQ ID NOs: 101-120. In some embodiments, the TRAC guide RNA comprises the sequence of any one of SEQ ID NOs: 107, 111, and 117-120. 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 the sequence of any one of SEQ ID NOs: 101-120. In some embodiments, the TRAC guide RNA comprises a guide sequence comprising the sequence of any one of SEQ ID NOs: 107, 111, and 117-120. 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 the sequence of any one of SEQ ID NOs: 1101-1120. In some embodiments, the TRAC guide RNA comprises the sequence of any one of SEQ ID NOs: 1107, 1111, and 1117-1120. 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 the sequence of any one of SEQ ID NOs: 2101-2120, 3003, and 3004. In some embodiments, the TRAC guide RNA comprises the sequence of any one 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.

[0522] In some embodiments, the TRAC guide RNA is a single guide RNA (sgRNA) comprising the sequence of any one of the sgRNA sequences listed in Table 2.

[0523] Additional embodiments of TRAC guide RNAs are provided herein, including, for example, exemplary modifications to the guide RNA.

[0524] 1. Genetic modification of TRAC

[0525] In some embodiments, the methods and compositions disclosed herein are genetically modified to at least one nucleotide of a TRAC gene in a cell. In some embodiments, the genetic modification of TRAC reduces or eliminates the expression of TRAC protein on the surface of a genetically modified cell (or engineered cell). Genetic modification encompasses a modified population produced by contact with a genome editing system (e.g., an editor population produced by Cas9 and TRAC guide RNA, or an editor population produced by BC22 and TRAC guide RNA).

[0526] In some embodiments, the genetic modification comprises at least one nucleotide within genomic coordinates chr14: 22547505-22551621 or chr14: 22547462-22551621. In some embodiments, the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any of the genomic coordinates listed in Table 2.

[0527] In some embodiments, the genetic modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: 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 contiguous nucleotides within genomic coordinates selected from the group consisting of: 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 contiguous nucleotides within genomic coordinates selected from the group consisting of: 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 contiguous nucleotides within genomic coordinates selected from the group consisting of: chr14: 22547481-22547505; chr14: 22547471-22547495; chr14: 22547470-22547494; or chr14: 22547462-22547486.

[0528] In some embodiments, the genetic modification comprises at least one insertion / deletion, at least one C to T substitution, or at least one A to G substitution within genomic coordinates selected from the group consisting of: 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 insertion / deletion, at least one C to T substitution, or at least one A to G substitution within genomic coordinates selected from the group consisting of: chr14: 22547481-22547505; chr14: 22547471-22547495; chr14: 22547470-22547494, or chr14: 22547462-22547486.

[0529] In some embodiments, the modification of TRAC 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 of TRAC comprises the insertion of 1,2,3,4 or 5 or more nucleotide in the target sequence. In some embodiments, the modification of TRAC comprises the disappearance of 1,2,3,4 or 5 or more nucleotide in the target sequence. In other embodiments, the modification of TRAC comprises the insertion of 1,2,3,4,5,6,7,8,9,10,15,20 or 25 or more nucleotide in the target sequence. In other embodiments, the modification of TRAC comprises the disappearance of 1,2,3,4,5,6,7,8,9,10,15,20 or 25 or more nucleotide in the target sequence. In some embodiments, the modification of TRAC comprises insertion / deletion, which is usually defined as the insertion or deletion less than 1000 base pairs (bp) in the art. In some embodiments, the modification of TRAC comprises an insertion / deletion that results in a frameshift mutation in the target sequence. In some embodiments, the modification of TRAC comprises the 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 of TRAC comprises one or more of a nucleotide insertion, deletion, or substitution caused by incorporating a template nucleic acid. In some embodiments, the modification of TRAC comprises inserting a donor nucleic acid into the target sequence. In some embodiments, the modification of TRAC is not transient.

[0530] In some embodiments, the methods and compositions disclosed herein use an RNA-guided DNA binder (e.g., a Cas enzyme) to modify a TRAC gene in a cell. In some embodiments, the RNA-guided DNA binder is Cas9. In some embodiments, the RNA-guided DNA binder cuts within the TRAC gene, wherein the TRAC guide RNA targets a TRAC genomic target sequence comprising at least 10 contiguous nucleotides of chr14: 22547505-22551621 or chr14: 22547462-22551621.

[0531] In some embodiments, the genetic modification of TRAC results in the utilization of an out-of-frame stop codon. In some embodiments, the genetic modification of TRAC results in exon skipping during splicing. In some embodiments, the genetic modification of TRAC results in reduced TRAC protein expression in cells. In some embodiments, the modification of the TRAC gene results in reduced or eliminated TRAC protein expression on the cell surface.

[0532] In some embodiments, TRAC expression on the cell surface is reduced due to genetic modification of TRAC. In some embodiments, TRAC expression on the cell surface is absent due to genetic modification of TRAC.

[0533] 2. Efficacy of TRAC guide RNA

[0534] In some embodiments, when delivering or expressing together with other components forming RNP, determine the effect of TRAC gRNA.In some embodiments, TRAC gRNA is expressed together with the DNA binding agent (such as Cas protein, for example Cas9) that RNA instructs.In some embodiments, TRAC gRNA is delivered to or expressed in the cell line of the DNA nuclease (such as Cas nuclease or nickase, for example Cas9 nuclease or nickase) that has been stably expressed RNA instructs.In some embodiments, TRAC gRNA is delivered to in the cell as a part for RNP.In some embodiments, TRAC gRNA is delivered to in the cell together with the mRNA of the DNA nuclease (such as Cas nuclease or nickase, for example Cas9 nuclease or nickase) that coding RNA instructs.

[0535] As described herein, the use of RNA-guided DNA nucleases and TRAC guide RNAs disclosed herein can cause double-strand breaks in DNA, which can generate errors in the form of insertion / deletion (indel) mutations after repair by the cellular machinery. Many mutations caused by indels change the reading frame or introduce premature stop codons and, therefore, produce non-functional proteins.

[0536] In some embodiments, the efficacy of a specific TRAC gRNA is determined based on an in vitro model. In some embodiments, the in vitro model is a HEK293 cell (HEK293_Cas9) stably expressing Cas9. In some embodiments, the in vitro model is a peripheral blood mononuclear cell (PBMC). In some embodiments, the in vitro model is a T cell, such as a primary human T cell. About using primary cells, commercially available primary cells can be used to provide greater consistency between experiments. In some embodiments, the number of off-target sites deleted or inser...

Claims

1. An engineered cell having reduced or eliminated surface expression of HLA-A relative to an unmodified cell, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises at least one nucleotide within the genomic coordinates chr6:29942540-29945459.

2. The engineered cell of claim 1, having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in an HLA-A gene, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 1, or wherein the genetic modification comprises at least one nucleotide within the genomic coordinate targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 66, 61, 2-60, 62-65, 67-80.

3. An engineered cell having reduced or eliminated surface expression of HLA-A relative to an unmodified cell, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495 and chr6: 29944470-29944494.

4. An engineered human cell having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809.

5. An engineered human cell having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-29942913; chr6: 29944471-29944495 and chr6: 29944470-29944494.

6. An engineered human cell having reduced or eliminated surface expression of HLA-A relative to unmodified cells, comprising a genetic modification in the HLA-A gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29942889-29942913; chr6: 29944471-29944495; chr6: 29944266-29944290; chr6: 29942785-29942809.

7. The engineered cell of any one of claims 1-6, wherein the HLA-A expression is reduced or eliminated by a genome editing system that binds to an HLA-A genomic target sequence, the HLA-A genomic target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of chr6: 29942891-29942915; chr6: 29942609-29942633; chr6: 29944266-29944290; chr6: 29942889-299 42913; chr6:29944471-29944495 and chr6:29944470-29944494, or selected from chr6:29942891-29942915; chr6:29942609-29942633; chr6:29942889-29942913; chr6:29944471-29944495; chr6:29944266-29944290; chr6:29942785-29942809.

8. The engineered cell of any one of claims 1-7, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 1.

9. The engineered cell of any one of claims 1-8, wherein the cell is homozygous for HLA-C.

10. The engineered cell of any one of claims 1-9, wherein the cell is homozygous for HLA-B and homozygous for HLA-C.

11. 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 19, 20, 21, 22, 23 or 24 contiguous nucleotides of a sequence selected from SEQ ID NO: 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 of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in Table 1; v. at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi. a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

12. A method for preparing an engineered human cell having reduced or eliminated surface expression of an HLA-A protein relative to an unmodified cell, the method comprising contacting the cell 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 contiguous nucleotides of a sequence selected from SEQ ID NO: 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 of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in Table 1; v. at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi. a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

13. A method for reducing surface expression of HLA-A protein in human cells relative 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 contiguous nucleotides of a sequence selected from SEQ ID NO: 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 of 10 contiguous nucleotides ± 10 nucleotides comprising the genomic coordinates listed in Table 1; v. at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi. a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

14. The composition or method of any one of claims 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.

15. The composition or method of any one of claims 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.

16. The composition or method of any one of claims 11-13, wherein the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO:

61.

17. The composition or method of any one of claims 11-13, wherein the HLA-A guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO:

66.

18. A cell population comprising the engineered cells of any one of claims 1-10, or engineered cells produced by the method of any one of claims 12-17.

19. A pharmaceutical composition comprising (a) an engineered cell as described in any one of claims 1-10; an engineered cell produced by a method as described in any one of claims 12-17 or by using a composition as described in claim 11; or (b) a cell population as described in claim 18.

20. An engineered human cell having reduced or eliminated surface expression of TRAC relative to an unmodified cell, comprising a genetic modification in the TRAC gene, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr14:22547505-22551621 or chr14:22547462-22551621.

21. The engineered cell of claim 20, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 2, or wherein the genetic modification comprises at least one nucleotide within the genomic coordinate targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120.

22. The engineered cell of claim 20 or 21, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: chrl4: 22550574-22550598; chrl4: 22550544-22550568; chrl4: 22547505-22547529; or chrl4: 22547525-22547549; chrl4: 22547674-22547698.

23. An engineered human cell having reduced or eliminated TRAC expression relative to unmodified cells, comprising a genetic modification in the TRAC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr14: 22550574-22550598; chr14: 22550544-22550568; chr14: 22547505-22547529; chrl4: 22547525-22547549 or chr14: 22547674-22547698.

24. The engineered cell of any one of claims 20-23, wherein the TRAC expression is reduced or eliminated by a genome editing system that binds to a TRAC target sequence comprising at least 5 contiguous nucleotides within genomic coordinates selected from the group consisting of: chrl4: 22550574-22550598; chr14: 22550544-22550568; chr14: 22547505-22547529; chrl4: 22547525-22547549 or chr14: 22547674-22547698.

25. A composition comprising: a) a TRAC guide RNA comprising a guide sequence, wherein the guide sequence i) targets a TRAC genomic target sequence; or ii) a DNA binder guided by the guide RNA to induce a double-strand break (DSB) or a single-strand break (SSB) in the TRAC genomic target sequence, wherein the TRAC genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates selected from the following: chr14: 22550574-22550598; chr14: 22550544-22550568; chr14: 22547505-22547529; chr14: 22547525-22547549 or chr14: 22547674-22547698.

26. 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 the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23 or 24 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; v) at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

27. The composition of claim 26, for use in altering a DNA sequence within a TRAC locus in a cell.

28. The composition of claim 26, which is used to reduce or eliminate the expression of TRAC protein in a cell.

29. A method of making an engineered human cell having reduced or eliminated surface expression of a TRAC protein relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23 or 24 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; v) at least 20, 21, 22, 23 or 24 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

30. A method of reducing surface expression of a TRAC protein in a human cell relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 111, 107, 101-106, 108-110, and 112-120; ii) at least 20, 21, 22, 23, 24 or 25 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 2; v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

31. The composition or method of any one of claims 25-30, wherein the TRAC guide RNA comprises a guide sequence of any one of SEQ ID NOs: 111, 107, 101, 102, and 103.

32. The composition or method of any one of claims 25-30, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO:

107.

33. The composition or method of any one of claims 25-30, wherein the TRAC guide RNA comprises a guide sequence comprising the sequence of SEQ ID NO:

111.

34. A pharmaceutical composition comprising the engineered cell of any one of claims 20-24, or an engineered cell produced by using the composition of claim 25 or 26 or by the method of any one of claims 25-33.

35. An engineered cell having reduced or eliminated surface expression of TRBC relative to an unmodified cell, comprising a genetic modification in the TRBC locus, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr7:142791756-142802543.

36. An engineered cell having reduced or eliminated surface expression of TRBC relative to an unmodified cell, comprising a genetic modification in a TRBC locus, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721 or (c) chr7: 142801104-142802543, or wherein the genetic modification comprises at least one nucleotide within the genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 215, 201-214 and 216-265.

37. The engineered cell of claim 35 or 36, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 3.

38. The engineered cell of claim 35 or 36, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 142791761-142791785; chr7: 14279 1820-142791844; chr7:142791939-142791963: chr7:142791940-142791964 or chr7:142792004-142792028; or (c) chr7:142801104-142801124; chr7:142802103-142802127 or chr7:142802106-142802130.

39. The engineered cell of claim 35 or 36, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: chr7: 142792690-142792714; chr7: 142802103-142802127 and chr7: 142802106-14280213.

40. An engineered cell having reduced or eliminated TRBC expression relative to an unmodified cell, comprising a genetic modification in a human TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the following genomic coordinates: (a) chr7: 142791862-142793149; (b) chr7: 142791756-142792721; or (c) chr7: 142801104-142802543.

41. An engineered human cell having reduced or eliminated TRBC expression relative to an unmodified cell, comprising a genetic modification in the TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: (a) chr7:142792690-142792714 or chr7:142792693-142792717; or (b) chr7:142791756-142791780; chr7:1427917 61-142791785; 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.

42. An engineered human cell having reduced or eliminated TRBC expression relative to an unmodified cell, comprising a genetic modification in the TRBC gene, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chr7: 142792690-142792714; chr7: 142802103-142802127 and chr7: 142802106-14280213.

43. The engineered cell of any one of claims 35-42, wherein the TRBC expression is reduced or eliminated by a genome editing system that binds to a TRBC target sequence comprising at least 5 contiguous nucleotides within a genomic coordinate selected from the group consisting of: (a) chr7: 142792690-142792714 or chr7: 142792693-142792717; or (b) chr7: 142791756-142791780; chr7: 14279176 1-142791785; 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.

44. The engineered cell of any one of claims 35-43, wherein the TRBC expression is reduced or eliminated by a genome editing system that binds to a TRBC target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates selected from the group consisting of: chr7: 142792690-142792714; chr7: 142802103-142802127 and chr7: 142802106-14280213.

45. A composition comprising (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 the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24 or 25 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 3; v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

46. ​​The composition of claim 45, for use in altering a DNA sequence within the TRBC locus in a cell.

47. The composition of claim 45, for use in reducing or eliminating the expression of TRBC protein in a cell.

48. A method of making an engineered human cell having reduced or eliminated surface expression of a TRBC protein relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24 or 25 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 3; v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

49. A method of reducing surface expression of a TRBC protein in a human cell relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 215, 201-214, and 216-265; ii) at least 20, 21, 22, 23, 24 or 25 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 3; v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

50. The method or composition of any one of claims 45-49, wherein the TRBC guide RNA comprises a guide sequence of any one of SEQ ID NOs: 215, 216, 223, 224, 229, 230, 246, 259, and 260.

51. The method or composition of any one of claims 45-50, wherein the TRBC guide RNA comprises the guide sequence of any one of SEQ ID NOs: 215, 216, 224, 229, 246, 259, and 260.

52. The method or composition of any one of claims 45-51, wherein the TRBC guide RNA comprises a guide sequence of any one of SEQ ID NOs: 215, 259, and 260.

53. A pharmaceutical composition comprising the engineered cell of any one of claims 35-44, or an engineered cell produced by the method of any one of claims 46-52, or by using the composition of claim 45.

54. An engineered cell having reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA gene, wherein the genetic modification comprises at least one nucleotide within the genomic coordinates selected from the group consisting of: (a) chrl6: 10877363-10907788 or (b) chrl6: 10906515-10908136.

55. The engineered cell of claim 54, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from any one of the genomic coordinates listed in Table 4.

56. The engineered cell of claim 54, wherein the genetic modification comprises at least one nucleotide within a genomic coordinate selected from the group consisting of: (a) chrl6: 10907504-10907528; chrl6: 10906643-10906667; chrl6: 10907508-10907532; chrl6: 10907539-10907559; chrl6: 10895658-10895682; chrl6: 10895668-10895692; chrl6: 1089 5750—10895774; chrl6: 10895753-10895777; chrl6: 10895754-10895778; chrl6: 10898684-10898708; chrl6: 10901529—10 901553; chrl6: 10902121-10902145; chrl6: 10902701-10902725; chrl6: 10904726-10904750; chrl6: 10904760-10904784; chrl6: 10906493-10906517; chrl6: 10906515-10906539; chrl6: 10906631-10906655; chrl6: 10906636-10906660; chrl6: 1 0906770-10906794; chrl6: 10906788-10906812; chrl6: 10906789-10906813; chrl6: 10906816-10906840; chrl6: 10907148 -10907172; chrl6:10907254-10907278; chrl6:10907331-10907355; chrl6:10907477-10907501; chrl6:10907497-10907521; chrl6:10907503-10907527; chrl6:10907574-10907598; or (b) chrl6:10906889-10906913 or chrl6:10907504-10907528.

57. An engineered cell as described in claim 54, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: chrl6: 10907504-10907528; chrl6: 10906643-10906667; chrl6: 10895658-10895682; chrl6: 10902701-10902725; chrl6: 10906493-10906517; chrl6: 10906631-10906655; chrl6: 10907477-10907501; chrl6: 10907497-10907521 or chrl6: 10907508-10907532.

58. An engineered cell having reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA locus, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: (a) chrl6: 10907504-10907528; chrl6: 10906643-10906667; chrl6: 10907508-10907532; chrl6: 10907539-10907559; chrl6: 10895658-10895682; chrl6: 1089566 8-10895692; chrl6: 10895750-10895774; chrl6: 10895753-10895777; chrl6: 10895754-10895778; chrl6: 10898684-1089 8708; chrl6: 10901529-10901553; chrl6: 10902121-10902145; chrl6: 10902701-10902725; chrl6: 10904726-10904750; ch rl6: 10904760-10904784; chrl6: 10906493-10906517; chrl6: 10906515-10906539; chrl6: 10906631-10906655; chrl6: 10 906636-10906660; chrl6: 10906770-10906794; chrl6: 10906788-10906812; chrl6: 10906789-10906813; chrl6: 10906816- 10906840; chrl6: 10907148-10907172; chrl6: 10907254-10907278; chrl6: 10907331-10907355; chrl6: 10907477-109075 01; chrl6: 10907497-10907521; chrl6: 10907503-10907527; chrl6: 10907574-10907598; or (b) chrl6: 10907504-10907528.

59. An engineered cell having reduced or eliminated surface expression of MHC class II relative to an unmodified cell, comprising a genetic modification in the CIITA locus, wherein the genetic modification comprises an insertion / deletion, a C to T substitution, or an A to G substitution within the genomic coordinates selected from the group consisting of: chrl6: 10907504-10907528; chrl6: 10906643-10906667; chrl6: 10895658-10895682; chrl6: 10902701 -10902725; chrl6:10906493–10906517; chrl6:10906631-10906655; chrl6:10907477-10907501; and chrl6:10907497–10907521; chrl6:10907504-10907528; chrl6:10907508-10907532.

60. The engineered cell of any one of claims 54-59, wherein the MHC class II expression 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 the group consisting of: (a) chrl6: 10907504-10907528; chrl6: 10906643-10906667; chrl6: 10907508-10907532; chrl6: 10907539-10907559; chrl6: 10895658-10895682; chrl6: 10895668-10895692; 50-10895774; chrl6: 10895753-10895777; chrl6: 10895754-10895778; chr16: 10898684-10898708; chr16: 10901529-10901553; chr16: 10 902121-10902145; chr16: 10902701-10902725; chr16: 10904726-10904750; chr16: 10904760-10904784; chr16: 10906493-10906517; chr1 6: 10906515-10906539; chr1 6: 10906631-10906655; chr1 6: 10906636-10906660; chr16: 10906770-10906794; chr16: 10906788-10906812; chr16: 10906789-10906813; chr16: 10906816-1 0906840; chr16: 10907148-10907172; chr16: 10907254-10907278; chr16: 10907331-10907355; chr16: 10907477-10907501; chr1 6:10907497-10907521; chr1 6:10907503-10907527; chr16:10907574-10907598: or chr16:10907504-10907528.

61. The engineered cell of any one of claims 54-60, wherein the MHC class II expression 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 the group consisting of: chr16: 10907504-10907528; chr16: 10906643-10906667; chr16: 10895658-10895682; chr16: 10902701-10902725; chr1 6: 10906493-10906517; chr1 6: 10906631-10906655; chr1 6:10907477-10907501; chr16:10907497-10907521 or chr16:10907508-10907532.

62. 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 comprises: i) a guide sequence selected from the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or ii) at least 20, 21, 22, 23, 24 or 25 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 4; or v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

63. A composition as claimed in claim 62, for use in altering a DNA sequence within a CIITA gene in a cell.

64. A composition as claimed in claim 62, for reducing or eliminating the expression of CIITA in a cell.

65. A method of making an engineered human cell having reduced or eliminated surface expression of an MHC class II protein relative to an unmodified cell, the method comprising contacting the cell 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 the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or ii) at least 20, 21, 22, 23, 24 or 25 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 4; or v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides from the sequence of (iv); or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

66. A method of reducing surface expression of MHC class II proteins in human cells relative 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 the group consisting of SEQ ID NOs: 301, 422, 302-421, and 423-576; or ii) at least 20, 21, 22, 23, 24 or 25 contiguous 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 contiguous nucleotides ± 10 nucleotides of the genomic coordinates listed in Table 4; or v) at least 20, 21, 22, 23, 24 or 25 contiguous nucleotides from the sequence of (iv): or vi) a guide sequence that is at least 95%, 90% or 85% identical to a sequence selected from (iv).

67. The method or composition of any one of claims 62-66, wherein the CIITA guide RNA comprises a guide sequence of any one 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.

68. The method or composition of any one of claims 62-67, wherein the CIITA guide RNA comprises a guide sequence of any one of SEQ ID NOs: 301, 422, 302, 320, 372, 414, 419, 462, and 463.

69. A pharmaceutical composition comprising the engineered cell of any one of claims 54-61, or an engineered cell produced by using the composition of claim 62 or the method of any one of claims 65-68.

70. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 1-69, wherein the genetic modification comprises 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 contiguous nucleotides within the genomic coordinates.

71. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 1-70, wherein the genetic modification comprises an insertion / deletion.

72. The engineered cell of any one of claims 1-71, wherein the genetic modification comprises the insertion of a heterologous coding sequence.

73. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 1-72, wherein the genetic modification comprises a substitution of at least one A to G within the genomic coordinates.

74. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 1-73, wherein the genetic modification comprises a substitution of at least one C to T within the genomic coordinates.

75. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 1-74, wherein the cell has a genetic modification in the CIITA gene.

76. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-75, wherein the cell has reduced expression of TRAC protein on the surface of the cell.

77. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-76, wherein the cell has reduced expression of TRBC protein on the surface of the cell.

78. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-77, wherein the cell has reduced expression of MHC class II molecules on the surface of the cell.

79. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-78, wherein the engineered cell is an immune cell.

80. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-79, wherein the cell is a stem cell.

81. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 1-80, wherein the cell is a primary cell.

82. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 1-81, wherein the cell is a stem cell.

83. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-82, wherein the cell is engineered with a genome editing system.

84. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 7-83, wherein the genome editing system comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

85. The engineered cell, cell population, pharmaceutical composition or method of claim 84, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid is Neisseria meningitidis Cas9 (NmeCas9).

86. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 84-85, wherein the RNA-guided DNA-binding agent or the RNA-guided DNA-binding agent encoded by the nucleic acid has double-stranded endonuclease activity.

87. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 84-86, wherein the RNA-guided DNA-binding agent or the RNA-guided DNA-binding agent encoded by the nucleic acid has nickase activity.

88. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 84-86, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid comprises a dCas9 DNA binding domain.

89. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 84-85, wherein the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is an A to G base editor.

90. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 84-85, wherein the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is a C to T base editor.

91. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-90, wherein the guide RNA is provided to the cell in a vector.

92. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-91, wherein the RNA-guided DNA binding agent is provided to the cell in a vector, optionally in the same vector as the guide RNA.

93. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-92, wherein the exogenous nucleic acid is provided to the cell in a vector.

94. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 91-93, wherein the vector is a viral vector.

95. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 91-93, wherein the vector is a non-viral vector.

96. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-95, wherein the guide RNA is provided to the cell in a lipid nucleic acid assembly composition, optionally in the same lipid nucleic acid assembly composition as the RNA-guided DNA binder.

97. The engineered cell, cell colony, pharmaceutical composition or method of any one of claims 1-96, wherein the exogenous nucleic acid is provided to the cell in a lipid nucleic acid assembly composition.

98. The engineered cell, cell colony, pharmaceutical composition or method of claim 96 or 97, wherein the lipid nucleic acid assembly composition is a lipid nanoparticle (LNP).

99. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-98, wherein the guide RNA is a single guide RNA.

100. The engineered cell, cell population, pharmaceutical composition, or method of any one of claims 1-99, wherein the guide RNA comprises a 5' end modification or a 3' end modification.

101. The method or composition of any one of claims 1-100, wherein the guide RNA comprises: A guide sequence, wherein the guide sequence comprises: Nucleotides modified with 2'-O-Me 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; A shortened repeat / anti-repeat region wherein nucleotides 38-48 and 53-63 are deleted relative to SEQ ID NO:900, comprising: 2'-O-Me modified nucleotides at nucleotides 25, 29, 30, 31, 32, 37, 49-52, 64, 65, 69, 70, and 73; A shortened hairpin 1 region wherein nucleotides 86 and 91 are deleted relative to SEQ ID NO:900, 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 1 region and the shortened hairpin 2 region; The shortened hairpin 2 region, wherein nucleotides 112-120 and 127-135 are deleted relative to SEQ ID NO: 900, comprises: 2′-O-Me modified nucleotides at nucleotides 104, 110, 111, 122-125, 142 and 143, PS linkages between nucleotides 141-142 and 142-143, wherein one or two of nucleotides 144-145 are optionally deleted relative to SEQ ID NO:

900.

102. A method of administering to a subject in need thereof an engineered cell, cell population, pharmaceutical composition as described in any one of claims 1-10, 18-24, 34-44, 53-61, 69, and 70-100.

103. A method of administering to a subject an engineered cell, cell population, or pharmaceutical composition as described in any one of claims 1-10, 18-24, 34-44, 53-61, 69, and 70-100 as adoptive cell transfer (ACT) therapy.

104. A method of treating a disease or condition comprising administering to a subject in need thereof an engineered cell, cell population, or pharmaceutical composition as described in any one of claims 1-10, 18-24, 34-44, 53-61, 69, and 70-100.

105. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-10, 18-24, 34-44, 53-61, 69 and 70-100 for administration to a subject as adoptive cell transfer (ACT) therapy.

106. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-10, 18-24, 34-44, 53-61, 69 and 70-100 for use in treating a subject having cancer.

107. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-10, 18-24, 34-44, 53-61, 69 and 70-100 for use in treating a subject suffering from an infectious disease.

108. The engineered cell, cell population, pharmaceutical composition or method of any one of claims 1-10, 18-24, 34-44, 53-61, 69 and 70-100 for use in treating a subject suffering from an autoimmune disease.

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