Methods for editing HLA-a in cells pre-screened for the absence of one or both alleles of HLA-h*01

Abstract

The present disclosure provides, among other things, methods and compositions for detecting HLA-H subtypes present in cells and / or for the use of such cells in modifying an HLA- A gene. The present disclosure specifically includes, among other things, the unexpected discovery that guide sequences for CRISPR editing of HLA-A can also target a particular subtype of HLA-H referred to herein as HLA-H* 01, which targeting can result in a deleterious chromosomal deletion. The present disclosure thus provides, among other things, methods and compositions useful for determining HLA-H status, altering HLA-A expression in cells that lack HLA-H*01, and identifying and / or selecting cells suitable for such use, as well as cells and other products produced therewith.

Description

METHODS FOR EDITING HLA-A IN CELLS PRE-SCREENED FOR THE ABSENCE OF ONE OR BOTH ALLELES OF HLA-H*01CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application No.: 63 / 535,028, filed August 28, 2023, the entire content of which is hereby incorporated by reference.BACKGROUND

[0002] Allogeneic cell therapies are being developed for the treatment of many diseases. However, human leukocyte antigen (HLA) molecules expressed on cell surfaces can induce immune rejection during allogeneic cell therapy.SUMMARY

[0003] Modification of HLA genes can reduce immune responses that would otherwise be triggered by immunogenic HLA molecules. The present disclosure provides, among other things, methods and compositions for detecting HLA-H alleles present in cells and / or for use in modifying an HLA-A gene. The present disclosure includes the recognition that the HLA-H locus and HLA-A locus are present on the same chromosome and are characterized by sequence similarity, as a result of which certain approaches for modifying HLA-A sequence and / or expression can result in deleterious off-target and / or unintended effects. The present disclosure specifically includes, among other things, the unexpected discovery that guide sequences for CRISPR editing of HLA-A can also target a particular allele of HLA-H referred to herein as HLA-H*01, which targeting can result in a deleterious chromosomal deletion. The present disclosure thus provides, among other things, methods and compositions useful for determining HLA-H status (e.g., the presence or absence of HLA-H*01, including without limitation HLA- H*01 heterozygosity and / or homozygosity), altering HLA-A expression in cells that lack HLA- H*01, and identifying and / or selecting cells suitable for such use, as well as cells and other products produced therewith. The present disclosure further includes the unexpected discovery of sequences (e.g., primers and / or probes) that distinguish HLA-H*01, and thereby permit detection of the HLA-H* 01 allele in cells.

[0004] In at least one aspect, the present disclosure provides a method of altering the expression of HLA-A in a population of cells, including providing a population of cells that have been pre-screened for the absence of one or both alleles of HLA-H*01, and contacting said population of cells with (i) a guide RNA targeting HLA-A and (ii) a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease, thereby effecting one or more double stranded breaks (DSBs) within the HLA-A gene that result in altered expression of HLA-A in the population of cells. In certain embodiments, the cells have been pre-screened for the absence of one allele of HLA-H*01. In certain embodiments, the cells have been pre-screened for the absence of two alleles of HLA-H*01. In certain embodiments, the pre-screening includes amplifying, detecting, and / or sequencing a portion of HLA-H corresponding to or including at least 15 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the method includes sequencing, optionally where the sequencing includes determining the sequence of at least a portion of the genome of the cells that corresponds to or includes at least 15 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the pre-screening includes contacting DNA extracted from the population of cells with at least one primer or probe which specifically hybridizes to a HLA-H*01 allele at a region including at least 15 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2; and detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2.

[0005] In certain embodiments, the guide RNA is a sgRNA. In certain embodiments, the guide RNA includes a spacer / targeting sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 20 contiguous nucleotides of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or to a sequence complementary thereto. In certain embodiments, the guide RNA includes a spacer / targeting sequence including SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0006] In certain embodiments, the CRISPR-associated Cas endonuclease is Cas9. In certain embodiments, the Cas9 is selected from the group of S. pyogenes, N. meningitides, S. wadsworthensis, F. alocis, L. johnsonii, C. lari, C. diptheriae, P. lavamentivorans, M. gallisepticum, S. aureus, L. pneumophila Paris, T. denticola, S. pseudintermedius, S. thermophilus, and N. cinerea.

[0007] In certain embodiments, the population of cells includes human immune cells. In certain embodiments, the human immune cells include T cells, B cells, monocytes, macrophages, or NK cells, or combinations thereof. In certain embodiments, the population of cells includes human stem cells. In certain embodiments, the human stem cells include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic stem cells (HSCs). In certain embodiments, the population of cells includes beta cells, cardiomyocytes, or neurons. In certain embodiments, fewer than 2% of the cells in the population of cells having altered expression of HLA-A include a chromosomal deletion at Chr6p21.3.

[0008] In at least one aspect, the present disclosure provides a method of determining the HLA-H status of a population of cells derived from a human subject, including: providing DNA extracted from the cells; contacting the DNA with a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2; and detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2; where hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells include an HLA-H*01 allele, and where the absence of hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells do not include an HLA-H*01 allele. In certain embodiments, the primer or probe includes at least 10 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the primer or probe includes at least 10 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 5 or SEQ ID NO: 6. In certain embodiments, the probe includes a detectable label. In certain embodiments, the detectable label is a fluorescent label or radiolabel. In certain embodiments, detecting hybridization of the probe to SEQ ID NO: 1 or SEQ ID NO: 2 includes detecting association of the detectable label with the DNA. In certain embodiments, detecting hybridization of the primer to SEQ ID NO: 1 or SEQ ID NO: 2 includes detecting amplification of an HLA-H*01 amplification product. In certain embodiments, the HLA-H*01 amplification product is detected using an amplicon specific probe, a fluorescent probe, by detecting the corresponding amplicon size on an electrophoresis gel, or by sequencing the amplicon by NGS. In certain embodiments, the DNA is contacted with a forward primer, a reverse primer, and a polymerase for a period of  time sufficient to amplify a sequence including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] In at least one aspect, the present disclosure provides a method of determining the HLA-H status of a population of cells derived from a human subject, including providing DNA extracted from the cells; contacting the DNA with a forward primer, a reverse primer, and a polymerase, where the forward primer or the reverse primer specifically hybridizes to an HLA- H*01 allele at a region including at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2; amplifying a sequence including at least 10 nucleotides of SEQ ID NO:1 or SEQ ID NO:2 by PCR; and detecting an amplicon including at least 10 nucleotides of SEQ ID NO:1 or SEQ ID NO:2, optionally where the amplicon is detected by gel electrophoresis (PCR), a fluorescent probe (qPCR), a hybridizing probe specific to the amplicon (ddPCR) or via NGS sequencing; where detection of the amplicon including at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells include an HLA-H*01 allele, and where the absence of the amplicon including at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells do not include an HLA-H*01 allele. In certain embodiments, the DNA is contacted with the forward primer, the reverse primer, and the polymerase for a period of time sufficient to amplify a sequence including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2.

[0010] In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 1, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 2. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 3, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 4. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 5, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 6. In certain embodiments, the reverse primer includes SEQ ID NO: 6, and where the forward primer includes SEQ ID NO: 7.

[0011] In certain embodiments, the DNA is genomic DNA. In certain embodiments, the cells are human immune cells. In certain embodiments, the human immune cells include T cells, B cells, monocytes, macrophages, or NK cells, or combinations thereof. In certain embodiments, the population of cells includes human stem cells. In certain embodiments, the human stem cells include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic  stem cells (HSCs). In certain embodiments, the population of cells includes beta cells, cardiomyocytes, or neurons.

[0012] In at least one aspect, the present disclosure provides a method of selecting a population of cells suitable for editing with a guide RNA targeting HLA-A and a CRISPR- associated Cas endonuclease, including providing DNA extracted from the population of cells; contacting the DNA with a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2; detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2, where the absence of hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells do not include an HLA-H*01 allele and thereby are suitable for editing with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease; and selecting the population of cells that do not include HLA-H*01 for editing with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease.

[0013] In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 5 or SEQ ID NO: 6.

[0014] In certain embodiments, the probe includes a detectable label. In certain embodiments, the detectable label is a fluorescent label or radiolabel. In certain embodiments, detecting hybridization of the probe to SEQ ID NO: 1 or SEQ ID NO: 2 includes detecting association of the detectable label with the DNA.

[0015] In certain embodiments, the DNA is contacted with a forward primer, a reverse primer, and a polymerase for a period of time sufficient to amplify a sequence including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 1, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 2. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 3, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 4. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 5, or the reverse primer includes at  least 15 contiguous nucleotides of SEQ ID NO: 6. In certain embodiments, the reverse primer includes SEQ ID NO: 6, and where the forward primer includes SEQ ID NO: 7.

[0016] In certain embodiments, detecting hybridization of the primer to SEQ ID NO: 1 or SEQ ID NO: 2 includes detecting amplification of an HLA-H*01 amplification product. In certain embodiments, the HLA-H*01 amplification product is detected using an amplicon specific probe, a fluorescent probe, by detecting the corresponding amplicon size on an electrophoresis gel, or by sequencing the amplicon by NGS. In certain embodiments, the DNA is genomic DNA.

[0017] In certain embodiments, where the method further includes contacting the selected population of cells with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease. In certain embodiments, the CRISPR-associated Cas endonuclease is Cas9. In certain embodiments, the Cas9 is selected from the group of S. pyogenes, N. meningitides, S. wadsworthensis, F. alocis, L. johnsonii, C. lari, C. diptheriae, P. lavamentivorans, M. gallisepticum, S. aureus, L. pneumophila Paris, T. denticola, S. pseudintermedius, S. thermophilus, and N. cinerea. In certain embodiments, the guide RNA is a sgRNA. In certain embodiments, the guide RNA includes a spacer / targeting sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 20 contiguous nucleotides of SEQ ID NO: 8 or to a sequence complementary thereto. In certain embodiments, the guide RNA includes a spacer / targeting sequence including SEQ ID NO: 8.

[0018] In certain embodiments, the cells are human immune cells. In certain embodiments, the human immune cells include T cells, B cells, monocytes, macrophages, or NK cells, or combinations thereof. In certain embodiments, the population of cells includes human stem cells. In certain embodiments, the human stem cells include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic stem cells (HSCs). In certain embodiments, the population of cells includes beta cells, cardiomyocytes, or neurons.

[0019] In at least one aspect, the present disclosure provides a method of identifying a subject suitable as a donor of cells to be edited with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease, the method including providing DNA extracted from a cell sample derived from the subject; contacting the DNA with a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region including all or a portion of SEQ ID  NO: 1 or SEQ ID NO: 2; and detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2, where the absence of hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the subject does not include an HLA-H*01 allele and thereby is suitable as a donor of cells to be edited with a guide RNA targeting HLA-A and a CRISPR- associated Cas endonuclease.

[0020] In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 5 or SEQ ID NO: 6.

[0021] In certain embodiments, the probe includes a detectable label. In certain embodiments, the detectable label is a fluorescent label or radiolabel. In certain embodiments, detecting hybridization of the probe to SEQ ID NO: 1 or SEQ ID NO: 2 includes detecting association of the detectable label with the DNA.

[0022] In certain embodiments, the DNA is contacted with a forward primer, a reverse primer, and a polymerase for a period of time sufficient to amplify a sequence including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 1, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 2. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 3, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 4. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 5, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 6. In certain embodiments, the reverse primer includes SEQ ID NO: 6, and where the forward primer includes SEQ ID NO: 7.

[0023] In certain embodiments, detecting hybridization of the primer to SEQ ID NO: 1 or SEQ ID NO: 2 includes detecting amplification of an HLA-H*01 amplification product. In certain embodiments, the HLA-H*01 amplification product is detected using an amplicon specific probe, a fluorescent probe, by detecting the corresponding amplicon size on an electrophoresis gel, or by sequencing the amplicon by NGS. In certain embodiments, the DNA is genomic DNA.

[0024] In certain embodiments, where the method includes obtaining a population of human immune cells from a subject identified as a suitable donor. In certain embodiments, the human immune cells include T cells, B cells, monocytes, macrophages, or NK cells, or combinations thereof. In certain embodiments, the population of cells includes human stem cells. In certain embodiments, the method further includes obtaining a population of induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells) or hematopoietic stem cells (HSCs) from a subject identified as a suitable donor. In certain embodiments, the population of cells includes beta cells, cardiomyocytes, or neurons.

[0025] In certain embodiments, where the method further includes contacting the obtained population with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease. In certain embodiments, the CRISPR-associated Cas endonuclease is Cas9. In certain embodiments, the Cas9 is selected from the group of S. pyogenes, N. meningitides, S. wadsworthensis, F. alocis, L. johnsonii, C. lari, C. diptheriae, P. lavamentivorans, M. gallisepticum, S. aureus, L. pneumophila Paris, T. denticola, S. pseudintermedius, S. thermophilus, and N. cinerea. In certain embodiments, the guide RNA is a sgRNA. In certain embodiments, the guide RNA includes a spacer / targeting sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 20 contiguous nucleotides of SEQ ID NO: 8 or to a sequence complementary thereto. In certain embodiments, the guide RNA includes a spacer / targeting sequence including SEQ ID NO: 8.

[0026] In at least one aspect, the present disclosure provides a population of cells including an edited HLA-A gene, where the edited HLA-A gene includes one or more insertions and / or deletions relative to a wild-type HLA-A gene sequence, and where fewer than 1% of the cells include a chromosomal deletion at Chr6p21.3. In certain embodiments, the one or more insertions and / or deletions decrease expression of HLA-A. In certain embodiments, the cells are heterozygous for HLA-H*01. In certain embodiments, the cells do not include HLA-H*01. In certain embodiments, the cells include HLA-H*02. In certain embodiments, the cells are human immune cells. In certain embodiments, the human immune cells include T cells, B cells, monocytes, macrophages, or NK cells, or combinations thereof. In certain embodiments, the population of cells includes human stem cells. In certain embodiments, the human stem cells include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic  stem cells (HSCs). In certain embodiments, the population of cells includes beta cells, cardiomyocytes, or neurons.

[0027] In at least one aspect, the present disclosure provides a composition or kit including (i) a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) a polymerase. In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments, the primer or probe includes at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 5 or SEQ ID NO: 6. In certain embodiments, the probe includes a detectable label. In certain embodiments, the detectable label is a fluorescent label or radiolabel. In certain embodiments, the composition or kit includes a forward primer and a reverse primer for amplification of an HLA-H*01 sequence including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 1, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 2. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 3, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 4. In certain embodiments, the forward primer includes at least 15 contiguous nucleotides of SEQ ID NO: 5, or the reverse primer includes at least 15 contiguous nucleotides of SEQ ID NO: 6. In certain embodiments, the reverse primer includes SEQ ID NO: 6, and wherein the forward primer includes SEQ ID NO: 7. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a schematic of chromosome 6 showing the relative location of HLA-A and HLA-H on chromosome 6. Pseudogene HLA-H is 55 kb from the telomeric side of HLA-A. White, gray, striped, and black boxes show expressed genes, gene candidates, non-coding genes, and pseudogenes, respectively.

[0029] FIG. 2 is a karyogram showing a chromosome 6 pair following HLA-A editing in HLA-H*01 positive cells, in which one of the chromosomes has a substantial terminal deletion referred to as a 6p21.3 deletion. The illustrated deletion is representative of a deletion that can  result from editing of HLA-A in a cell comprising an HLA-H*01 allele, e.g., when utilizing a guide RNA of the present disclosure.

[0030] FIG. 3 is a sequence alignment between a portion of the genomic sequences of HLA-H*01 and HLA-H*02.

[0031] FIG. 4 is a graph showing the percentage of CD8+ cells from Donor “1” or Donor “2” that express HLA-A3 and HLA-A2 alleles after editing at the HLA-A gene, as measured by flow cytometry (see also Table 2).

[0032] FIG. 5 is a sequence alignment of HLA-H*01, HLA-H*02, HLA-A (HLA-A*02), and the targeting portion of six numerically designated sgRNAs. For illustrative purposes, the reverse complement of guides marked with an asterisk (G018998 and G019000) are shown in this figure, as these guides target the HLA-A nucleic acid strand complementary to that shown in the alignment.

[0033] FIG. 6A is an image of a DNA gel showing PCR product bands of HLA-H*01, one in each of lanes 3 and 4 of the DNA gel, and a corresponding DNA ladder in lane M of the gel. Detection of HLA-H*01 bands as shown in lanes 3 and 4 was observed from an HLA-H*01 positive donor.

[0034] FIG. 6B is an image of a DNA gel showing the absence of PCR product bands of HLA-H*01. PCR product was run in lanes 3 and 4. A corresponding DNA ladder is shown in lane M of the gel. The absence of HLA-H*01 bands was observed in PCR product from an HLA-H*01 negative donor.

[0035] FIG. 7A is a graph showing the average number of detected 6p21.3 chromosomal deletion events in cell samples from HLA-H*01 positive donors. Two hundred HLA-A-edited cells from each of three donors were assessed. Unedited cells are provided as a negative control.

[0036] FIG. 7B is a graph showing the average number of detected 6p21.3 chromosomal deletion events in cell samples from HLA-H*01 negative donors. Two hundred HLA-A-edited cells from each of three donors were assessed. Unedited cells are provided as a negative control.

[0037] FIG. 8 is a graph showing the average frequency of indels at the HLA-H locus after editing the HLA-A gene in donor cells that were homozygous or heterozygous for HLA- H*01 (Donors 3 and 4, respectively), or that did not carry HLA-H*01 (Donor 5).

[0038] FIG. 9 is an alignment of the HLA-H*01 and HLA-H*02 allele sequences. SEQ ID NO: 7 (nucleotides 385-404 of HLA-H*01 and nucleotides 383-402 HLA-H*02) is shown in  bold and italics; SEQ ID NO: 8 (nucleotides 474-493 of HLA-H*01 and nucleotides 472-491 of HLA-H*02) is shown in bold and italics; SEQ ID NO: 1 (see nucleotides 602-643 of HLA- H*01) is underlined; SEQ ID NO: 10 (i.e., from nucleotides 606-626 for the HLA-H*01) is in bold, in italics, and underlined; SEQ ID NO: 9 corresponds to nucleotides 716-735 of HLA- H*01 and nucleotides 714-733 of HLA-H*02, and is shown in bold and italics. DETAILED DESCRIPTION

[0039] The human leukocyte antigen (HLA) super-locus is a genomic region of chromosome 6 that includes the six classical HLA genes (i.e., HLA-A, HLA-B, HLA-C, HLA- DR, HLA-DP, and HLA-DQ) and at least 132 protein coding genes with diverse roles in fundamental molecular and cellular processes including, e.g., immunity. The HLA super-locus is among the most polymorphic regions in the human genome and has been associated with more than 100 different diseases, including common diseases, such as diabetes, rheumatoid arthritis, psoriasis, asthma, and various other autoimmune disorders.

[0040] HLA genes are also important in the success of allogeneic therapies, such as “off- the-shelf” allogeneic therapies. Allogeneic therapies that utilize, e.g., engineered immune cells or stem cells, may be useful for the treatment of, e.g., many cancers and genetic diseases, but are hindered by the alloreactive immune responses against immunogenic HLA molecules. The success of allogeneic therapies can be increased by selection and / or engineering of therapeutic cells in a manner that decreases the likelihood of such anti-HLA immune responses. The present disclosure includes compositions and methods useful for reducing HLA-A gene expression (e.g., using CRISPR / Cas9 gene editing) as one means of producing cells advantageous for allogeneic therapy. Cells genetically modified for reduced HLA-A expression, optionally in combination with other advantageous characteristics, are less likely to induce an anti-HLA immune response and promote therapeutic success. Moreover, by reducing or eliminating one potential source of immunogenicity, the present disclosure can expand the pool of allogeneic donor cells that are available for treatment of individual subjects.

[0041] The present disclosure includes the unexpected discovery that genetic modification of HLA-A using a CRISPR / Cas gRNA can, in certain instances, result in a deleterious chromosomal deletion that removes a significant portion of chromosome 6. This large deletion, which can be referred to as a 6p21.3 chromosomal deletion, decreases cell  viability and fitness and / or reduces the therapeutic efficacy of cells carrying the deletion. It was further discovered that this deletion specifically occurred in cells that carry a particular subtype of HLA-H, which is also positioned on chromosome 6. In some embodiments, the present disclosure provides, among other things, methods and compositions for determining whether a cell is homozygous positive, heterozygous positive, or negative for the detrimental allele of HLA-H (HLA-H*01). Methods for selecting cells for HLA-A genetic modification, and / or genetically modifying HLA-A using a CRISPR / Cas gRNA, are also provided. DEFINITIONS

[0042] A, An, The, Or: As used herein, “a”, “an”, and “the” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” discloses embodiments of exactly one element and embodiments including more than one element. As used herein, the terms “or” and “and / or”, as conjunctions in a list of at least two elements, encompass and disclose embodiments in which the listed elements are included in the alternative, together, or in any combination.

[0043] About: As used herein, term “about”, when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0044] Administration: As used herein, the term “administration” typically refers to administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition.

[0045] Allogeneic: As used herein, the term “allogeneic” refers to any material derived from one subject and transplanted or introduced to another subject, and to processes characterized by such transplantation or introduction. Examples of allogeneic therapy include allogeneic T cell therapy and allogeneic stem cell therapy.

[0046] Amplicon or amplicon molecule: As used herein, the term “amplicon” or “amplicon molecule” refers to a nucleic acid molecule generated by transcription from a template nucleic acid molecule, or a nucleic acid molecule having a sequence complementary  thereto, or a double-stranded nucleic acid including any such nucleic acid molecule. Transcription can be initiated from a primer, for example, during a PCR process.

[0047] Amplification: As used herein, the term “amplification” refers to the use of a template nucleic acid molecule in combination with various reagents to generate further nucleic acid molecules from the template nucleic acid molecule, which further nucleic acid molecules may be identical to or similar to (e.g., at least 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to) a segment of the template nucleic acid molecule and / or a sequence complementary thereto.

[0048] Amplification reaction mixture: As used herein, the terms “amplification reaction mixture” or “amplification reaction” refer to a template nucleic acid molecule together with reagents sufficient for amplification of at least a portion of the template nucleic acid molecule.

[0049] Binding: As used herein, the term “binding” refers to a non-covalent association between or among two or more agents. “Direct” binding involves physical contact between agents; indirect binding involves physical interaction by way of physical contact with one or more intermediate agents. Binding between two or more agents can occur and / or be assessed in any of a variety of contexts, including where interacting agents are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier agents and / or in a biological system or cell).

[0050] Hybridization: As used herein, hybridization refers to pairwise binding between multiple bases of a first nucleic acid and of a second nucleic acid across a region of complementarity. In various embodiments, hybridization occurs or is understood to occur where a first nucleic acid and a second nucleic acid are at least 75% complementary (e.g., 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 100% complementary). A first nucleic acid and a second nucleic acid can be referred to as hybridized when pairwise binding between bases of the nucleic acids produced a double-stranded region, e.g., a double stranded region of at least 5, at least 10, at least 15, or at least 20, or more nucleotides and / or a double stranded region representing at least 40% (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more) of one or more both of the nucleic acids. Hybridization does not necessarily require perfect complementarity between sequences, and does not necessarily require pairwise binding of every base within a region (e.g., within a region of complete or partial complementarity,  and / or within a double-stranded region formed between, a first nucleic acid and a second nucleic acid). Pairwise binding of nucleic acids (e.g., within a double-stranded region) can be contiguous, partially contiguous, discontiguous, and / or partially discontiguous. Those of skill in the art will appreciate that hybridization occurs under various well-known conditions, and that stringency of hybridization can be controlled through modulation of conditions such as temperature and buffer (e.g., salt concentration) as needed for various applications (e.g., to achieve specific hybridization of a primer with a first sequence but not a second sequence). Hybridization conditions can be selected to achieve hybridization specificity in which hybridization specifically occurs between sequences that differ (i.e., have non-complementary bases) at no more than, e.g., 1, 2, 3, 4, or 5 positions, e.g., over the length of a primer or probe, or a length of at least or about, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

[0051] Biological Sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a biological source is or includes an organism, such as an animal or human. In some embodiments, a biological sample is or includes biological tissue or fluid. In some embodiments, a biological sample can be or include cells, tissue, or bodily fluid. In some embodiments, a biological sample can be or include blood, blood cells, cell-free DNA, free floating nucleic acids, ascites, biopsy samples, surgical specimens, cell-containing body fluids, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, gynecological fluids, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as a ductal lavages or broncheoalveolar lavages, aspirates, scrapings, or bone marrow. In some embodiments, a biological sample is or includes cells obtained from a single subject or from a plurality of subjects. A sample can be a “primary sample” obtained directly from a biological source, or can be a “processed sample” (e.g., a sample prepared from a primary sample, e.g., by a process such as isolation, e.g., of mRNA, DNA, or protein, by a process that modifies the primary sample’s chemical structure, and / or by a process that produces a new or different composition that represents one or more components or properties of the primary sample). A biological sample can also be referred to as a “sample.”

[0052] Corresponding to: As used herein, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. Examples of corresponding sequences can include, e.g., two distinct sequences that represent the same region of a gene but are from two distinct alleles of the gene and differ at, e.g., 12, 3, 4, 5, or more nucleotide positions. In some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of skill in the art appreciate that residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered or labeled) including the scheme of a related reference sequence (even if, e.g., such designation does not reflect literal numbering of the provided sequence). By way of illustration, if a reference sequence includes a particular amino acid motif at positions 100-110, and a second related sequence includes the same motif at positions 110-120, the motif positions of the second related sequence can be said to “correspond to” positions 100-110 of the reference sequence. Those of skill in the art appreciate that corresponding positions can be readily identified, e.g., by alignment of sequences, and that such alignment is commonly accomplished by any of a variety of known tools, strategies, and / or algorithms, including without limitation software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE. In some embodiments, two sequences can be identified as corresponding if they are identical or if they share substantial identity, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In various embodiments, a nucleic acid sequence can correspond to a sequence that is identical or substantially identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the complement of the nucleic acid sequence.

[0053] Detectable moiety: The term “detectable moiety” as used herein refers to any element, molecule, functional group, compound, fragment, or other moiety that is detectable. In some embodiments, a detectable moiety is provided or utilized alone. In some embodiments, a detectable moiety is provided and / or utilized in association with (e.g., joined to) another agent. Examples of detectable moieties include, but are not limited to, various ligands, radionuclides  (e.g.,3H,14C,18F,19F,32P,35S,135I,125I,123I,64Cu,187Re,111In,90Y,99mTc,177Lu,89Zr etc.), fluorescent dyes, chemiluminescent agents, bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles, nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels, biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0054] Downstream and Upstream: As used herein with respect to DNA, the term “downstream” means that a first DNA region is closer, relative to a second DNA region, to the 3′ terminus of a nucleic acid that includes the first DNA region and the second DNA region. As used herein, the term “upstream” means a first DNA region is closer, relative to a second DNA region, to the 5′ terminus of a nucleic acid that includes the first DNA region and the second DNA region.

[0055] Expression: As used herein, “expression” refers individually and / or cumulatively to one or more biological process that result in production from a nucleic acid sequence of an encoded agent, such as a polypeptide. Expression specifically includes either or both of transcription and translation. A cell that produces the encoded agent can be said to express the encoded agent.

[0056] Genetic Modification: As used herein, the term “genetic modification” refers to a change at the DNA level, e.g., induced by a CRISPR / Cas gRNA and Cas system, e.g., a CRISPR / Cas9 gRNA and Cas9 system. A genetic modification can be or include an insertion, deletion, or substitution (i.e., base sequence substitution, i.e., mutation), typically within a defined sequence or genomic locus. A genetic modification changes the nucleic acid sequence of the DNA. A genetic modification may be at a single nucleotide position. A genetic modification may be at multiple nucleotides, e.g., 2, 3, 4, 5 or more nucleotides, typically in close proximity to each other, e.g., contiguous nucleotides. A genetic modification of a gene can reduce or eliminate expression of one or more, or all, products encoded by the gene. A genetic modification can be in a coding sequence, e.g., an open reading frame (ORF). As used herein, a genetic modification can be used to prevent translation of an endogenous full-length protein having an amino acid sequence of the full-length protein prior to genetic modification of the defined sequence or genomic locus. Prevention of translation of a full-length protein or gene product includes prevention of translation of a protein or gene product of any length. Translation of an endogenous full-length protein can be prevented, for example, by a frameshift mutation  that results in the generation of a premature stop codon or by generation of a nonsense mutation. A genetic modification can include a modification that inhibits protein activity that may not correspondingly decrease protein level as determined by a protein detection assay, e.g., ELISA or western blot.

[0057] Guide RNA: “Guide RNA”, “gRNA”, and simply “guide” are used herein interchangeably to refer to the guide that directs an RNA-guided DNA binding agent to a target DNA and can be either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally occurring sequence, or a trRNA sequence with modifications or variations compared to naturally occurring sequences.

[0058] Guide Sequence: As used herein, a “guide sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. A “guide sequence” may also be referred to as a “targeting sequence,” or a “spacer sequence.” A guide sequence can be about 20 nucleotides in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs, or guide sequence can be about 24 nucleotides in length, e.g., in the case of Neisseria meningitidis (Nme Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. In some embodiments, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20, 21, 22, 23, 24, 25 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides. In some embodiments, the guide  sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.

[0059] Target sequences for RNA-guided DNA binding agents include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence’s reverse compliment), as a nucleic acid substrate for an RNA-guided DNA binding agent is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0060] In some embodiments, the targeting sequence is in an open reading frame or in a genome, e.g., a viral genome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a targeting sequence and its corresponding target sequence in a genome is at least 80%, 85%, preferably 90%, or 95%, or is 100%. For example, in some embodiments, the targeting sequence comprises a sequence that is at least 80%, 85%, preferably 90%, or 95%, or is 100% identical or complementary to 20 contiguous nucleotides of a sequence. In some embodiments, the guide sequence and the target region may contain at least one mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches within the duplex formed between the guide and the target sequence, where the total length of the target sequence is 16, 17, 18, 19, 20 nucleotides, or more. In some embodiments, the guide sequence and the target sequence may contain 1-4 mismatches where the guide sequence comprises at least 20 nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides. That is, the guide sequence and the target region may form a duplex region having 16, 17, 18, 19, 20 base pairs, or more. In certain embodiments, the duplex region may include 1, 2, 3, or 4 mismatches such that guide strand and target sequence are not fully complementary. For example, a guide strand and target sequence may be complementary over a 20 nucleotide region, including 2 mismatches, such that the guide sequence and target sequence are 90% complementary providing a duplex region of 18 base pairs  out of 20. More tolerated mismatch positions are known in the art, for example, PAM distal mismatches tend to be better tolerated than PAM proximal matches. Mismatch tolerances at other positions are known in the art (see, e.g., Sternberg et al., 2015, Nature:527:110-113)

[0061] In some embodiments, the target sequence is in an open reading frame or a in a genome, e.g., a viral genome, and is complementary to the targeting sequence. In some embodiments, the degree of complementarity or identity between a targeting sequence and its corresponding target sequence in a genome is at least 80%, 85%, preferably 90%, or 95%. For example, in some embodiments, the guide sequence comprises a sequence 24 contiguous nucleotides of a sequence. In some embodiments, the targeting sequence and the target sequence may be 100% complementary or identical. In other embodiments, the targeting sequence and the target sequence may contain at least one mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence. For example, the targeting sequence and the target sequence may contain 1-2, preferably no more than 1 mismatch, where the total length of the target sequence is 19, 20, 21, 22, preferably 23, or 24, nucleotides, or more. In some embodiments, the targeting sequence and the target sequence may contain 1-2 mismatches where the targeting sequence comprises at least 24 nucleotides. In some embodiments, the targeting sequence and the target sequence may contain 1-2 mismatches where the targeting sequence comprises 24 nucleotides. That is, the targeting sequence and the target sequence may form a duplex region having 24 base pairs, or more. In certain embodiments, the duplex region may include 1-2 mismatches such that targeting sequence and target sequence are not fully complementary. Mismatch positions are known in the art, for example, PAM distal mismatches tend to be better tolerated than PAM proximal matches. Mismatch tolerances at other positions are known in the art (see, e.g., Edraki et al., 2019. Mol. Cell, 73:1-13).

[0062] Target sequences for RNA-guided DNA binding agents, as defined by the targeting sequence of a guide RNA, may be present on either the positive or negative strand. Tables and other disclosures provided herein may recite genomic coordinates or position within a nucleotide sequence as a target sequence. It is understood that the guide can be complementary to either the positive or negative strand of the DNA as defined by the genomic coordinates or position within a nucleotide sequence. The sequence to which the guide is complementary depends on the presence of an appropriate PAM for the RNA guided DNA binding protein on the opposite strand. Thus, in some embodiments, when the targeting sequence binds the reverse  complement of a target sequence, i.e., the targeting sequence is identical to certain nucleotides of the sense (positive) strand of the target sequence, when the PAM is present in the sense strand, except for the substitution of U for T in the targeting sequence.

[0063] RNA-guided DNA binding agent: As used herein, the term “RNA-guided DNA binding agent” refers to a polypeptide or complex of polypeptides 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. In some embodiments, the RNA-guided DNA binding agent has nuclease activity, e.g., cleavase activity. Exemplary RNA-guided DNA binding agents include Cas cleavases and inactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease,” as used herein, encompasses Cas cleavases and dCas DNA binding agents. In some embodiments the Cas cleavase encompasses a dCas DNA binding agent modified to permit DNA cleavage, e.g., via fusion with a FokI domain. Cas cleavases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases.

[0064] Improve, increase, inhibit, decrease or reduce: As used herein, the terms “improve”, “increase”, “inhibit”, “decrease” and “reduce”, and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference.

[0065] Isolated: As used herein, “isolated” refers to a substance and / or entity that has been (a) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (b) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated substances and / or entities are at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance and / or entity is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance and / or entity may still be considered “isolated” or “pure” after having been combined  with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance and / or entity is calculated without including such carriers or excipients. To give but one example, in some embodiments, cells (e.g., a population of cells, such as a population of edited cells) can be referred to as “isolated” when (a) the cells are not associated with some or all of the components with which they were associated in an organism or culture from which they were derived; or (b) the cells are substantially free of other cell types (i.e., substantially free of cells that are not of the isolated type). To give another example, a biological polymer such as a polypeptide or polynucleotide that occurs in nature can be referred to as “isolated” when (a) by virtue of its origin or source of derivation is not associated with some or all of the components with which it was associated in its native state in nature; (b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; (c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an “isolated” polypeptide. Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an “isolated” polypeptide to the extent that it has been separated from other components (a) with which it is associated in nature; and / or (b) with which it was associated when initially produced.

[0066] Nucleic Acid Extract: As used herein, the term “nucleic acid extract” refers to a composition that includes nucleic acid isolated from a lipid nucleic acid assembly composition. The present disclosure includes that a nucleic acid extract can be the direct product of a process of nucleic acid extraction (a “primary extract”) or can be a composition produced or obtained by processing of a primary extract, e.g., by addition or removal of one or more agents, e.g., by dilution or concentration of a primary extract and / or by combination of a primary extract with reagents to facilitate analysis.

[0067] Primer: As used herein, the term primer, or oligonucleotide primer, refers to a nucleic acid molecule used, capable of being used, or for use in, generating amplicons from a template nucleic acid molecule. Under transcription-permissive conditions (e.g., in the presence of nucleotides and a DNA polymerase, and at a suitable temperature and pH), a primer can  provide a point of initiation of transcription from a template to which the primer hybridizes. Typically, a primer is a single-stranded nucleic acid between 5 and 200 nucleotides in length. Those of skill in the art will appreciate that optimal primer length for generating amplicons from a template nucleic acid molecule can vary with conditions including temperature parameters, primer composition, and transcription or amplification method. A pair of primers, as used herein, refers to a set of two primers that are respectively complementary to a first strand and a second strand of a template double-stranded nucleic acid molecule. First and second members of a pair of primers may be referred to as a “forward” primer and a “reverse” primer, respectively, with respect to a template nucleic acid strand, in that the forward primer is capable of hybridizing with a nucleic acid strand complementary to the template nucleic acid strand, the reverse primer is capable of hybridizing with the template nucleic acid strand, and the position of the forward primer with respect to the template nucleic acid strand is 5′ of the position of the reverse primer sequence with respect to the template nucleic acid strand. It will be understood by those of skill in the art that the identification of a first and second primer as forward and reverse primers, respectively, is arbitrary inasmuch as these identifiers depend upon whether a given nucleic acid strand or its complement is utilized as a template nucleic acid molecule.

[0068] Probe: As used herein, the term “probe,” or “oligonucleotide probe,” refers to a nucleic acid molecule used, capable of being used, or for use in, producing a detectable signal indicative of the presence of a target nucleic acid sequence with which the probe can hybridize. A probe can include a detectable label. Typically, a probe is a single-stranded nucleic acid between 5 and 200 nucleotides in length.

[0069] Polynucleotide and nucleic acid: As used herein, the terms “polynucleotide” and “nucleic acid” refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar- phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2’ methoxy, 2’ halide, or a 2’-O-(2-methoxyethyl) (2’-O-moe) substitutions. An RNA may comprise one or more deoxyribose nucleotides, e.g., as modifications, and  similarly a DNA may comprise one or more ribonucleotides. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5- methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza- pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5- methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6- methylaminopurine, O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; US Pat. No. 5,378,825 and PCT No. WO 93 / 13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11thed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional nucleosides with 2’ methoxy substituents, or polymers containing both conventional nucleosides and one or more nucleoside analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Nucleic acid includes “unlocked nucleic acid” enables the modulation of the thermodynamic stability and also provide nuclease stability. RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

[0070] Polypeptide: As used herein, the term “polypeptide” refers to a multimeric compound comprising amino acid residues that can adopt a three-dimensional conformation. Polypeptides include but are not limited to enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies, etc. Polypeptides may, but do not necessarily, comprise post-translational modifications, non-natural amino acids, prosthetic groups, and the like.

[0071] Reference: As used herein, “reference” refers to a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or characteristic representative thereof, is compared with a reference, an agent, sample, sequence, subject, animal,  or individual, or population thereof, or a measure or characteristic representative thereof. In some embodiments, a reference is a measured value. In some embodiments, a reference is an established standard or expected value. In some embodiments, a reference is a historical reference. A reference can be quantitative of qualitative. Typically, as would be understood by those of skill in the art, a reference and the value to which it is compared represent assessments under comparable conditions. Those of skill in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison. In some embodiments, an appropriate reference may be an agent, sample, sequence, subject, animal, or individual, or population thereof, under conditions those of skill in the art will recognize as comparable, e.g., for the purpose of assessing one or more particular variables (e.g., presence or absence of an agent or condition), or a measure or characteristic representative thereof.

[0072] Subject: As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human, rat, or mouse). In some embodiments, a subject is suffering from a disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject is not suffering from a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject that has been tested for a disease, disorder, or condition, and / or to whom therapy has been administered. In some instances, a human subject can be interchangeably referred to as a “patient” or “individual.” A subject administered an agent associated with treatment of a disease, disorder, or condition with which the subject is associated can be referred to as a subject in need of the agent, i.e., as a subject in need thereof.

[0073] Target Sequence: As used herein, a “target sequence” refers to a sequence of nucleic acid in a target genome or open reading frame, in either the positive or the negative strand, that has complementarity to the guide sequence of the gRNA, i.e., that is sufficiently complementary to the guide sequence to permit specific binding of the guide sequence. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent), within the  target sequence. The specific length of the target sequence and the number of mismatches possible between the target sequence and the guide sequence depend, for example, on the identity of the Cas nuclease being directed by the gRNA.

[0074] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool). HLA-A

[0075] HLA-A is one of three major types of human major histocompatibility complex (MHC) class I transmembrane proteins. HLA-A proteins, as a part of MHC class I molecules, present short polypeptides to the immune system. HLA-A is a significant determinant of immunogenicity in allogeneic cell therapy. The present disclosure includes that genetic modification of HLA-A to reduce or eliminate HLA-A expression can improve the suitability and utility of cells for use in allogeneic therapy. Thus, cells successfully edited for reduced HLA-A expression are advantageous for the development of allogeneic therapies.

[0076] The HLA-A gene is located on the short arm of chromosome 6, specifically at location 6p21.3 (as shown in FIG.1; for an exemplary HLA-A sequence (HLA-A*02:01:01:01),  see SEQ ID NO: 16). In various embodiments, an HLA-A gene can include a nucleotide sequence having at least 85% sequence identity with SEQ ID NO: 16 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 16). In some embodiments, an HLA-A gene can include a sequence including SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. HLA-H

[0077] The HLA-H gene is one of many genes and pseudogenes encompassed by the highly polymorphic HLA super-locus. The HLA-H gene is located 55 kb from the telomeric side of the HLA-A gene. Alleles of HLA-H can have significant regions of identity with HLA-A. HLA-H*01 and HLA-H*02 are two alleles of HLA-H.

[0078] In various embodiments, an HLA-H*01 gene can include a nucleotide sequence having at least 99% sequence identity with SEQ ID NO: 17 (e.g., at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7, at least 99.8, at least 99.9, or 100% identity with SEQ ID NO: 17). In some embodiments, an HLA- H*01 gene can include a sequence comprising SEQ ID NO: 1 and / or SEQ ID NO: 2, and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, no more than 5 sequence differences, or no more than 6 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 1 and / or SEQ ID NO: 2, respectively. In some embodiments, an HLA-H*01 gene can include a sequence comprising SEQ ID NO: 3 and / or SEQ ID NO: 4, and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 3 and / or SEQ ID NO: 4, respectively. In some embodiments, an HLA-H*01 gene can include a sequence comprising SEQ ID NO: 5 and / or SEQ ID NO: 6, and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence  differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 5 and / or SEQ ID NO: 6, respectively.

[0079] HLA-H*01 and HLA-H*02 contain regions having a high degree of sequence similarity, as set forth in the alignment provided in Fig.9. In various embodiments, an HLA- H*02 gene can include a nucleotide sequence having at least 99% sequence identity with SEQ ID NO: 18 (e.g., at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7, at least 99.8, at least 99.9, or 100% identity with SEQ ID NO: 18). In various embodiments, an HLA-H*02 gene does not include a nucleic acid sequence comprising any one or more of SEQ ID NOs: 1-6, or a sequence differing by less than 2, less than 3, less than 4, less than 5, or less than 6 differences e.g., nucleotide insertions, deletions, and / or substitutions) from, one or more, or all, of SEQ ID NOs: 1-6.

[0080] In some embodiments, an HLA-H*01 gene and / or an HLA-H*02 gene can include a sequence comprising SEQ ID NO: 7, and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 7.

[0081] In some embodiments, an HLA-H*01 gene and / or an HLA-H*02 gene can include a sequence comprising SEQ ID NO: 8, and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 8.

[0082] In some embodiments, an HLA-H*01 gene and / or an HLA-H*02 gene can include a sequence comprising SEQ ID NO: 9, and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 9. Determining HLA-H Status

[0083] The present disclosure provides, among other things, methods and compositions useful for determining the HLA-H status of a sample (e.g., the presence, absence, heterozygosity, and / or homozygosity of HLA-H*01 in one or more genomes thereof). In various embodiments, determination of HLA-H status includes distinguishing between HLA-H*01 and HLA-H*02 alleles and / or genomes including the same. The present disclosure includes a number of non- exclusive approaches to determining HLA-H*01 status. Certain approaches are useful for  distinguishing whether HLA-H*01 alleles are present or absent. Certain approaches are useful for distinguishing presence, absence, and / or heterozygosity for HLA-H*01. In various embodiments, determining HLA-H*01 status can include amplifying, sequencing, and / or detecting hybridization to SEQ ID NO: 1, or a portion thereof (see, e.g., FIG. 3). Samples

[0084] The present disclosure includes determination of the HLA-H status of sample, where a sample can be or refer to, e.g., a genome, cell, or population of cells. In some embodiments, a sample can be or refer to a nucleic acid sample, e.g., comprising nucleic acid extracted or otherwise derived from a cell or population of cells. A sample can be representative of a source from which it is derived, e.g., a subject, population of subjects, or population of cells (e.g., a cell culture, cell bank, stored cell sample, or other cell stock). Cells of the present disclosure can be, be present in, and / or be derived (e.g., isolated) from a biological sample. In various embodiments, HLA-H status is determined from nucleic acid extracted from a sample. The present disclosure therefore includes determining HLA-H status in a wide range of sample types, and / or nucleic acid extracts thereof.

[0085] In some embodiments, a sample comprises a plurality of isolated cells. In some embodiments, cells present in a sample are predominantly of a single type (e.g., 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% of the same type). In some embodiments, cells present in a sample include a mixture of cell types, such as a mixture of cell types reflective of the source of the sample. In some embodiments the cells are identical or nearly identical, e.g., a clonal cell population. In some embodiments, cells can be immune cells. In some embodiments, cells can be lymphocytes (e.g., B cells and / or T cells), natural killer cells, myeloid cells (e.g., monocytes, macrophages, dendritic cells eosinophils, mast cells, basophils, and / or granulocytes cells) or combinations thereof. In certain embodiments, cells are T cells, natural killer cells, or macrophages. In certain embodiments, cells are T cells. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, T-helper 1 type T cells, gamma delta T cells, and / or T-helper 2 type T cells. In some embodiments, cells are antigen presenting cells, e.g., professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, and / or Langerhans cells) and / or  other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and / or oligodendrocytes).

[0086] In some embodiments, the cells are stem cells, e.g., induced pluripotent stem cells, embryonic stem cells (ES cells), or hematopoietic stem cells (HSCs). In some embodiments, cells are iPSC-derived cells. In some embodiments, the iPSC-derived cells are differentiated from iPSCs (e.g., β cells, cardiomyocytes, neurons, T cells, B cells, macrophages, monocytes, and / or variants thereof). In some embodiments, cells are of a type known to naturally express HLA-A.

[0087] In some embodiments, a sample can be derived from one or more tissues of a subject. In some embodiments, a sample is derived from lymphoid tissue (e.g., bone marrow tissue, thymus tissue, spleen tissue, lymph node tissue, small intestine tissue, cardiovascular tissue, neural tissue, and / or pancreatic tissue). In some embodiments, a sample can be or include blood and / or a blood product such as plasma or serum. In some embodiments, a sample can be derived from saliva, blood, plasma, serum, lymph fluid, nasal discharge, and / or aspirate. In some embodiments, a sample can be obtained by surgery or autopsy of a subject. In certain embodiments, the sample is a human sample (e.g., a patient sample).

[0088] In some embodiments, a population of cells is clonal. In some embodiments, the population of cells is a primary cell line of any one or more of the tissues herein disclosed. In some embodiments, a sample is derived from or comprises a cell culture.

[0089] In some embodiments, HLA-H status can be determined using nucleic acid (e.g., DNA) extracted from a sample. Techniques for DNA extraction are known to those of skill in the art.

[0090] In some embodiments, the sample comprises cells derived from a subject. In some embodiments, a subject is a human subject. In some embodiments, a subject is a non-human mammal. In some embodiments, a subject is, e.g., a primate, sheep, dog, cat, cow, horse, pig, or chicken. Primers and Probes

[0091] The present disclosure includes primers and / or probes that specifically hybridize to, and / or are useful to amplify a portion of, HLA-H*01. As will be appreciated by those of skill in the art, such reagents are useful for PCR-based and probe-based methods of determining the  HLA-H status of sample. In some embodiments, altering the expression of HLA-A in a population of cells includes pre-screening the cells for the absence of one or both alleles of HLA- H*01. In some embodiments, the pre-screening includes the step of contacting DNA extracted from the population of cells with at least one primer or probe which specifically hybridizes to an HLA-H*01 allele.

[0092] The present disclosure specifically includes primers and probes that hybridize to a portion of the sequence of HLA-H*01 that distinguishes the allele from other HLA-H alleles, e.g., HLA-H*02, at least in that they specifically hybridize with nucleic acids having the sequence of HLA-H*01 to a greater extent than with nucleic acids having the sequence of HLA- H*02. As provided herein, a primer or probe can be characterized as specifically hybridizing HLA-H*01 if, when contacted with equal numbers of HLA-H*01 and HLA-H*02 sequences under given hybridization conditions, it hybridizes HLA-H*01 with at least two-fold greater affinity or frequency (e.g., at least two-fold, at least three-fold, at least four-fold, at least five- fold, fifty-fold, at least 100-fold, or at least 1000-fold greater affinity or frequency) relative to HLA-H*02. As provided herein, a primer or probe that specifically hybridizes to HLA-H*01 can have at least one (e.g., at least one, at least two, at least three, at least four, at least five, or at least six) positions with a nucleotide that is identical (or perfectly complementary) to the corresponding position of HLA-H*01, but differs from the corresponding position of HLA- H*02. As provided herein, a primer or probe can be characterized as specifically hybridizing to HLA-H*01 if, when contacted with equal numbers of HLA-H*01 and HLA-H*02 sequences under given hybridization conditions, it hybridizes HLA-H*01 with at least two-fold greater affinity or frequency (e.g., at least two-fold, at least three-fold, at least four-fold, at least five- fold, fifty-fold, at least 100-fold, or at least 1000-fold greater affinity or frequency) relative to HLA-H*02.

[0093] In some embodiments, a primer or probe that specifically hybridizes to HLA- H*01 can have or include a region that comprises all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2 (wherein said region is not identical to the corresponding region of HLA-H*02). In some embodiments, a primer or probe that specifically hybridizes HLA-H*01 can have or include the sequence of a region of at least 10 contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., a region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 contiguous nucleotides of SEQ ID NO: 1 or SEQ  ID NO: 2), and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

[0094] In some embodiments, a primer or probe that specifically hybridizes to HLA- H*01 can have or include a region that comprises all or a portion of SEQ ID NO: 3 or SEQ ID NO: 4 (wherein said region is not identical to the corresponding region of HLA-H*02). In some embodiments, a primer or probe that specifically hybridizes to HLA-H*01 can have or include the sequence of a region of at least 10 contiguous nucleotides of SEQ ID NO: 3 or SEQ ID NO: 4 (e.g., a region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of SEQ ID NO: 3 or SEQ ID NO: 4), and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

[0095] In some embodiments, a primer or probe that specifically hybridizes to HLA- H*01 can have or include a region that comprises all or a portion of SEQ ID NO: 5 or SEQ ID NO: 6 (wherein said region is not identical to the corresponding region of HLA-H*02). In some embodiments, a primer or probe that specifically hybridizes HLA-H*01 can have or include the sequence of a region of at least 15 contiguous nucleotides of SEQ ID NO: 5 or SEQ ID NO: 6 (e.g., a region of 15, 16, or 17 contiguous nucleotides of SEQ ID NO: 5 or SEQ ID NO: 6), and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 5 or SEQ ID NO: 6, respectively.

[0096] In some embodiments, a first primer that specifically hybridizes HLA-H*01 can be paired in a PCR reaction with a second primer that hybridizes to HLA-H*01 upstream or downstream of the first primer. Accordingly, in some embodiments, the present disclosure also includes, for pairing with a primer that specifically hybridizes HLA-H*01, a primer that has or includes (i) the sequence of a region of at least 10 contiguous nucleotides of SEQ ID NO: 17 or its complement (e.g., a region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 27, 28, 29, or 30 contiguous nucleotides of SEQ ID NO: 17 or its complement), or (ii) a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or  substitutions) as compared to SEQ ID NO: 17 or its complement, respectively. In various embodiments, such a pair of primers can be a forward primer and reverse primer suitable for amplification of an HLA-H*01 amplicon. In some embodiments, the amplicon is an amplicon of about 50 to about 1,000 nucleotides (e.g., about 100 to about 750 nucleotides or about 100 to about 500 nucleotides). In some embodiments, the second primer can have or include the sequence of a region of at least 15 contiguous nucleotides of SEQ ID NO: 7 (e.g., a region of 15, 16, 17, 18, 19, or 20 contiguous nucleotides of SEQ ID NO: 7), and / or a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 7.

[0097] In some embodiments, a probe that specifically hybridizes HLA-H*01 can be used to detect HLA-H*01 status alone or in combination with any primer pair that amplifies a nucleic acid region of HLA-H*01 that includes the sequence to which the probe hybridizes. In some embodiments, the primer pair can be used to amplify a region of HLA-H*01 comprising 1, 2, 3, 4, 5, 6 or more sequence differences relative to HLA-H*02, and the probe can be used to determine whether the amplified region comprises a HLA-H*01 sequence, for example, in a ddPCR assay. Accordingly, in some embodiments, the present disclosure also includes, for pairing with a probe that specifically hybridizes to HLA-H*01, a primer pair in which each of the two primers comprises (i) the sequence of a region of at least 10 contiguous nucleotides of SEQ ID NO: 17 or its complement (e.g., a region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 27, 28, 29, or 30 contiguous nucleotides of SEQ ID NO: 17 or its complement), or (ii) a corresponding sequence that has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 17 or its complement, respectively. In various embodiments, such a pair of primers can be a forward primer and reverse primer suitable for amplification of an HLA-H*01 amplicon. In some embodiments, the amplicon is an amplicon of about 50 to about 1,000 nucleotides (e.g., about 100 to about 750 nucleotides or about 100 to about 500 nucleotides), where the amplicon includes the sequence to which the probe hybridizes. In some embodiments, the primer pair comprises a forward primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 8 (e.g., a region of 15, 16, 17, 18, 19, or 20 contiguous nucleotides of SEQ ID NO: 8), and a reverse primer comprising at least 15  contiguous nucleotides of SEQ ID NO: 9 (e.g., a region of 15, 16, 17, 18, 19, or 20 contiguous nucleotides of SEQ ID NO: 9). The foregoing primer pair can amplify a region of HLA-H*01 that can be distinguished from HLA-H*02, e.g., using a probe that specifically hybridizes to HLA-H*01, or by sequencing the resulting amplicons, e.g., using digital droplet PCR (ddPCR) or next generation sequencing (NGS).

[0098] In some embodiments, a probe can include a detectable label. In some embodiments, a detectable label permits detection of the association of the detectable label with the DNA. In some embodiments, at least one base is suitably labelled with a fluorescent label, a radiolabel, a colorimetric label, a heavy molecule, or a molecular handle for a biorthogonal reaction (e.g., biotin, streptavidin, or a click chemistry handle).

[0099] In some embodiments, the detectable label is a fluorescent label. In some embodiments, a fluorescent label is a compound with a fluorescent emission maximum between about 350 and 900 nm. In some embodiments, a fluorescent label can be, without limitation, 5- FAM (also called 5- carboxyfluorescein; also called Spiro(isobenzofuran-l(3H), 9'- (9H)xanthene)-5-carboxylic acid,3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro- Fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6- Hexachloro-Fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5-Tetrachloro-Fluorescein; ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)-5- carboxylic acid]); 6-Tetrachloro-Fluorescein; ([4,7,2',7'-tetrachloro-(3',6'- dipivaloylfluoresceinyl)-6- carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine); Xanthylium, 9-(2,4- dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA (6- carboxytetramethylrhodamine); 9- (2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5- ((2-aminoethyl)amino)naphthalene-l-sulfonic acid); 1,5-IAEDANS (5-((((2- iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); Cy5 (Indodicarbocyanine-5); Cy3 (Indo-dicarbocyanine-3); and BODIPY FL (2,6-ibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-proprionic acid); Quasar®-670 dye (Biosearch Technologies); Cal Fluor®Orange dye (Biosearch Technologies); Rox dyes; Max dyes (Integrated DNA Technologies), as well as suitable derivatives thereof.

[0100] In some embodiments, a fluorescent label is a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP,  AceGFP, ZsGreen1 ), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[0101] In some embodiments, a detectable label can be or include, without limitation, FAM®, 6-FAM (FLUORESCEIN), 6-FAM (NHS ESTER), 6-FAM (AZIDE), FLUORESCEIN DT, YAKIMA YELLOW®, VIC®, ABY®, JUN®, TET™, HEX™, JOE™, JOE™ (NHS ESTER), CY®3, CY®3.5, CY®5, CY®5.5, TAMRA™, TAMRA™ (NHS ESTER), 5 - TAMRA™ (AZIDE), ROX™, ROX™ (NHS ESTER), LC RED 610, TEXAS RED®, TEXAS RED®-X (NHS ESTER), TEX 615, LC RED 640, FLUORESCEIN, BEBO, MAX, MAX (NHS ESTER), OREGON GREEN 488, OREGON GREEN 514, ATTO™ 425, ATTO™ 465, ATTO™ 488, ATTO™ 520, ATTO™ 532, ATTO™ 550, ATTO™ 565, ATTO™ RHO101, ATTO™ 590, ATTO™ 594, ATTO™ 610, ATTO™ 633, ATTO™ 647N, ATTO™ 680, ATTO™ 700, ATTO™ 740, ATTO™ 532 (NHS ESTER), ATTO™ 550 (NHS ESTER), ATTO™ 565 (NHS ESTER), ATTO™ RHO101 (NHS ESTER), ATTO™ 590 (NHS ESTER), ATTO™ 633 (NHS ESTER), ATTO™ 647N (NHS ESTER), TYE™ 563, TYE™ 665, TYE™ 705, ALEXA FLUOR® 488 (NHS ESTER), ALEXA FLUOR® 532 (NHS ESTER), ALEXA FLUOR® 546 (NHS ESTER), ALEXA FLUOR® 555, ALEXA FLUOR® 594 (NHS ESTER), ALEXA FLUOR® 647 (NHS ESTER), ALEXA FLUOR® 650 (NHS ESTER), ALEXA  FLUOR® 750 (NHS ESTER), 5'IRDYE® 700, 5'IRDYE® 800, 5'IRDYE® 800CW (NHS ESTER),RHOD AMINE GREEN™-X (NHS ESTER), RHOD AMINE RED ™-X (NHS ESTER), WELLRED D2 DYE, WELLRED D3 DYE, WELLRED D4 DYE, LIGHTCYCLER® 610, LIGHTCYCLER® 640 (NHS ESTER), DY 415, DY 480, DY 610, DY 649, DY 682, DY 782, DY 750 (NHS ESTER), PET®, BODIPY FL, BODIPY 530 / 550, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY TRM-X, FITC, BODIPY R6G, CAL GOLD, CAL ORANGE, CAL RED, PULSAR-650, QUASAR-570, QUASAR 670, and NED™.

[0102] In various instances in which one or more probes includes a detectable moiety, the same probe may include a quenching moiety. Exemplary quenching moieties (e.g., for use in connection with qPCR) are known in the art. Exemplary quenching moieties include Black Hole Quencher®(e.g., BHQ®0, BHQ®1, BHQ®1-dt, BHQ®2, BHQ®3, BHQ®10), BlackBerry Quencher BBQ 650, TAMRA, Dabcyl, Dabcyl-dT, Eclipse, non-fluorescent quencher (NFQ), a G nucleotide or plurality of G nucleotides, QSY 7, QSY 9, QSY 21, QSY 35, ELLEQUENCHER, and IOWA BLACK. In various instances, a probe as described herein is double quenched. In various embodiments, a labeled oligonucleotide probe as described herein includes a detectable moiety on its 5' end and a quenching moiety on its 3' end. In various embodiments, a labeled oligonucleotide probe as described herein includes a detectable moiety on its 3' end and a quenching moiety on its 5' end.

[0103] In some embodiments, a detectable label is a radioactive label (i.e., a radiolabel). In some embodiments, the radiolabel is selected from a compound in which at least one of the atoms is a radioactive atom or radioactive isotope, wherein the radioactive atom or isotope spontaneously emits gamma rays or energetic particles, for example alpha particles or beta particles, or positrons. Examples of such radioactive atoms include, but are not limited to,3H (tritium),14C,nC,150,18F,35S,123I, and125I. In some embodiments, a detectable label is a molecule that substantially increases the mass of the primer or probe. In some embodiments, a label that substantially increases the mass of the primer of probe includes a polymer, a protein, a polypeptide, an antibody or variant thereof. In some embodiments, a probe is composed of RNA. In some embodiments, a probe includes one or more modified bases. In some embodiments, a probe may be useful in the detection, identification, and isolation of particular gene sequences (e.g., a “capture probe”).

[0104] Those of skill in the art will appreciate from the present disclosure that for primers and probes disclosed herein that specifically hybridize with HLA-H*01, corresponding primers and probes can be made and used that specifically hybridize with HLA-H*02 by reference to corresponding sequences of HLA-H*02 as set forth, e.g., in SEQ ID NO: 18. Accordingly, to the extent the present disclosure provides methods and compositions for detection and / or identification of HLA-H*01, the present disclosure also provides corresponding methods and compositions for detection and / or identification of HLA-H*02, irrespective of the extent to which such compositions and methods are otherwise expressly set forth herein. Hybridization-Based Approaches for Determining HLA-H status

[0105] In some embodiments, primers and / or probes of the present disclosure that specifically hybridize with a particular HLA-H allele, e.g., HLA-H*01, can be used to determine HLA-H status of a sample. For example, HLA-H status can be revealed by determining whether a primer or probe specific for HLA-H*01 (e.g., comprising 10-15 nucleotides or more of SEQ ID NO: 1 or SEQ ID NO: 2) specifically hybridizes with sample nucleic acid.

[0106] In certain embodiments, a first primer that specifically hybridizes with HLA- H*01 can be used in combination with a second primer (e.g., where the primers are a forward primer and a reverse primer, or vice versa), such that amplification of product from HLA-H*01 demonstrates hybridization of the selective primer with HLA-H*01. Those of skill in the art will appreciate that a first primer that specifically hybridizes to a particular allele (e.g., HLA-H*01) can be used in combination with any second primer that amplifies a product when paired with the first primer in determining HLA-H status. For the avoidance of doubt, there is no need for the second primer to hybridize in an allele-specific manner because sufficient specificity is conferred by the first primer. Indeed, as provided herein, HLA-H status can be determined in some embodiments by use of a single allele-specific primer alone. Those of skill in the art will therefore appreciate that, in various embodiments provided herein, a second primer need not be expressly identified and / or could be readily selected by those of skill in the art.

[0107] A variety of hybridization-based methods that provide a signal indicative of hybridization of a primer to a nucleic acid present in a sample are known to those of skill in the art, and can be used to detect the specific hybridization of a primer to an HLA-H*01 sequence. Certain such methods can utilize Polymerase Chain Reaction (PCR)-based amplification in  which one or more primers are used to amplify a portion of a template nucleic acid molecule (e.g., genomic DNA extracted from a sample). Methods of PCR and steps thereof are well known in the art. A method of PCR can include, in some instances, steps of (a) providing a template nucleic acid, (b) contacting the template with at least (i) polymerase, (ii) free deoxynucleotides, and (iii) at least one forward oligonucleotide primer and one reverse oligonucleotide primer, and (c) incubating the reaction under conditions sufficient to achieve amplification of that portion of the template nucleic acid between and including the sequences of the primers.

[0108] Examples of suitable thermostable DNA polymerases include, e.g., Thermus aquaticus polymerase (Taq), Thermus thermophilus polymerase (Tth), Thermus species NH polymerase (TspNH), Thermus brockianus polymerase (Tbr) (all obtainable for example from GeneSys Limited, Farnborough, U.K.), Pyrococcus furiosus polymerase (Pfu) (obtainable from Stratagene), 9°N7 exo-DNA polymerase, and Thermococcus litoralis DNA polymerase (obtainable from New England Biolabs as VENT™ DNA polymerase).

[0109] Incubation of a PCR reaction can include amplification cycles, where each cycle includes phases of (i) denaturation, (ii) annealing, and (iii) extension. In at least some example PCR reactions, denaturing includes incubating a reaction mixture to 94°C or higher for 15 seconds to 2 minutes; denaturing includes incubating the reaction mixture at approximately 40- 60°C for approximately 15-60 seconds; extension includes incubating the reaction mixture at a temperature in the range of 70-74°C for approximately 1-2 minutes. A series of denaturing, annealing, and extension can be referred to as an amplification cycle. Amplification cycles can be repeated for 2 to 60 or more cycles, e.g., at least 10 cycles, 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles, or 50 cycles, or any range therebetween. In certain embodiments, amplification of a polynucleotide of HLA-H*01 can include, e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 cycles. In certain embodiments, amplification of a nucleic acid sequence can include, e.g., 5-60 cycles, 5-50 cycles, 5-40 cycles, 5-30 cycles, 5-20 cycles, 10-60 cycles, 10-50 cycles, 10-40 cycles, 10-30 cycles, or 10-20 cycles. Those of skill in the art will be able to select an appropriate number of cycles based, e.g., on the particular approach and conditions utilized. In some examples, PCR can be isothermal.

[0110] In certain instances, an amplification reaction is performed with or in the presence of reagents that enable or facilitate detection of the amplification of one or more amplicons. Certain such methods are known in the art as quantitative PCR (qPCR). In various methods of quantitative-PCR (qPCR) or real-time-PCR (rtPCR), including dye-based and probe-based methods or combinations thereof, the signal generated can be used to quantitatively or semi- quantitatively determine the absolute or relative level, amount, or concentration of any of one or more amplicons or nucleic acids. In various methods of qPCR or rtPCR, analysis may be non- quantitative or qualitative. Absolute quantitation is a rigorous technique for quantification. Absolute quantification can utilize the addition of external standards in every reaction to determine the absolute amount of the target nucleic acid of interest. Quantification may utilize a standard curve of external standard dilutions, which can be generated and used to determine the concentration of target. Relative quantitation requires calculation of the ratio between the amount of target template and a reference template in a sample. One approach is the comparative Ct method in which the Ct value of an amplification reaction including a sample of interest is compared to a control, with or without normalization. Qualitative analysis can utilize end-point data acquired after the PCR has reached plateau phase. End-point analysis can be relative, competitive, or comparative.

[0111] In certain examples of amplification-based approaches of determining HLA-H status, an intercalating dye is present in an amplification reaction. Such intercalating dyes may be referred to as amplification-dependent detectable moieties. Intercalating dyes are detectable moieties, e.g., fluorescent moieties, that become detectable or become more detectable when intercalated with or bound to double-stranded DNA. In certain such examples, a signal (or level of signal) is detectable when the dye is intercalated with or bound to double-stranded DNA. Thus, as the number of amplicons (and nucleic acids complementary thereto) increases in an amplification reaction, fluorescence (e.g., momentary, mean, median, mode, or maximum fluorescence) can increase. Such means of amplification may be referred to as dye-based methods. The use of a dye-based method is not exclusive of combination with other methods, such as a probe-based method.

[0112] Examples of intercalating dyes or amplification-dependent detectable moieties include, without limitation, ethidium bromide, proflavine, SYBR® Green (e.g., I or II), SYBR® Gold, EVAGREEN®, YO (Oxazole Yellow) and related intercalating dyes, TO (Thiazole  Orange) and related intercalating dyes, PG (PicoGreen) and related intercalating dyes, indoles and related intercalating dyes, imidazoles and related intercalating dyes, Cyanine dyes, SYTO®- 9, SYTO®-13, SYTO®-16, SYTO®-60, SYTO®-62, SYTO®-64, SYTO®-82, POPO™-3, TOTO®-3, BOBO-3, PO-PRO™-3, TO-PRO™-3, YO-PRO™-1, SYTOX®, YOYO™-1, YO- PRO™-1, BOXTO™, BEBO™, BETO™, and others known in the art.

[0113] In some embodiments, a primer that specifically hybridizes with HLA-H*01 can be used in asymmetric PCR or in a primer extension reaction. Asymmetric amplification reactions may be used to preferentially amplify one strand representing the target sequence that is used for detection. In some cases, the presence and / or amount of the amplification product itself may be used to determine the presence of HLA-H*01 in a cell or population of cells.

[0114] Various protocols and reagents for PCR are known in the art. Various techniques for PCR are described, e.g., in: PCR: A Practical Approach, M. J. McPherson, et al., IRL Press (1991); PCR Protocols: A Guide to Methods and Applications, by Innis, et al., Academic Press (1990); and PCR Technology: Principals and Applications for DNA Amplification, H. A. Erlich, Stockton Press (1989); U.S. Pat. No.4,683,195; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,800,159; U.S. Pat. No. 4,965,188; U.S. Pat. No.4,889,818; U.S. Pat. No. 5,075,216; U.S. Pat. No. 5,079,352; U.S. Pat. No. 5,104,792; U.S. Pat. No. 5,023,171; U.S. Pat. No. 5,091,310; and U.S. Pat. No.5,066,584, each of which is herein incorporated by reference. Various specific PCR amplification applications are available in the art (for reviews, see for example, Erlich, 1999, Rev Immunogenet, 1: 127-34; Prediger 2001, Methods Mol. Biol. 160: 49-63; Jurecic et al, 2000, Curr. Opin. Microbiol. 3: 316-21; Triglia, 2000, Methods Mol. Biol.130: 79-83; MaClelland et al., 1994, PCR Methods Appl. 4: S66-81; and Abramson and Myers, 1993, Current Opinion in Biotechnology 4: 41-47; each of which is incorporated herein by reference).

[0115] Numerous methods of identifying the presence of an amplicon following an amplification reaction are known in the art, which methods may be quantitative, semi- quantitative, non-quantitative, or qualitative. Detection and / or identification of an amplicon can be achieved, e.g., by gel electrophoresis methods (including without limitation, autoradiography, ethidium bromide staining, and silver staining), chromatographic separation, capillary electrophoresis hybridization analysis, comparative hybridization analysis, chromogenic hybridization (e.g., CISH), oligonucleotide hybridization analysis, microarray hybridization analysis, fluorescence hybridization analysis (e.g., FISH), Southern blot analysis, heteroduplex  mobility assay (HMA), restriction fragment length polymorphism (RFLP) analysis, RNAase mismatch analysis, surface plasmon resonance analysis, and single strand conformational polymorphism (SSCP) analysis. Detection and / or identification of an amplicon (with or without a probe-based signal or dye-based signal) can instead or additionally include a mass spectrometry step utilizing, e.g., tandem mass spectrometry, matrix assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption / ionization-time of flight (SELDI- TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption / ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, or secondary ion mass spectrometry (SIMS)).

[0116] In some embodiments, amplicons are detected by sequencing. Various methods of sequencing are known in the art and include, without limitation, sequencing methods. In some embodiments, a sequencing-based approach to determining the HLA-H status of a sample can include a sequencing method such as high-throughput sequencing, deep sequencing, next generation sequencing, massively parallel DNA sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, duplex sequencing, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real- time sequencing, reverse-terminator sequencing, massively parallel signature sequencing (MPSS), direct sequencing, random shotgun sequencing, Sanger sequencing, targeted sequencing, exon sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, nanopore sequencing, sequencing by synthesis, sequencing by ligation, mass spectrometry sequencing, polymerase sequencing, RNA polymerase (RNAP) sequencing, microscopy-based sequencing, capillary electrophoresis-based sequencing, micropore-based sequencing, nanoball sequencing, dye-terminator sequencing, ion semiconductor sequencing, quantum-dot-labeled polymerase sequencing, parallelized single molecule real-time DNA sequencing, true single-molecule sequencing, and the like), and methods that utilize mass spectrometry (e.g., tandem mass spectrometry, matrix assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption / ionization-time of flight (SELDI-TOF) mass spectrometry,  quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption / ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, or secondary ion mass spectrometry (SIMS)).

[0117] Thus, in some embodiments, methods of detecting and / or identifying an amplicon can include hybridization analysis, comparative hybridization analysis, chromogenic hybridization (e.g., CISH), oligonucleotide hybridization analysis, microarray hybridization analysis, fluorescence hybridization analysis (e.g., FISH), Southern blot analysis, heteroduplex mobility assay (HMA), restriction fragment length polymorphism (RFLP) analysis, RNAase mismatch analysis, surface plasmon resonance analysis, single strand conformational polymorphism (SSCP) analysis, polymerase chain reaction (PCR)-based methods (including, without limitation, quantitative PCR, real-time PCR, reverse-transcriptase-PCR analysis (RT- PCR), multiplex PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex ligation-dependent probe amplification (MLPA), and emulsion PCR), and / or sequencing methods. In some embodiments, a sequencing-based approach to determining HLA- H status of a sample can include a sequencing method such as high-throughput sequencing, deep sequencing, next generation sequencing, massively parallel DNA sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, duplex sequencing, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, massively parallel signature sequencing (MPSS), direct sequencing, random shotgun sequencing, Sanger sequencing, targeted sequencing, exon sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, nanopore sequencing, sequencing by synthesis, sequencing by ligation, mass spectrometry sequencing, polymerase sequencing, RNA polymerase (RNAP) sequencing, microscopy-based sequencing, capillary electrophoresis-based sequencing, micropore-based sequencing, nanoball sequencing, dye-terminator sequencing, ion semiconductor sequencing, quantum-dot-labeled polymerase sequencing, parallelized single molecule real-time DNA sequencing, true single-molecule sequencing, and the like. In some embodiment, the methods can utilize mass spectrometry (e.g., tandem mass spectrometry, matrix assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry,  electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption / ionization- time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption / ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, and secondary ion mass spectrometry (SIMS)), any or all of which may be used alone or in conjunction with gel electrophoresis methods (including without limitation, e.g., autoradiography, ethidium bromide staining, and silver staining), chromatographic separation, and / or capillary electrophoresis. In some embodiments, HLA-H*01 status is detected by measuring the amplification product (e.g., amplicon) size on an electrophoresis gel. In some embodiments, electrophoretic detection of an HLA-H*01 amplicon includes detection of a signal from an intercalating dye (e.g., Sybr Green, Sybr Gold, ethidium bromide, or variants thereof).

[0118] In certain embodiments, amplicons and / or nucleic acid sequences are detected and / or quantified based on hybridization with a probe including a detectable label. In various such instances, nucleic acids hybridized to a probe having a detectable label can be separated, isolated, or purified from other nucleic acids by, e.g., chromatography or gel electrophoresis. In various such instances, separated, isolated, or purified nucleic acids may be quantified and / or sequenced. In some embodiments, the detection of radiolabeled primers or probes includes the use of any known method in the art including, but not limited to, scintillation counting and autoradiography (as described in Clark, D. P., et al., Manipulation of Nucleic Acids, Molecular Biology, Third Edition, 2019, pg. 132-166, doi: 10.1016 / B978-0-12-813288-3.00005-7, herein incorporated by reference in its entirety).

[0119] In certain examples of amplification-based approaches of determining HLA-H status, one or more probes are present in the amplification reaction and are labeled with (a) a detectable moiety (e.g., a fluorescent moiety). In certain examples of amplification-based approaches of determining HLA-H status, one or more probes are present in the amplification reaction and are labeled with (a) a detectable moiety (e.g., a fluorescent moiety) and (b) a quenching moiety capable of quenching the signal of the detectable moiety of (a). In such an amplification reaction, the signal of the detectable moiety is quenched by the quenching moiety, the two being associated with the same single probe molecule (e.g., with the fluorescent moiety at the 5' end of the probe and the quenching moiety at the 3' end of the probe) and therefore in  sufficiently close proximity to allow quenching. When the detectable moiety and quenching moiety are physically close to one another, the overall level of fluorescent output is low. During amplification, polymerase activity can cleave a probe that is hybridized to a template nucleic acid molecule (e.g., the original nucleic acid sequence or an amplicon produced from the original nucleic acid sequence) at a position 3' of a nascent amplicon, separating the detectable moiety from the quenching moiety. As a result, the detectable signal of the detectable moiety is no longer quenched or no longer substantially quenched by the quenching moiety associated with the single probe molecule. Non-limiting examples of detectable moieties and quenching moieties are provided above. The use of a probe-based method is not exclusive of combination with other methods, such as a dye-based methods.

[0120] In some embodiments, the detection of a fluorescent label is preferably accomplished using a light (e.g., fluorescence) detector such as a charge couple device (CCD) camera or intensified CCD camera systems or the like. In some embodiments, high-sensitivity fluorescence detection is achieved using laser excitation and total internal reflection optics. The present disclosure also encompasses reaction mixtures including a nucleic acid from or derived from a sample together with one or more primers or probes as described herein. Reaction mixtures of the present disclosure can include, e.g., at least one primer or probe of the present disclosure, optionally in combination with one or more of an intercalating dye, a PCR buffer, water, deoxynucleoside triphosphates (dNTPs), and / or magnesium. Sequencing-Based Approaches for Determining HLA-H status

[0121] HLA-H status can be determined by sequencing of all or a portion of HLA-H (e.g., of HLA-H*01, HLA-H*02, or both). Sequencing-based approaches can distinguish, e.g., samples in which all HLA-H alleles are HLA-H*01 alleles (e.g., HLA-H*01 homozygotes), samples in which a portion (e.g., half) of HLA-H alleles are HLA-H*01 alleles (e.g., HLA-H*01 heterozygotes or carriers), and samples in which no HLA-H alleles are HLA-H*01 alleles (e.g., HLA-H*01 non-carriers, e.g., HLA-H*02 homozygotes). It is to be appreciated from the present disclosure that various sequence-based approaches for determining HLA-H status do not require the use of a primer or probe that specifically hybridizes a particular HLA-H allele (e.g., HLA- H*01). Instead, in such embodiments, the sequencing information itself reveals the HLA-H status of the sample.

[0122] In some embodiments, a sequencing-based approach to determining HLA-H status of a sample can include a sequencing method such as high-throughput sequencing, deep sequencing, next generation sequencing, massively parallel DNA sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, duplex sequencing, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, massively parallel signature sequencing (MPSS), direct sequencing, random shotgun sequencing, Sanger sequencing, targeted sequencing, exon sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, nanopore sequencing, sequencing by synthesis, sequencing by ligation, mass spectrometry sequencing, polymerase sequencing, RNA polymerase (RNAP) sequencing, microscopy-based sequencing, capillary electrophoresis-based sequencing, micropore-based sequencing, nanoball sequencing, dye-terminator sequencing, ion semiconductor sequencing, quantum-dot-labeled polymerase sequencing, parallelized single molecule real-time DNA sequencing, true single-molecule sequencing, and the like.

[0123] Sequence techniques can utilize a primer or pair of primers that amplify a targeted region of interest, or can be based on genomic methods that are target agnostic. For example, for determination of HLA-H status as provided herein, a primer for use in sequencing can be a single forward sequencing primer for a region of interest. In various embodiments, a region of interest can be or correspond to any primer or probe hybridization site provided herein (e.g., a region comprising at least 15 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 as provided herein). In various embodiments, a forward sequencing primer can hybridize at a position having a 3´ terminus that is upstream of a region of interest, e.g., by 1-1000 nucleotides (e.g., 1-5, 1-10, 1- 20, 1-30, 1-40, 1-50, 1-100, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-800, or 1-900 nucleotides). In various embodiments, a pair of primers for use in sequencing can include a forward primer and a reverse primer that amplify an amplicon that includes a region of interest and has a total size of, e.g., 10-1000 nucleotides (e.g., 10-20, 10-30, 10-40, 10-50, 10-100, 10- 200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, or 10-900 nucleotides). In various embodiments, a sequencing primer or pair of sequencing primers can include, solely to provide an illustrative and non-limiting example, one or both of SEQ ID NOs: 8 and 9.  Altering HLA-A Expression using CRISPR-Associated Cas Endonucleases

[0124] The present disclosure provides methods and compositions useful for altering HLA-A expression. Those of skill in the art will appreciate that such edited cells and sequences can be advantageously used alone or as tools and / or intermediate compositions for production of therapeutic cells, e.g., where an HLA-A genetic modification can be used in combination with further genotypic or phenotypic characteristics that together provide or further advance the development of therapeutic cells for use in subjects.

[0125] The present disclosure includes methods and compositions for genetic modification to reduce or eliminate HLA-A expression using a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease. In some embodiments, the present disclosure includes a guide RNA that targets HLA-A such that the CRISPR-associated Cas endonuclease effects one or more double stranded breaks (DSBs) within the HLA-A gene. DSBs lead to non-homologous ending joining (NHEJ) during repair, leading to a modification, e.g., a mutation, in an HLA-A gene. In some embodiments, NHEJ leads to a deletion or insertion of a nucleotide(s), which induces a frame shift or nonsense mutation in an HLA-A gene. Thus, DSBs result in altered expression of HLA-A.

[0126] In some embodiments, cells are contacted with a nucleic acid molecule (e.g., an mRNA molecule) encoding a CRISPR-associated Cas endonuclease and a guide RNA targeting HLA-A. In certain embodiments, cells contacted with such a nucleic acid molecule express the CRISPR-associated Cas endonuclease. In some embodiments, cells are contacted with a CRISPR-associated Cas endonuclease and a guide RNA targeting HLA-A. In some embodiments, cells are contacted with a ribonucleoprotein complex (RNP) that includes a CRISPR-associated Cas endonuclease together with a guide RNA targeting HLA-A. A gRNA together with a Cas nuclease can be called a Cas RNP (e.g., a Cas9 RNP). In some embodiments, an RNP comprises Type-I, Type-II, or Type-III components.

[0127] In some embodiments, a CRISPR-associated Cas endonuclease is Cas9. In some embodiments, a Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes. In some embodiments, a Cas nuclease is the Cas9 nuclease from Neisseria meningitidis. In some embodiments, a Cas nuclease is the Cas9 nuclease from Sutterella wadsworthensis. In some embodiments, a Cas nuclease is the Cas9 nuclease from Filifactor alocis. In some embodiments, a Cas nuclease is a Cas9 nuclease from Lactobacillus johnsonii. In some embodiments, a Cas  nuclease is a Cas9 nuclease from Campylobacter lari. In some embodiments, a Cas nuclease is a Cas9 nuclease from Corynebacterium diphtheriae. In some embodiments, a Cas nuclease is a Cas9 nuclease from Parvibaculum lavamentivorans. In some embodiments, a Cas nuclease is a Cas9 nuclease from Mycoplasma gallisepticum. In some embodiments, a Cas nuclease is a Cas9 nuclease from Staphylococcus aureus. In some embodiments, a Cas nuclease is a Cas9 nuclease from Legionella pneumophila Paris. In some embodiments, a Cas nuclease is a Cas9 nuclease from Treponema denticola. In some embodiments, a Cas nuclease is a Cas9 nuclease from Staphylococcus pseudintermedius. In some embodiments, a Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In some embodiments, a Cas nuclease is a Cas9 nuclease from Neisseria cinerea.

[0128] Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 protein comprises one RuvC domain, and / or one HNH domain. In some embodiments, the Cas9 protein comprises more than one RuvC domain or more than one HNH domain. In some embodiments, the Cas9 protein is a wild type Cas9.

[0129] In some embodiments, a Cas nuclease can be from a Type-I CRISPR / Cas system. In some embodiments, a Cas nuclease can be a component of the Cascade complex of a Type-I CRISPR / Cas system. In some embodiments, a Cas nuclease can be a Cas3 protein. In some embodiments, a Cas nuclease can be from a Type-III CRISPR / Cas system. In some embodiments, a Cas nuclease can have an RNA cleavage activity.

[0130] In some embodiments, chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1. In some embodiments, a Cas nuclease may be a modified nuclease.

[0131] In some embodiments, Cas proteins provided herein can induce a double strand break in target DNA.

[0132] In some embodiments, the guide RNA is a dual guide RNA (dgRNA). In some embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally or naturally occurring components or configurations that  are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified gRNA synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.” Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); and (iv) modification of the oligonucleotide to provide exonuclease stability, e.g., with 2’ O-me, 2’ halide, or 2’ deoxy substituted ribose; or inverted abasic terminal nucleotide, or replacement of phosphodiester with phosphorothioate. In some embodiments, the modification is at the 3' end or 5' end of the oligonucleotide.

[0133] Chemical modifications such as those listed above can be combined to provide modified gRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In some embodiments, up to 15% of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 5' end of the RNA. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 3' end of the RNA.

[0134] In some embodiments, the gRNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, 10%, 15%, preferably at least 20%, 25%, 30%, 35%, 40%, 45%, or 50%) of the positions in a modified gRNA are modified nucleosides or nucleotides. In some embodiments, at least 5% of the positions in the modified guide RNA are modified nucleotides or nucleosides. In some embodiments, at least 10% of the positions in the modified guide RNA are modified nucleotides or nucleosides. In some embodiments at least 15% of the positions in the modified gRNA are modified nucleotides or nucleosides. In some embodiments preferably at least 20% of the positions in the modified gRNA are modified nucleotides or nucleosides. In some embodiments, no more than 65% of the positions in the modified gRNA are modified nucleotides. In some embodiments, no more than 55% of the positions in the modified gRNA are modified nucleotides. In some embodiments, no  more than 50% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 10-70% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 20-70% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 30-60% of the positions in the modified gRNA are modified nucleotides and the nuclease is a Spy Cas9 nuclease. In some embodiments, about 30-70% of the positions in the modified gRNA are modified nucleotides and the nuclease is an Nme Cas9 nuclease.

[0135] Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[0136] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[0137] Examples of modified phosphate groups include, phosphorothioate, borano phosphate esters, methyl phosphonates, phosphoroamidates, phosphodithioate, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged  phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

[0138] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications, e.g., an amide linkage. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, carboxymethyl, carbamate, amide, thioether. Further examples of moieties which can replace the phosphate group can include, without limitation, e.g., ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

[0139] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[0140] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e., a sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g., replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion.

[0141] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycol (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2' hydroxyl group modification can be 2'-O-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2' hydroxyl can be  connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can include "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative). 2' modifications can include hydrogen (i.e. deoxyribose sugars); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2- amino (wherein amino can be, e.g., as described herein), -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.

[0142] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g., L- nucleosides. As used herein, a single abasic sugar is not understood to result in a discontinuity of a duplex.

[0143] In certain embodiments, 2’ modifications include, for example, 2’-OMe, 2’-F, and / or 2’-H.

[0144] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the  nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally occurring and synthetic derivatives of a base.

[0145] In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA or tracr RNA. In embodiments comprising an sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, or internal nucleosides may be modified, or the sgRNA may be chemically modified throughout. Certain embodiments comprise a 5' end modification. Certain embodiments comprise a 3' end modification. Certain embodiments comprise a 5’ end modification and a 3’ end modification.

[0146] HLA-A contains significant sequence similarity to HLA-H, particularly the HLA- H*01 allele. In some embodiments, the guide RNA targets a sequence that is present in HLA-A and HLA-H*01 but is not present in HLA-H*02. In some embodiments, the guide RNA comprises a spacer / targeting sequence having greater similarity to HLA-H*01 than to HLA- H*02. For example, in some embodiments, the spacer / targeting sequence can be 100% identical to HLA-A and HLA-H*01, and can contain 1, 2, 3, 4, 5, 6 or more mismatches with respect to HLA-H*02. In other embodiments, the spacer / targeting sequence can contain 1 mismatch with respect to HLA-H*01, and can contain 2, 3, 4, 5, 6 or more mismatches with respect to HLA- H*02. In other embodiments, the spacer / targeting sequence can contain 2 mismatches with respect to HLA-H*01, and can contain 3, 4, 5, 6 or more mismatches with respect to HLA-H*02. In other embodiments, the spacer / targeting sequence can contain 3 mismatches with respect to HLA-H*01, and can contain 4, 5, 6 or more mismatches with respect to HLA-H*02. In other embodiments, the spacer / targeting sequence can contain 4 mismatches with respect to HLA- H*01, and can contain 5, 6 or more mismatches with respect to HLA-H*02.

[0147] In some embodiments, the guide RNA comprises a spacer / targeting sequence that is or comprises a region capable of specifically hybridizing to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4.

[0148] In some embodiments, the guide RNA comprises a spacer / targeting sequence that is or comprises at least 20 contiguous nucleotides of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the guide RNA comprises a spacer / targeting sequence that includes no more than 1, no more than 2, no  more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the guide RNA is or includes a spacer / targeting sequence comprising SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or a sequence complementary thereto.

[0149] In some embodiments, the guide RNA includes a spacer / targeting sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to at least 10 nucleotides (e.g., at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 nucleotides) of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the guide RNA includes a spacer / targeting sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to at most 20 nucleotides (e.g., at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, or at most 5 nucleotides) of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0150] Means of delivering (i) a guide RNA targeting HLA-A and (ii) a CRISPR- associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease to a cell are known in the art. For example, lipid nanoparticles (LNPs) are a well-known means for delivery of nucleotide and protein cargo, and may be used for delivery of guide RNAs and Cas9 to cells in vivo and in vitro. In some embodiments, LNPs deliver nucleic acid, protein, or nucleic acid together with protein. In some embodiments, a gRNA is delivered via an LNP. In some embodiments, a LNP delivers a gRNA and a Cas9 protein, or a gRNA and an mRNA encoding Cas9. In some embodiments, the composition further comprises a uracil glycosylase inhibitor (UGI) or an mRNA encoding a UGI.

[0151] In some embodiments, DNA or RNA vectors encoding a guide RNA of the present disclosure are provided. In some embodiments, in addition to a guide RNA, a vector can include, e.g., promoters, enhancers, regulatory sequences, and / or a nucleic acid encoding a CRISPR-associated Cas endonuclease, which can be a nuclease such as Cas9.

[0152] In some embodiments, cells can be contacted with vectors encoding a guide RNA and / or a CRISPR-associated Cas endonuclease in the form of naked nucleic acid vectors. In  some embodiments, cells can be contacted with vectors encoding a guide RNA and / or a CRISPR-associated Cas endonuclease in the form of nucleic acid complexed with an agent such as a liposome or poloxamer. In some embodiments, cells can be contacted with viral vectors (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus) comprising nucleic acids encoding a guide RNA and / or a CRISPR-associated Cas endonuclease. Methods and compositions for non- viral delivery of vectors encoding a guide RNA and / or a CRISPR-associated Cas endonuclease include lipofection, virosomes, liposomes, immunoliposomes, LNPs, polycation or lipid:nucleic acid conjugates, naked nucleic acid (e.g., naked DNA / RNA), artificial virions, and agent- enhanced uptake of DNA. Such considerations are well known in the art. Applications and Cell Products

[0153] The present disclosure includes the unexpected finding that genetic modification of HLA-A using a CRISPR-associated Cas endonuclease and a guide RNA targeting HLA-A, e.g., as disclosed herein, can produce a significant and deleterious deletion on Chromosome 6 when an HLA-H*01 allele is also present. However, this was not observed when other HLA-H alleles were present, such as HLA-H*02. Accordingly, the present disclosure includes applications for genetic modification of HLA-A using a CRISPR-associated Cas endonuclease and a guide RNA as disclosed herein, where the modified genomes and / or cells are not homozygous for HLA-H*01, are heterozygous for HLA-H*01, or do not include an HLA-H*01 allele (e.g., are homozygous for HLA-H*02). For the avoidance of doubt, the present disclosure recognizes a particular utility of cells heterozygous for HLA-H*01 in that such cells will have reduced risk or frequency of the observed chromosomal deletion at Chr6p21.3, and distinct utility in subjects for whom such cells are found to constitute the best-suited population for a needed allogeneic therapy. The present disclosure therefore includes methods and compositions for, among other things, the determination of HLA-H status, selection of cells for HLA-A genetic modification, genetic modification of such cells, and resulting genetically modified cell populations. Methods for selecting a subject suitable for supplying donor cells to be edited at the HLA-A locus are also provided.

[0154] The present disclosure provides methods and compositions useful for determining HLA-H status. In various embodiments, these methods can include hybridization, amplification, and / or sequencing of a portion of HLA-H that includes at least 10 contiguous nucleotides of SEQ  ID NO: 1 or SEQ ID NO: 2 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2), which, in some embodiments, can include at least 10 contiguous nucleotides of SEQ ID NO: 3 or SEQ ID NO: 4 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of SEQ ID NO: 3 or SEQ ID NO: 4), and / or at least 10 contiguous nucleotides of SEQ ID NO: 5 or SEQ ID NO: 6 (e.g., 10, 11, 12, 13, 14, 15, 16, or 17 contiguous nucleotides of SEQ ID NO: 5 or SEQ ID NO: 6). The present disclosure includes the recognition that such a region is of particular utility for determination of HLA-H status (e.g., distinguishing HLA-H*01 from HLA-H*02).

[0155] In certain embodiments, the present disclosure provides methods of selecting a population of cells suitable for editing with a guide RNA targeting HLA-A and a CRISPR- associated Cas endonuclease. Such methods can include providing DNA extracted from the population of cells, contacting the DNA with a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2, and detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the absence of hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells do not include an HLA-H*01 allele and thereby are suitable for editing with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease. The method can, in some embodiments, further comprise selecting the population of cells that do not include HLA-H*01 for editing with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease. The present disclosure also provides methods of identifying a subject suitable as a donor of cells to be edited with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease. Such methods can include providing DNA extracted from a cell sample derived from the subject; contacting the DNA with a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2; and detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2. The absence of hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the subject does not include an HLA-H*01 allele and thereby is suitable as a donor of cells to be edited with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease.

[0156] The present disclosure further provides that cells determined (e.g., pre-screened) to have no HLA-H*01 alleles or only one HLA-H*01 allele can be advantageously selected for  HLA-A genetic modification and / or can be genetically modified to reduce expression of HLA-A, while reducing or eliminating chromosomal deletion events in chromosome 6, relative to cells having two HLA-H*01 alleles. Identification of cells suitable for HLA-A genetic modification to reduce expression of HLA-A provides an advantageous insight for use in the process of reducing the immunogenicity of cells, e.g., for allogeneic gene therapy.

[0157] The present disclosure further includes embodiments in which cells identified as having a desired HLA-H status (e.g., no HLA-H*01 alleles or only one HLA-H*01 allele) are genetically modified by contact with (i) a guide RNA targeting HLA-A and (ii) a CRISPR- associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease, causing double stranded breaks (DSBs) within the HLA-A gene that result in reduced expression of HLA-A. In certain embodiments, the present disclosure includes a method of altering the expression of HLA-A in a population of cells that includes providing a population of cells that have been pre-screened for the absence of one or both alleles of HLA-H*01, and contacting the population of cells with (i) a guide RNA targeting HLA-A and (ii) a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease, thereby effecting one or more double stranded breaks (DSBs) within the HLA-A gene that results in altered expression of HLA-A in the population of cells. In various embodiments, HLA-A genetic modification can be achieved using a guide RNA that includes a spacer / targeting sequence that has the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., nucleotide insertions, deletions, and / or substitutions) as compared to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0158] Cells produced by such methods, and / or progeny thereof, are characterized by certain advantages, including reduced expression of HLA-A, and a significantly reduced presence and / or frequency of the observed chromosomal deletion at Chr6p21.3 (e.g., as compared to chromosomal deletion at Chr6p21.3 in a similarly modified reference cell population with a higher frequency of HLA-H*01 alleles, e.g., a population of cells homozygous for the HLA-H*01 allele).

[0159] In some embodiments, HLA-A genes of cells genetically modified as set forth herein, and / or progeny thereof, include one or more insertions and / or deletions (e.g., 1, 2, 3, 4, 5,  6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions and / or deletions) relative to a wild-type HLA-A sequence. In various embodiments, as set forth herein, the insertions and / or deletions in HLA-A decrease expression of HLA-A. In various embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least of 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) of cells and / or HLA-A genes present in a population of cells genetically modified as set forth herein, and / or progeny thereof, include one or more insertions and / or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions and / or deletions) relative to a wild-type HLA-A sequence. In various embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least of 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100%) of cells and / or HLA-A genes present in a population of cells genetically modified as set forth herein, and / or progeny thereof, are characterized by reduced expression of HLA-A. In various embodiments, reduction of HLA-A protein and / or mRNA levels in a cell or population of cells can be, e.g., at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least of 50%, at least 55%, at least 60%, 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 100%).

[0160] In various embodiments, a reduced number of cells (i.e., HLA-H*01-deficient cells, e.g., cells lacking one or both alleles of HLA-H*01, or cells including one or both alleles of HLA-H*02) that have been genetically modified at an HLA-A locus as set forth herein include a chromosomal deletion at Chr6p21.3, relative to similarly modified reference cells that are homozygous for HLA-H*01. In some embodiments, fewer than 5% (e.g., fewer than 4.5%, fewer than 4%, fewer than 3.5%, fewer than 3%, fewer than 2.5%, fewer than 2%, fewer than 1.5%, fewer than 1%, or fewer than 0.5%) of cells genetically modified as set forth herein, and / or progeny thereof, include a chromosomal deletion at Chr6p21.3. In some embodiments, fewer than 3% of cells genetically modified as set forth herein include a deletion at Chr6p21.3. In some embodiments, fewer than 2.5% of cells genetically modified as set forth herein include a deletion at Chr6p21.3. In some embodiments, fewer than 2% of cells genetically modified as set  forth herein include a deletion at Chr6p21.3. In some embodiments, fewer than 1.5% of cells genetically modified as set forth herein include a deletion at Chr6p21.3. In some embodiments, fewer than 1% of cells genetically modified as set forth herein include a deletion at Chr6p21.3. In some embodiments, fewer than 0.5% of cells genetically modified as set forth herein include a deletion at Chr6p21.3. In some embodiments, the present disclosure provides a population of cells including an edited HLA-A gene, wherein the edited HLA-A gene includes one or more insertions and / or deletions relative to a wild-type HLA-A gene sequence, and wherein fewer than 2% (e.g., fewer than 2%, fewer than 1.5%, fewer than 1%, fewer than 0.5% etc.) of the cells include a chromosomal deletion at Chr6p21.3.

[0161] In some embodiments, cells genetically modified as set forth herein are characterized by fewer chromosomal deletions at Chr6p21.3 than reference HLA-A genetically modified cells that have a higher frequency of HLA-H*01 alleles. For example, if cells genetically modified as set forth herein did not include an HLA-H*01 allele, reference genetically modified cells can be cells of the same cell type genetically modified according to the same protocol but heterozygous or homozygous for HLA-H*01. For example, if cells genetically modified as set forth herein were heterozygous for HLA-H*01, reference genetically modified cells can be cells of the same cell type genetically modified according to the same protocol but homozygous for HLA-H*01. In various embodiments, cells genetically modified at the HLA-A locus as set forth herein are characterized by a frequency of chromosomal deletions at Chr6p21.3 that is less than that observed in reference cells by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In various embodiments, cells genetically modified as set forth herein are characterized a frequency of chromosomal deletions at Chr6p21.3 that is less than that observed in reference cells by at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold, or at least ten-fold.

[0162] In some embodiments, a cell genetically modified as set forth herein can be isolated from a population of genetically modified cells. In various embodiments, a cell characterized by a genetic of modification of HLA-A that reduces expression of HLA-A (e.g., by at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and which does not include a deletion at Chr6p21.3 can be selected for further study or use. For  example, a selected cell can be further modified to have additional characteristics useful for administration to human subjects for treatment of particular diseases. Additionally, a population of cells characterized by a genetic of modification of HLA-A (e.g., by introduction of indels in the HLA-A locus) and which do not include a deletion at Chr6p21.3 can be selected for further study or use. For example, such a population of cells can be further modified to have additional characteristics useful for administration to human subjects for treatment of particular diseases.

[0163] Various cell types can be genetically modified by the methods and compositions provided herein. In various embodiments, cells genetically modified for reduced expression of HLA-A can be cells useful for allogeneic therapy. In various embodiments, cells genetically modified for reduced expression of HLA-A as provided herein can be immune cells. In some embodiments, cells can be lymphocytes (e.g., B cells and / or T cells), natural killer cells, myeloid cells (e.g., monocytes, macrophages, dendritic cells eosinophils, mast cells, basophils, and / or granulocytes cells). In certain embodiments, cells are T cells, natural killer cells, or macrophages. In certain embodiments, cells are T cells. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, T-helper 1 type T cells, gamma delta T cells, and / or T-helper 2 type T cells. In some embodiments, cells are antigen presenting cells, e.g., professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, and / or Langerhans cells) and / or other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and / or oligodendrocytes). In some embodiments, cells are stem cells, e.g., induced pluripotent stem cells, embryonic stem cells (ES cells) or hematopoietic stem cells (HSCs). In some embodiments, cells are iPSC-derived cells. In some embodiments, the iPSC-derived cells are differentiated from iPSCs (e.g., β cells, cardiomyocytes, neurons, T cells, B cells, macrophages, monocytes, and / or variants thereof). In some embodiments, the cells are of a type known to naturally express HLA-A. In some embodiments, the cells are human cells. In some embodiments, the cells are of a non-human mammal. In some embodiments, the cells are of a non-human primate, sheep, dog, cat, cow, horse, pig, or chicken. Kits

[0164] In some embodiments, the present disclosure provides kits and compositions that include a primer or probe of the present disclosure that specifically hybridizes to HLA-H*01 at a region including at least 15 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2, and a polymerase.  In some embodiments, a kit or composition includes a primer or probe of the present disclosure that specifically hybridizes to HLA-H*01 at a region including at least 15 nucleotides of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, , a kit or composition includes a primer or probe of the present disclosure that specifically hybridizes to HLA-H*01 at a region including at least 15 nucleotides of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, , a kit or composition includes a primer or probe of the present disclosure that specifically hybridizes to HLA-H*01 at a region including at least 15 nucleotides of SEQ ID NO: 7. In some embodiments, a kit or composition includes a forward primer as set forth herein and a reverse primer as set forth herein for amplification of an HLA-H*01 sequence that includes all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2.

[0165] In some embodiments, a kit or composition includes a forward primer and a reverse primer for amplification of an HLA-H*01 sequence including all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, a kit or composition includes a forward primer and a reverse primer for amplification of an HLA-H*01 sequence including all or a portion of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, a kit or composition includes a forward primer and a reverse primer for amplification of an HLA-H*01 sequence including all or a portion of SEQ ID NO: 5 or SEQ ID NO: 6.

[0166] In some embodiments, a kit or composition includes a probe as set forth herein, e.g., including a detectable label as set forth herein. In some embodiments, the detectable label is, e.g., a fluorescent label or a radiolabel.

[0167] In various embodiments, a composition or kit can include one or more reagents for determining HLA-H status, which can include, for example, one or more of a PCR buffer, deoxynucleoside triphosphates (dNTPs), and / or magnesium. In various embodiments, a composition or kit can include one or more reagents for HLA-A gene editing, which can include, for example a guide RNA provided herein and / or a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease.

[0168] In various embodiments, a composition or kit can include instructions for determining HLA-H status. In various embodiments, a composition or kit can include instructions for genetically modifying HLA-A. In various embodiments, a composition or kit can include instructions for determining HLA-H status and genetically modifying HLA-A in  cells determined not to include an HLA-H*01 allele, or found to be heterozygous for HLA- H*01.  Listing of Sequences         EXAMPLES

[0170] The following Examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way. The present Examples provide exemplary protocols and reagents useful in determining the HLA-H status. The present Examples also provide exemplary protocols and reagents useful in altering the expression HLA-A. Example 1: T cell editing at the HLA-A locus

[0171] Several features of T cell editing at the HLA-A locus were assessed in an allogeneic T cell system for impact on protein knockdown via flow cytometry and on chromosomal integrity via karyotyping. T cells were edited using S. pyogenes Cas9 (SpCas9) and sgRNAs targeting HLA-A, TRAC, and CIITA, followed by AAV6-mediated delivery of a chimeric antigen receptor targeting CD30 having homology arms flanking the TRAC gRNA cut site. 1.1. T cell editing

[0172] T cells with the genotype indicated in Table 1 were isolated using human donor apheresis and were prepared and activated one day after thaw as described in Example 7.3. LNPs were formulated as described in Example 7.2 with the lipid molar ratio and RNA ratio indicated in this Example. Table 1. T cell donor genotypes

[0173] Samples were activated and edited on the schedule indicated in Table 2. Prior to editing, T cells were centrifuged, resuspended and plated at 1,000,000 cells / well in 1 ml / well in TCGM with 2.5% human AB serum on D0 / D1 and resuspended at 500,000 cells / well in 1 ml / well in TCGM with 2.5% human AB serum on D3 / D4. LNPs were prepared as described in Example 7.2. LNP composition A (LNP A) had the following molar ratio of lipids (50 Lipid A: 39.5 cholesterol: 9 DSPC: 1.5 PEG2k-DMG), and a ratio of gRNA to SpyCas9 mRNA of 1:2 by  weight. LNP composition B (LNP B) had the following molar ratio of lipids (35 Lipid A: 47.5 cholesterol: 15 DSPC:2.5 PEG2k-DMG), and a ratio of gRNA to SpyCas9 mRNA of 1:1 by weight. LNPs were incubated with 2.5 μg / ml ApoE (Peprotech, Cat. 350-02) in TCGM with 2.5% human AB serum at 37°C for about 5 minutes. LNP A compositions were added to T cells at 5 µg total RNA cargo / mL. LNP B compositions were added to T cells at 1 μg total RNA cargo / ml. All edits for a given sample used the same LNPs (all LNPA or all LNPB) throughout the experiment. Between edits, cells were incubated at 37°C. HLA-A LNPs contain SpCas9 mRNA and gRNA G018995. Cells were also treated with LNPs containing S. pyogenes Cas9 (SpCas9) mRNA and guide RNA targeting the TRAC or CIITA genomic loci. “CAR AAV” in Table 2 indicates samples were treated with AAV6 virus encoding a chimeric antigen receptor at a multiplicity of infection (MOI) of 1e5. Samples labeled D0A, D0B, D1#1 and D4#1 were treated with 0.25 µM of DNA protein kinase inhibitor (DNApki) (Compound 4 in WO / 2022 / 221696) at the same time as AAV administration. Table 2. Editing schedule  1.2. Flow cytometry

[0174] T cells were analyzed by flow cytometry eleven days after thaw to determine surface biomarker expression for each target gene following editing, using a cocktail of antibodies targeting HLA-A2 (Invitrogen, 17-9876-42), HLA-A3 (BD Biosciences, 747776), HLA-DR, DP, DQ (Biolegend, 361712), CD3 (Biolegend, 317336), CD4 (Biolegend, 317434), CD8 (Biolegend, 301046), and human IgG Fc (Biolegend, 410708), and ViaKrome 808 Fixable Viability Dye (Beckman Coulter, C36628). T cells were subsequently washed and analyzed on a Cytoflex instrument (Beckman Coulter). Data analysis was performed using FlowJo software package (v.10.6.1). T cells were gated on size, viability, CD4 or CD8 expression, and expression of markers. Flow cytometry data showing the percentage of CD8+ cells staining positive for HLA-A2 or HLA-A3 are shown in Table 3 and FIG. 4. Table 3. Percentage of CD8+ cells positive for HLA-A3 or HLA-A2 in Donor 1 or Donor 2Example 2: Karyotype and ddPCR Analysis following Editing of HLA-A 2.1. Karyotype Analysis

[0175] Karyotype analysis was performed at KromaTiD, Inc. (Longmont, CO, USA) to assess chromosomal structure. The integrity of chromosome 6 was assessed after HLA-A editing, as HLA-A resides on chromosome 6 band p21.3. On day 11 or 12 post thaw, edited cells were harvested and cryopreserved for karyotyping and ddPCR. Samples were prepared using standard  G-banding techniques. Two hundred metaphase spreads per sample were analyzed for structural abnormalities in chromosome 6. Industry-standard protocols were used to score events (ISCN 2020: An International System for Human Cytogenomic Nomenclature, 2020). Table 4 shows the number of chromosome 6p21.3 abnormalities detected per sample. Samples from Donor 2 (homozygous for HLA-H*01) had a higher number of deletions compared to samples from Donor 1 (heterozygous for HLA-H*01). In addition, samples where HLA-A was edited on day 4 have a higher number of deletions compared to samples where HLA-A was edited on day 1 or day 0. Figure 2 shows a karyotype image of chromosome 6 with a 6p21.3 deletion. Table 4. Number of deletions detected by karyotype analysis2.2. Droplet Digital PCR Analysis

[0176] Droplet Digital PCR (ddPCR) was performed to determine copy number variation (CNV) of the short arm of human chromosome 6 (6p) following T cell editing The NEDD9 gene, with a cytogenetic location of 6p24, was used as a marker for the distal portion of the short arm of human chromosome 6. The MRPL18 gene, with a cytogenetic location of 6q25.3, was used as a reference control on the long arm of human chromosome 6.

[0177] Genomic DNA (gDNA) was isolated from cryopreserved edited or unedited cells using the DNeasy Blood and Tissue Kits (Qiagen, Cat# 69506) according to the manufacturer’s instructions and diluted using molecular grade distilled water (Gibco, Cat# 46-00-CI). For the ddPCR assay, about 20 ng of gDNA from the editedcells of Donor 2 (homozygous for HLA- H*01) was mixed with 2× ddPCR Supermix for Probes (Bio-Rad, Cat# 1863024). Validated primers and probes for NEDD9 (Bio-Rad, Assay ID: dHsaCP1000467) and MRPL18 (Customized assay design, Bio-Rad) were added to final concentrations of 900 nM and 250 nM, respectively. Samples were processed with an Automated Droplet Generator (Bio-Rad, Cat# 1864101). Droplet fluorescence was measured using a QX200™ Droplet Reader (Bio-Rad, Cat# 1864003). The measured copy number variation (CNV) for each treatment was obtained using the QuantaSoft™ Software, Regulatory Edition (Bio-Rad, Cat# 1864011). The CNV was calculated using this formula: Copy Number Variation (CNV) = (NEDD9 copies / μl / MRPL18 copies / μl) * 2. Table 5 shows the CNV of the tested samples in triplicates. Unedited cells from Donor 2 (homozygous for HLA-H*01) have a mean CNV of 2.01, indicating that on average there are two intact copies of the short arm of chromosome 6 per cell. Cells edited for HLA-A had a reduction in mean CNV, indicating a loss in the short arm of chromosome 6. This reduction was heightened in cells where HLA-A editing was performed on Day 4 of the engineering process. Table 5. ddPCR analysis showing the copy number variations of Chr6p in tested samples  Example 3: Identification of pseudogene HLA-H*01 as an off-target locus

[0178] Without wishing to be bound by theory, it was hypothesized that the creation of a double strand break at a second, off-target, locus on the same arm of chromosome 6 as HLA-A could contribute to the loss of Chr6p21.3 observed on the karyotype analysis (illustrated in FIG. 2). In order to identify the homology of guides targeting HLA-A to off-target sites within other HLA genes, a bioinformatic assessment was performed using Spy cleavase guides targeting HLA-A (e.g., targeting the HLA-A*02 allele). Six guides targeting HLA-A as shown in Table 6, including G018995 used for HLA-A editing in the previous Examples, were all found to contain spacer sequences with 100% identity to both HLA-A and HLA-H*01, but not HLA-H*02, as shown in FIG. 5.

[0179] HLA-H is a pseudogene, which is linked to HLA-A. There are two major HLA-H alleles, HLA-H*01 and HLA-H*02. HLA-H*01 and HLA-H*02 have a percent identity of 99.31% as shown, e.g., in the alignment in FIG. 5 and are also highly similar to HLA-A. Table 6. Guides targeting HLA-AExample 4: Using a PCR assay to distinguish between HLA-H*01 and HLA-H*02

[0180] A PCR assay was designed to test whether donors were HLA-H*01 positive or negative.

[0181] Genomic DNA was isolated using the protocol of the Zymo Quick-DNA 96 kit (Zymo, Cat. # 50444139) for extraction. The PCR reaction was run using a master mix of 10 µl Q5 Hot Start Master mix (NEB, Cat. # M0494X), 6 µl molecular grade water (Corning, Cat. # 46-000-CI), 1 µl of forward primer, 1 µl of reverse primer, and 2 µl of genomic DNA per well.  The forward and reverse primers used are shown in Table 7. 18 µl of the final master mix and an additional 2 µl of genomic DNA were added to each well. Plates were spun at 1200 rpm for 1 minute and subsequently run on a Bio-Rad thermal cycler T100 (Bio-Rad, Cat. # 1861096). Table 7. Forward and reverse primer sequences for amplifying HLA-H*01

[0182] The PCR product was analyzed on a 2% E-get (Thermofisher, Cat. # G401002) using a Power Snap Electrophoresis Device (Invitrogen, Cat. # G8100) for 8 minutes. A clear band was present at 250 bp for HLA-H*01 positive donors, and no band was present in HLA- H*01 negative donors. Results are shown in FIGs. 6A and 6B. Example 5: Assessing chromosomal deletion events following HLA-A gene editing in donor cells pre-screened for HLA-H*01 expression

[0183] The number of chromosome 6p21.3 deletions was assessed in three HLA-H*01 positive and three HLA-H*01 negative donors in HLA-A edited or unedited T cells. T cells were edited as described in Example 1. 200 HLA-A edited or unedited T cells per donor were karyotyped as in Example 1. Results for HLA-H*01 positive donors are shown in FIG.7A and Table 8A. Results for HLA-H*01 negative donors are shown in FIG. 7B and Table 8B.

[0184] We observed that in HLA-H1- cells, the HLA-A editing resulted in a significant reduction of Chr6p21.3 deletions compared to when HLA-editing was performed in HLA-H1+ cells (FIGs. 7A and 7B). Each dot in FIG. 7A and FIG. 7B represents one donor and 200 cells karyotyped for each donor.

[0185] This study demonstrates that the presence of HLA-H*01 can lead to excision of a 55 kb region in chromosome 6 and loss of Chr6p21.3 in 2-4% of edited cells. Deletion of a portion of chromosome 6 was observed near the HLA-A / H sites in about 3% of cells. This study suggests that the detrimental effects of a chromosomal deletion such as that observed herein can be mitigated by selecting HLA-H*01 negative donor cells.  Table 8A: Chromosomal deletion number of HLA-A edited and unedited cells in HLA- H*01 positive donors.Table 8B: Chromosomal deletion number of HLA-A edited and unedited cells in HLA- H*01 negative donors.Example 6: Next generation sequencing to identify HLA-H*01 homozygous and HLA-H*01 heterozygous donors 6.1. Cell Preparation

[0186] Cells from 3 additional donor subjects (Donor 3, Donor 4, and Donor 5) were screened by PCR to determine HLA-H status. Donor 3 was identified to be homozygous for HLA-H*01, Donor 4 was identified to be heterozygous for HLA-H*01 / HLA-H*02, and Donor 5 was HLA-H*01 negative (homozygous for HLA-H*02). Cells were edited using sgRNA targeting HLA-A (G018995; SEQ ID NO:10). Genomic DNA (gDNA) was isolated from the cells, and idel formation at the HLA-H locus was assessed by next-generation sequencing.

[0187] Cells were centrifuged down as cell pellet at 500g for 5 minutes in a deep-well 96 well plate and either frozen to -80°C for future use, or genomic DNA (gDNA) was directly isolated using the Zymo quick DNA kit (Cat. D3012). 500 µl of genomic lysis buffer was then added directly to the cell pellet. The pellet was resuspended, and the mixture was transferred to the wells of a Silicon-A™ Plate on a collection plate for centrifugation at 2,500 - 5,000 x g for 5 minutes. Cells were washed and centrifuged twice before being transferred to a Silicon-A™ Plate onto an elution plate. Following washing the cells were eluted and the gDNA was  quantified using the nanodrop (Thermo Scientific™ 912A1100) and then utilized for Amplicon- seq PCR reaction. 6.2 Next-Generation Sequencing

[0188] NGS was used to assess indel formation at the HLA-H locus. Donors positive for HLA-H*01 had high HLA-H indel formation as shown in FIG. 8 and Table 9. Donors positive for HLA-H*01 were characterized by a mean percent indel above 90%. Table 9. HLA-H indels in donors homozygous, heterozygous, or negative for HLA-H*01Example 7: Materials and Methods 7.1. In vitro transcription (IVT) of mRNA

[0189] Capped and polyadenylated mRNA was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. The linearized plasmid DNA containing a T7 promoter and a sequence for transcription was linearized by restriction endonuclease digestion followed by heat inactivation of the reaction mixture, and purified from enzyme and buffer salts. Messenger RNA was synthesized and purified using standard techniques known in the art.

[0190] Messenger RNA was generated from plasmid DNA encoding an open reading frame.

[0191] When the sequences cited in this paragraph are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which can be modified nucleosides as described above). Messenger RNAs used in the Examples include a 5' cap and a 3' polyadenylation sequence e.g., up to 100 nts. Guide RNAs were chemically synthesized using standard in vitro synthesis techniques with modified nucleotides.  7.2. Lipid nanoparticle formulation

[0192] In general, the lipid nanoparticle (LNP) components were dissolved in 100% ethanol at various molar ratios. The RNA cargos (e.g., Cas9 mRNA and sgRNA) were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg / mL. The LNPs used contained ionizable lipid ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), also called herein Lipid A, cholesterol, distearoylphosphatidylcholine (DSPC), and 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2K-DMG) (catalog # GM-020 from NOF, Tokyo, Japan) in a molar ratio as stated in each Example. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6. The LNPs were prepared with a mixture of Cas9 mRNA and a guide RNA.

[0193] The LNPs were prepared using a cross-flow technique utilizing impinging jet mixing of the lipid in ethanol with two volumes of RNA solution and one volume of water. First, the lipid in ethanol was mixed through a mixing cross with the two volumes of RNA solution. Then, a fourth stream of water was mixed with the outlet stream of the cross through an inline tee (See FIG. 2 in WO 2016 / 010840). The LNPs were held for at least 1 hour at room temperature, and further diluted with water (approximately 1:1 v / v). Diluted LNPs were buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) and concentrated as needed by methods known in the art. The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNPs were characterized to determine the encapsulation efficiency, polydispersity index, and average particle size. The final LNPs were stored at 4 °C or -80 °C until further use. 7.3. T cell preparation

[0194] T cells were isolated from commercially obtained donor apheresis and cryopreserved. Upon thaw, T cells were plated at a density of 2.0 x 10^6 cells / mL in T cell growth media (TCGM) composed of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. # A1048501) containing 2.5% human AB serum (GeminiBio, Cat. # 100-512), 1X Penicillin-Streptomycin (ThermoFisher, Cat. # 15140122), 1X Glutamax (ThermoFisher, Cat. # 35050061), and 10 mM HEPES (ThermoFisher, Cat. #  15630106) further supplemented with 200 U / mL recombinant human interleukin-2 (Peprotech, Cat. # 200-02), 5 ng / mL recombinant human interleukin-7 (Peprotech, Cat. # 200-07), and 5 ng / mL recombinant human interleukin-15 (Peprotech, Cat. # 200-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec, Cat. # 130-111-1). OTHER EMBODIMENTS

[0195] It will be appreciated that the scope of the present disclosure is to be defined by that which may be understood from the disclosure and claims rather than by the specific embodiments that have been presented by way of example. Elements described with respect to one aspect or embodiment of the present disclosure are also contemplated with respect to other aspects or embodiments of the present disclosure. Throughout the description, where compositions or methods are described as having, including, or comprising specific elements, compositions that consist essentially of, consist of, or do not comprise the recited elements are likewise hereby disclosed. Moreover, recitation of claim elements in connection with a particular independent claim support recitation of such elements in connection with other independent claims. All references cited herein are hereby incorporated by reference.

Claims

CLAIMS What is claimed is:

1. A method of altering the expression of HLA-A in a population of cells, comprising providing a population of cells that have been pre-screened for the absence of one or both alleles of HLA-H*01, and contacting said population of cells with (i) a guide RNA targeting HLA-A and (ii) a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease, thereby effecting one or more double stranded breaks (DSBs) within the HLA-A gene that results in altered expression of HLA-A in the population of cells.

2. The method of claim 1, wherein the cells have been pre-screened for the absence of one allele of HLA-H*01.

3. The method of claim 1, wherein the cells have been pre-screened for the absence of two alleles of HLA-H*01.

4. The method of any one of claims 1-3, wherein the pre-screening comprises amplifying, detecting, and / or sequencing a portion of HLA-H corresponding to or comprising at least 15 nucleotides of SEQ ID NO: 1 or SEQ ID NO:

2.

5. The method of claim 4, wherein the method comprises sequencing, optionally wherein the sequencing comprises determining the sequence of at least a portion of the genome of the cells that corresponds to or comprises at least 15 nucleotides of SEQ ID NO: 1 or SEQ ID NO:

2.

6. The method of claim 4, wherein the pre-screening comprises contacting DNA extracted from the population of cells with at least one primer or probe which specifically hybridizes to a HLA-H*01 allele at a region comprising at least 15 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2; and detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO:

2.

7. The method of any one of claims 1-6, wherein the guide RNA is a sgRNA.

8. The method of any one of claims 1-7, wherein the guide RNA comprises a spacer / targeting sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 20 contiguous nucleotides of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or to a sequence complementary thereto.

9. The method of any one of claims 1-8, wherein the guide RNA comprises a spacer / targeting sequence comprising or consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or to a sequence complementary thereto.

10. The method of any one of claims 1-9, wherein the CRISPR-associated Cas endonuclease is Cas9.

11. The method of claim 10, wherein the Cas9 is selected from the group consisting of S. pyogenes, N. meningitides, S. wadsworthensis, F. alocis, L. johnsonii, C. lari, C. diptheriae, P. lavamentivorans, M. gallisepticum, S. aureus, L. pneumophila Paris, T. denticola, S. pseudintermedius, S. thermophilus, and N. cinerea.

12. The method of any one of claims 1-11 wherein the population of cells comprises human immune cells.

13. The method of claim 12, wherein the human immune cells comprise T cells, B cells, monocytes, macrophages, or NK cells, or combinations thereof.

14. The method of any one of claims 1-11, wherein the population of cells comprises human stem cells.

15. The method of claim 14, wherein the human stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic stem cells (HSCs).

16. The method of any one of claims 1-11, wherein the population of cells comprises beta cells, cardiomyocytes, or neurons.

17. The method of any one of claims 1-16, wherein fewer than 2% of the cells in the population of cells having altered expression of HLA-A comprise a chromosomal deletion at Chr6p21.

3.

18. A method of determining the HLA-H status of a population of cells derived from a human subject, comprising: providing DNA extracted from the cells; contacting the DNA with a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2; and detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2; wherein hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells comprise an HLA-H*01 allele, and wherein the absence of hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells do not comprise an HLA- H*01 allele.

19. The method of claim 18, wherein the primer or probe comprises at least 10 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 1 or SEQ ID NO:

2.

20. The method of claim 18, wherein the primer or probe comprises at least 10 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 3 or SEQ ID NO:

4.

21. The method of claim 18, wherein the primer or probe comprises at least 10 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 5 or SEQ ID NO:

6.

22. The method of any one of claims 18-21, wherein the probe comprises a detectable label.

23. The method of claim 22, wherein the detectable label is a fluorescent label or radiolabel.

24. The method of claim 22 or 23, wherein detecting hybridization of the probe to SEQ ID NO: 1 or SEQ ID NO: 2 comprises detecting association of the detectable label with the DNA.

25. The method of any one of claims 18-24, wherein detecting hybridization of the primer to SEQ ID NO: 1 or SEQ ID NO: 2 comprises detecting amplification of an HLA-H*01 amplification product.

26. The method of claim 25, wherein the HLA-H*01 amplification product is detected using an amplicon specific probe, a fluorescent probe, by detecting the corresponding amplicon size on an electrophoresis gel, or by sequencing the amplicon by NGS.

27. The method of any one of claims 18-26, wherein the DNA is contacted with a forward primer, a reverse primer, and a polymerase for a period of time sufficient to amplify a sequence comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO:

2.

28. A method of determining the HLA-H status of a population of cells derived from a human subject, comprising: providing DNA extracted from the cells; contacting the DNA with a forward primer, a reverse primer, and a polymerase, wherein the forward primer or the reverse primer specifically hybridizes to an HLA-H*01 allele at a region comprising at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2; amplifying a sequence comprising at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 by PCR; and detecting an amplicon comprising at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the amplicon is detected by gel electrophoresis (PCR), a fluorescent probe (qPCR), a hybridizing probe specific to the amplicon (ddPCR) or via NGS sequencing; wherein detection of the amplicon comprising at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells comprise an HLA-H*01 allele, and wherein the absence of the amplicon comprising at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells do not comprise an HLA-H*01 allele.

29. The method of claim 28, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 1, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

2.

30. The method of claim 28 or 29, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 3, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

4.

31. The method of any one of claims 28-30, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 5, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

6.

32. The method of any one of claims 28-31, wherein the reverse primer comprises SEQ ID NO: 6, and wherein the forward primer comprises SEQ ID NO:

7.

33. The method of any one of claims 18-32, wherein the DNA is genomic DNA.

34. The method of any one of claims 18-33, wherein the cells are human immune cells.

35. The method of claim 34, wherein the human immune cells comprise T cells, B cells, monocytes, macrophages, NK cells, or combinations thereof.

36. The method of any one of claims 18-33, wherein the population of cells comprises human stem cells.

37. The method of claim 36, wherein the human stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic stem cells (HSCs).

38. The method of any one of claims 18-33, wherein the population of cells comprises beta cells, cardiomyocytes, or neurons.

39. A method of selecting a population of cells suitable for editing with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease, comprising: providing DNA extracted from the population of cells; contacting the DNA with a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2; detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the absence of hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the cells do not comprise an HLA-H*01 allele and thereby are suitable for editing with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease; and selecting the population of cells that do not comprise HLA-H*01 for editing with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease.

40. The method of claim 39, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 1 or SEQ ID NO:

2.

41. The method of claim 39, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 3 or SEQ ID NO:

4.

42. The method of claim 39, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 5 or SEQ ID NO:

6.

43. The method of any one of claims 39-42, wherein the probe comprises a detectable label.

44. The method of claim 43, wherein the detectable label is a fluorescent label or radiolabel.

45. The method of claim 43 or 44, wherein detecting hybridization of the probe to SEQ ID NO: 1 or SEQ ID NO: 2 comprises detecting association of the detectable label with the DNA.

46. The method of any one of claims 39-45, wherein the DNA is contacted with a forward primer, a reverse primer, and a polymerase for a period of time sufficient to amplify a sequence comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO:

2.

47. The method of claim 46, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 1, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

2.

48. The method of claim 46, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 3, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

4.

49. The method of claim 46, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 5, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

6.

50. The method of claim 46, wherein the reverse primer comprises SEQ ID NO: 6, and wherein the forward primer comprises SEQ ID NO:

7.

51. The method of any one of claims 46-50, wherein detecting hybridization of the primer to SEQ ID NO: 1 or SEQ ID NO: 2 comprises detecting amplification of an HLA-H*01 amplification product.

52. The method of claim 51, wherein the HLA-H*01 amplification product is detected using an amplicon specific probe, a fluorescent probe, by detecting the corresponding amplicon size on an electrophoresis gel, or by sequencing the amplicon by NGS.

53. The method of any one of claims 39-52, wherein the DNA is genomic DNA.

54. The method of any one of claims 39-53, further comprising contacting the selected population of cells with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease.

55. The method of claim 54, wherein the CRISPR-associated Cas endonuclease is Cas9.

56. The method of claim 55, wherein the Cas9 is selected from the group consisting of S. pyogenes, N. meningitides, S. wadsworthensis, F. alocis, L. johnsonii, C. lari, C. diptheriae, P. lavamentivorans, M. gallisepticum, S. aureus, L. pneumophila Paris, T. denticola, S. pseudintermedius, S. thermophilus, and N. cinerea.

57. The method of any one of claims 39-56, wherein the guide RNA is a sgRNA.

58. The method of any one of claims 39-57, wherein the guide RNA comprises a spacer / targeting sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 20 contiguous nucleotides of SEQ ID NO: 8 or to a sequence complementary thereto.

59. The method of any one of claims 39-58, wherein the guide RNA comprises a spacer / targeting sequence comprising or consisting of SEQ ID NO:

8.

60. The method of any one of claims 39-59, wherein the cells are human immune cells.

61. The method of claim 60, wherein the human immune cells comprise T cells, B cells, monocytes, macrophages, NK cells, or combinations thereof.

62. The method of any one of claims 39-59, wherein the population of cells comprises human stem cells.

63. The method of claim 62, wherein the human stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic stem cells (HSCs).

64. The method of any one of claims 39-59, wherein the population of cells comprises beta cells, cardiomyocytes, or neurons.

65. A method of identifying a subject suitable as a donor of cells to be edited with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease, the method comprising providing DNA extracted from a cell sample derived from the subject; contacting the DNA with a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2; and detecting hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the absence of hybridization of the primer or probe to SEQ ID NO: 1 or SEQ ID NO: 2 indicates that the subject does not comprise an HLA-H*01 allele and thereby is suitable as a donor of cells to be edited with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease.

66. The method of claim 65, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 1 or SEQ ID NO:

2.

67. The method of claim 65, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 3 or SEQ ID NO:

4.

68. The method of claim 65, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 5 or SEQ ID NO:

6.

69. The method of any one of claims 65-68, wherein the probe comprises a detectable label.

70. The method of claim 69, wherein the detectable label is a fluorescent label or radiolabel.

71. The method of claim 69 or 70, wherein detecting hybridization of the probe to SEQ ID NO: 1 or SEQ ID NO: 2 comprises detecting association of the detectable label with the DNA.

72. The method of any one of claims 65-71, wherein the DNA is contacted with a forward primer, a reverse primer, and a polymerase for a period of time sufficient to amplify a sequence comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO:

2.

73. The method of claim 72, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 1, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

2.

74. The method of claim 72, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 3, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

4.

75. The method of claim 72, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 5, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

6.

76. The method of claim 72, wherein the reverse primer comprises SEQ ID NO: 6, and wherein the forward primer comprises SEQ ID NO:

7.

77. The method of any one of claims 65-76, wherein detecting hybridization of the primer to SEQ ID NO: 1 or SEQ ID NO: 2 comprises detecting amplification of an HLA-H*01 amplification product.

78. The method of claim 77, wherein the HLA-H*01 amplification product is detected using an amplicon specific probe, a fluorescent probe, by detecting the corresponding amplicon size on an electrophoresis gel, or by sequencing the amplicon by NGS.

79. The method of any one of claims 65-78, wherein the DNA is genomic DNA.

80. The method of any one of claims 65-79, further comprising obtaining a population of human immune cells from a subject identified as a suitable donor.

81. The method of claim 80, wherein the human immune cells comprise T cells, B cells, monocytes, macrophages, NK cells, or combinations thereof.

82. The method of any one of claims 65-79, wherein the population of cells comprises human stem cells.

83. The method of claim 82, wherein the human stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic stem cells (HSCs).

84. The method of any one of claims 65-79, wherein the population of cells comprises beta cells, cardiomyocytes, or neurons.

85. The method of any one of claims 65-84, further comprising contacting the obtained population with a guide RNA targeting HLA-A and a CRISPR-associated Cas endonuclease or a nucleic acid encoding a CRISPR-associated Cas endonuclease.

86. The method of claim 85, wherein the CRISPR-associated Cas endonuclease is Cas9.

87. The method of claim 86, wherein the Cas9 is selected from the group consisting of S. pyogenes, N. meningitides, S. wadsworthensis, F. alocis, L. johnsonii, C. lari, C. diptheriae, P. lavamentivorans, M. gallisepticum, S. aureus, L. pneumophila Paris, T. denticola, S. pseudintermedius, S. thermophilus, and N. cinerea.

88. The method of any one of claims 65-87, wherein the guide RNA is a sgRNA.

89. The method of any one of claims 65-88, wherein the guide RNA comprises a spacer / targeting sequence at least 80%, 85%, 90%, or 95% identical to at least 20 contiguous nucleotides of SEQ ID NO: 8 or to a sequence complementary thereto.

90. The method of any one of claims 65-89, wherein the guide RNA comprises a spacer / targeting sequence comprising or consisting of SEQ ID NO:

8.

91. A population of cells comprising an edited HLA-A gene, wherein the edited HLA-A gene comprises one or more insertions and / or deletions relative to a wild-type HLA-A gene sequence, and wherein fewer than 2% of the cells comprise a chromosomal deletion at Chr6p21.

3.

92. The population of cells of claim 91, wherein the one or more insertions and / or deletions decrease expression of HLA-A.

93. The population of cells of claim 91 or 92, wherein the cells are heterozygous for HLA- H*01.

94. The population of cells of claim 91 or 92, wherein the cells do not comprise HLA-H*01.

95. The population of cells of any one of claims 91-94, wherein the cells comprise HLA- H*02.

96. The population of cells of any one of claims 91-95, wherein the cells are human immune cells.

97. The population of cells of claim 96, wherein the human immune cells comprise T cells, B cells, monocytes, macrophages, NK cells, or combinations thereof.

98. The population of cells of any one of claims 91-95, wherein the population of cells comprises human stem cells.

99. The population of cells of claim 98, wherein the human stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), or hematopoietic stem cells (HSCs).

100. The population of cells of any one of claims 91-95, wherein the population of cells comprises beta cells, cardiomyocytes, or neurons.

101. A composition or kit comprising (i) a primer or probe which specifically hybridizes to a HLA-H*01 allele at a region comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) a polymerase.

102. The composition or kit of claim 101, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 1 or SEQ ID NO:

2.

103. The composition or kit of claim 101, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 3 or SEQ ID NO:

4.

104. The composition or kit of claim 101, wherein the primer or probe comprises at least 15 contiguous nucleotides complementary to all or a portion of SEQ ID NO: 5 or SEQ ID NO:

6.

105. The composition or kit of any one of claims 101-104, wherein the probe comprises a detectable label.

106. The composition or kit of claim 105, wherein the detectable label is a fluorescent label or radiolabel.

107. The composition or kit of any one of claims 101-106 wherein the composition or kit comprises a forward primer and a reverse primer for amplification of an HLA-H*01 sequence comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO:

2.

108. The composition or kit of claim 107, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 1, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

2.

109. The composition or kit of claim 107, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 3, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

4.

110. The composition or kit of claim 107, wherein the forward primer comprises at least 15 contiguous nucleotides of SEQ ID NO: 5, or wherein the reverse primer comprises at least 15 contiguous nucleotides of SEQ ID NO:

6.

111. The composition or kit of claim 107, wherein the reverse primer comprises SEQ ID NO: 6, and wherein the forward primer comprises SEQ ID NO: 7.

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